NO320241B1 - Fragment of ICAM-1 and its use in in vitro diagnosis - Google Patents

Description

The present invention relates to a fragment of intracellular adhesion molecule-1 (ICAM-1) and its use in in vitro diagnosis.

Leukocytes must be able to attach to cell substrates in order to adequately defend the host against foreign invaders such as bacteria or viruses. An excellent overview of the defense system is provided by H.W. Eisen, (In: Microbiology, 3rd ed. Harper & Row, Philadelphia, PA (1980), pp. 290-295 and 381-418). They must be able to attach to endothelial cells so that they can migrate from the circulation to sites of inflammation. Furthermore, they must be attached to antigen-presenting cells, so that a normal specific immune response can occur, and finally they must be attached to suitable target cells so that virus-infected cells or tumor cells can be lysed.

Leukocyte surface molecules involved in effecting such adhesions have recently been identified using hybridoma technology. Briefly, monoclonal antibodies directed against human T cells (D. Davignon et al., Proe. Nati. Acad. Sei. USA, 78:4535-4539 (1981)) and mouse spleen cells (T. Springer et al. ., Eur. J. Immunol., 9:301-306 (1979)) which bound to leukocyte surfaces and inhibited the adhesion-related functions described above (T. Springer et al., Fed. Proe, 44:2660-2663 (1985 )).The molecules identified by these antibodies were named Mac-1 and lymphocyte function-associated antigen 1 (LFA-1). Mac-1 is a heterodimer found on macrophages, granulocytes and large granular lymphocytes. LFA-1 is a heterodimer that can be found on

most lymphocytes (T.A. Springer et al., Immunol. Rev., 68:111-135 (1982)). These two molecules, plus a third molecule, p150.95 (which has a tissue distribution similar to Mac-1) play a role in cellular adhesion (G. Keizer et al., Eur. J. Immunol., 15:1142-1147 (1985 )).

The leukocyte molecules described above were found to be members of a related family of glycoproteins (F. Sanchez-Madrid et al., J. Exper. Med., 158:1785-1803 (1983);

G. D. Keizer et al., Eur. J. Immunol., 15:1142-1147 (1985)).

This glycoprotein family consists of heterodimers with one alpha chain and one beta chain. Although the alpha chain in each of the antigen genes differed from one another, the beta chain was found to be the most highly conserved (F. Sanchez-Madrid et al., J. Exper. Med., 158:1785-1803 (1983)). The beta chain of the glycoprotein family (sometimes referred to as "CD18") was found to have a molecular weight of 95 kd, while the alpha chains were found to range from 150 kd to 180 kd (T. Springer, Fed. Proe, 44:2660-2663

(1985)). Although the alpha subunits of the membrane proteins do not have the extensive homology of the beta subunits, careful analysis of the alpha subunits of the glycoproteins has shown that there are significant similarities between them. Overviews of the similarities between the alpha and beta subunits of the LFA-1 related glycoproteins are provided by F. Sanchez-Madrid et al., (J. Exper. Med., 158:586-602 (1983); J. Exper. Med., 158:1785-1803

(1983)).

A group of individuals has been identified that cannot express normal amounts of any members of this adhesion protein family on their leukocyte cell surface (D.C. Anderson et al., Fed. Proe, 44:2671-2677 (1985); D.C. Anderson et al., J. Infect. Dis., 152:668-689 (1985)). Lymphocytes from these patients showed defects in vi tro similar to corresponding normal individuals whose LFA-1 molecular family had been counteracted by antibodies. Furthermore, these individuals could not produce a normal immune response due to that their cells were unable to adhere to cell substrates (D.C. Anderson et al., Fed. Proe, 44:2671-2677 (185); D.C. Anderson et al., J. Infect. Dis., 152:668- 689 (1985)). These data show that immune reactions are dampened when lymphocytes cannot attach normally due to lack of functional adhesion molecules in the LFA-1 family.

In summary, the ability of lymphocytes to maintain an animal's health and viability requires that they can attach to other cells (such as endothelial cells). This adhesion has been found to require cell-cell contacts that involve the presence of specific receptor molecules on the lymphocyte's cell surface. These receptors enable a lymphocyte to adhere to other lymphocytes or to endothelial cells and other non-vascular cells. The cell surface receptor molecules have been found to be highly related to each other. Humans whose lymphocytes lack these cell surface receptor molecules show chronic and recurrent infections, as well as other clinical symptoms including deficient antibody responses.

Since lymphocyte adhesion is part of the process by which foreign tissue is identified and rejected, an understanding of this process is of considerable value in the areas of organ transplantation, tissue transplantation, allergy and oncology.

The present invention relates to a diagnostic, applicable, water-soluble, functional derivative of ICAM-1 which is a fragment of ICAM-1, characterized in that it lacks the cytoplasmic domain and the transmembrane domain of ICAM-1, and it contains domain 1 or domains 1 and 2, or fields 1, 2 and 3.

The invention also includes the use of the derivative according to the invention for in vitro diagnosis of the presence and location of a tumor cell expressing a member of the LFA-1 molecular family in an individual suspected of having such a cell, by a) incubating a tissue sample from the individual in the presence of a mixture containing a detectable, labeled, water-soluble,

functional derivative according to claim 1, and

b) detection of said binding ligand which is bound to a member of the LFA-1 molecular family present in the tissue sample.

A method for in vitro diagnosis of the presence and location of a tumor cell expressing a member of the LFA-1 molecule family in a mammal suspected of having such a cell is described, comprising:

Ca) a tissue sample from the individual is incubated in the presence of a detectably labeled binding ligand capable of binding to a member of the LFA-1 family of molecules, which ligand is selected from the group consisting of ICAM-1 and a functional derivative of ICAM-1, and ( b) the binding ligand bound to a member of the LFA-1 molecule family present in the tissue sample is detected.

Reference is now made to the drawings:

Figure 1 shows in schematic form the adhesion between a normal cell and a cell lacking LFA-1. Figure 2 shows in schematic form the process of a normal/- normal cell adhesion. Figure 3 shows the kinetics of cellular aggregation in the absence (X) or presence of 50 ng/ml PMA (O). Figure 4 shows co-aggregation between LFA-1" and LFA-1<+-> cells. Carboxyfluorescein diacetate-labeled EBV-transformed cells (IO<4>) as shown in the figure were mixed with 10<5> unlabeled autologous cells (solid bars) or JY cells (open bars) in the presence of PMA. After 1.5 h, the labeled cells, in aggregates or free, were counted using the qualitative assay of Example 2. The percentage of labeled cells in aggregates is shown. One representative experiments of two are shown. Figure 5 shows immunoprecipitation of ICAM-1 and LFA-1 from JY cells. Triton X-100 lysates of JY cells (lanes 1 and 2) or control lysis buffer (lanes 3 and 4) were immunoprecipitated with antibody that could bind to ICAM-1 (lanes 1 and 3) or antibodies that could bind to LFA-1 (lanes 2 and 4).Part A shows results under reducing conditions; Part B shows results obtained under non-reducing conditions .Molecular weight standards were run in field S. Figure 6 shows the kinetics of IL-1 and gamma interferon effects on ICAM-1 expression on human skin fibroblasts. Human skin fibroblasts were grown to a density of 8 x 10 cells/0.32 cm well. IL-1 (10 U/ml, closed circles) or recombinant gamma interferon (10 U/ml, open squares) was added, and at the indicated time, the assay was cooled to 4°C, and an indirect binding assay was performed. The standard deviation did not exceed 10%. Figure 7 shows the concentration dependence of IL-1 and gamma interferon effects on ICAM-1. Human skin fibroblasts were grown to a density of 8 x 10<4> cells/0.32 cm<2>/well. IL-2 (open circle), recombinant human IL-1 (open square), recombinant mouse IL-1 (solid square), recombinant human gamma interferon (solid circles) and recombinant beta interferon (open triangle) were added in the indicated dilution and was incubated for 4 hours (IL-1) or 16 hours (beta and gamma interferon). The results given are the mean values from quadruplicate determinations; standard deviation did not exceed 10%. Figure 8 shows the nucleotide and amino acid sequence of ICAM-1 cDNA. The first ATG is at position 58. Translated sequences corresponding to tryptic ICAM-1 peptides are underlined. The hydrophobic putative signal peptide and transmembrane sequences are strongly underlined. N-linked glycosylation sites are boxed. A line is drawn over the polyadenylation signal AATAAA at position 2976. The sequence shown is for the HL-60 cDNA clone. Endothelial cell cDNA was sequenced over the majority of its length and showed only minor differences. Figure 9 shows the ICAM-1 homologous regions and the relationship with the immunoglobulin supergene family. (A) arrangement of 5 homologous regions (Dl-5). Two or more identical residues that matched are boxed. Residues conserved two or more times in NCAM regions, as well as residues conserved in regions in sets C2 and C1 were aligned with the internal ICAM-1 repeat units. The location of the predicted β-chains in the ICAM-1 region is marked with columns and lowercase letters above the alignments, and the known location for 6-chains in immunoglobulin C regions is marked with columns and uppercase letters below the alignment. The position of the putative disulfide bridge in ICAM-1 regions is indicated by S-S. (B-D) alignment of protein regions homologous to ICAM-1 regions; the proteins were initially aligned by examining NBRF databases using the FASTP program. The protein sequences are MAG, NCAM, T-cell receptor α-subunit V-region, IgM^-chain, and α-1-B-glycoprotein. Figure 10 shows a schematic comparison of the secondary structures of ICAM-1 and MAG. Figure 11 shows LFA-1-positive EBV-transformed B-lymphoblastoid cells that bind to ICAM-1 in planar membranes. Figure 12 shows LFA-1-positive T-lymphoblasts and T-lymphoma cells that bind to ICAM-1 in plastic-bound vesicles. Figure 13 shows the inhibition of binding of JY-B lymphoblastoid cells that bind to ICAM-1 in plastic-bound vesicles by pretreatment of cells or vesicles with monoclonal antibodies. Figure 14 shows the effect of temperature on binding of T-lymphoblasts to ICAM-1 in plastic-bound vesicles. Figure 15 shows requirements regarding divalent cations for binding of T-lymphoblasts to ICAM-1 in plastic bound vesicles. Figure 16 shows the effect of anti-adhesion antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to perception of the T-cell associated antigen 0KT3. "0KT3" indicates addition of antigen. Figure 17 shows the effect of anti-adhesion antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of the non-specific T-cell mitogen, concanavalin A. "CONA" denotes the addition of concanavalin A. Figure 18 shows the effect of anti -adhesion antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to recognition of the "keyhole limpet" hemocyanin antigen gene. The addition of keyhole limpet hemocyanin to the cells is indicated by "KLH". Figure 19 shows the effect of anti-adhesion antibodies on the ability of peripheral blood mononuclear cells to proliferate in response to perception of the tetanus toxoid antigen. The addition of tetanus toxoid antigen to the cells is indicated by

"BAIT".

Figure 20 shows the binding of monoclonal antibodies RR1/1, R6.5, LB2 and CL203 to ICAM-1 knockout mutants. Figure 21 shows the binding of ICAM-1 knockout mutants to LFA-1. Figure 22 shows the epitopes recognized by monoclonal anti-ICAM-1 antibodies RR1/1, R6.5, LB2 and CL203. Figure 23 shows the binding capacity of ICAM-1 region 2 mutants to LFA-1. Figure 24 shows the binding capacity of ICAM-1 region 3 mutants to LFA-1. Figure 25 shows the binding capacity of ICAM-1 region 1 mutants to LFA-1. Figure 26 shows the alignment of amino-terminal ICAM regions.

One aspect of the present invention concerns the discovery of a natural binding ligand for LFA-1. Molecules such as those in the LFA-1 family, which are involved in the process of cell adhesion, are referred to as "adhesion molecules".

The natural binding ligand is termed "intercellular adhesion molecule" or "ICAM-1". ICAM-1 is a 76-97 kd glycoprotein. ICAM-1 is not a heterodimer. The present invention is directed to ICAM-1 and its "functional derivatives". A "functional derivative" of ICAM-1 is a compound that has a biological activity (either functional or structural) that is substantially similar to a biological activity of ICAM-1. The term "functional derivatives" is intended to include the "fragments", "variants", "analogs" or "chemical derivatives" of a molecule. A "fragment" of a molecule such as ICAM-1 is intended to denote any polypeptide subset of the molecule. Fragments of ICAM-1 which have ICAM-1 activity and which are soluble (ie not membrane bound) are particularly preferred. A "variant" of a molecule such as ICAM-1 is intended to denote a molecule with substantially similar structure and function to either the entire molecule or a fragment thereof. A molecule is said to be "substantially similar" to another molecule if both molecules have substantially similar structures, or if both molecules have similar biological activity. On the condition that two molecules have equal activity, they are thus considered variants as this designation is used herein, even if the structure of one of the molecules is not found in the other, or if the sequence of amino acid residues is not identical. An "analogue" of a molecule such as ICAM-1 is intended to mean a molecule having substantially the same function as either the entire molecule or a fragment thereof. As used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties that are not normally part of the molecule. Such parts can improve the molecule's solubility, absorption, biological half-life, etc. The parts can alternatively reduce the molecule's toxicity, eliminate or dampen any undesirable side effects of the molecule, etc. Parts that can produce such effects are described in Remington's Pharmaceutical Sciences (1980). "Toxin-derivatized" molecules constitute a special class of "chemical derivatives". A "toxin derivatized" molecule is a molecule (such as ICAM-1 or an antibody) that contains a toxin moiety. The binding of such a molecule to a cell brings the toxin part into close proximity to the cell and thereby accelerates cell death. Any suitable toxin moiety may be used; however, it is preferred to use the toxins such as e.g. ricinus toxin, diphtheria toxin, radioisotopic toxins, membrane channel-forming toxins, etc. Methods for connecting such parts to a molecule are well known in the field.

An antigenic molecule such as ICAM-1, or members of the LFA-1 molecule family, is naturally expressed on the surface of lymphocytes. Thus, introduction of such cells into a suitable animal, such as by intraperitoneal injection, etc, will result in the formation of antibodies that can bind to ICAM-1 or members of the LFA-1 molecular family. If desired, the serum from such an animal can be removed and used as a source of polyclonal antibodies that can bind these molecules. However, it is preferred to remove splenocytes from such animals, to fuse such spleen cells with a myeloma cell line, and to allow such fusion cells to form a hybridoma cell that secretes monoclonal antibodies capable of binding ICAM-1 or members of the LFA-1 family of molecules.

The hybridoma cells obtained in the manner described herein can be screened by many different methods to identify desired hybridoma cells that secrete antibody that can bind either to ICAM-1 or to members of the LFA-1 family of molecules. In a preferred screening assay, such molecules are identified by their ability to inhibit the aggregation of Epstein-Barr virus-transformed cells. Antibodies which can inhibit such aggregation are then further examined to determine whether they inhibit such aggregation by binding to ICAM-1, or to a member of the LFA-1 molecule family. Any aid that can distinguish ICAM-1 from the LFA molecular family can be used in such an investigation. Thus, e.g. the antigen bound by the antibody is analyzed as by immunoblotting and polyacrylamide gel electrophoresis. If the bound antigen is a member of the LFA-1 family of molecules, the immunoprecipitated antigen will be found to be a dimer, whereas if the bound antigen is ICAM-1, a single molecular weight compound will have been immunoprecipitated. Alternatively, it is possible to distinguish such antibodies that bind to members of the LFA-1 family of molecules from those that bind ICAM-1 by examining the antibody's ability to bind to cells such as granulocytes, which express LFA-1, but not ICAM-1. The ability of an antibody (which is known to inhibit cell aggregation) to bind to granulocytes indicates that the antibody can bind LFA-1. Absence of such binding indicates an antibody capable of recognizing ICAM-1. The ability of an antibody to bind to a cell such as a granulocyte can be demonstrated by aids that are usually used by ordinary professionals. Such aids include immunoassays, cell agglutination, filter binding studies, antibody precipitation, etc.

Alternatively, the anti-aggregation antibodies can be identified by measuring their ability to bind differentially to cells expressing ICAM-1 (such as activated endothelial cells) and their inability to bind to cells not expressing ICAM-1. As will be readily understood by those of ordinary skill in the art, the above assays can be modified or performed in a different sequential order to provide different potential screening assays, each of which can identify and discriminate between antibodies that can bind to ICAM-1 and members of the LFA family of molecules.

A. Identification of the LFA-1 binding partner (ICAM-1)

1. Analyzes of LFA-1-dependent aggregation

Many Epstein-Barr virus-transformed cells show aggregation. This aggregation can be increased by the presence of phorbol esters. Such homotypic aggregation (ie, aggregation involving only one cell type) was found to be blocked by anti-LFA-1 antibodies (R. Rothlein et al., J. Exper. Med., 163:1132-1149 (1986)), which reference is incorporated herein by reference. Thus, the extent of LFA-1-dependent binding can be determined by assessing the extent of spontaneous or phorbol ester-dependent aggregate formation.

An agent that interacts with LFA-1-dependent aggregation can be identified using an assay that can determine whether the agent interacts with either the spontaneous aggregation or the phorbol ester-dependent aggregation of Epstein-Barr virus-transformed cells. Most Epstein-Barr virus-transformed cells can be used in such an analysis as long as the cells can express the LFA-1 receptor molecule. Such cells can be produced according to the technique of T.A. Springer et al., J. Exper Med., 160:1901-1918 (1984), which reference is incorporated herein by reference. Although any such cell may be used in the LFA-1-dependent binding assay of the present invention, it is preferred to use cells from the JY cell line (C.T. Terhost et al., Proe. Nati. Acad. Sei. USA, 73: 910 (1976)). The cells can be cultured in any suitable culture medium; however, it is most preferred to grow the cells in RMPI 1640 culture medium supplemented with 10% fetal calf serum and 50 µg/ml gentamycin (Gibco Laboratories, NY). The cells should be cultured under conditions suitable for mammalian cell propagation (ie at a temperature of typically 37°C, in an atmosphere of 5% CO 2 , at a relative humidity of 95%, etc).

2. LFA-1 binds to ICAM-1

Individuals have been identified among humans whose lymphocytes lack the LFA-1 family of receptor molecules (D.C. Anderson et al., Fed. Proe, 44:2671-2677 (1985); D.C. Anderson et al., J. Infect. Dis., 152: 668-689 (1985)). Such individuals are said to suffer from leukocyte adhesion deficiency (LAD). EBV-transformed cells from such individuals do not aggregate either spontaneously or in the presence of phorbol esters in the aggregation assay described above. When such cells were mixed with LFA-1 expressing cells, aggregation was observed (R. Rothlein et al., J. Exper. Med., 163:1132-1149 (1986)) (Figure 1). It is significant that these a Aggregates were not formed if these cells were incubated in the presence of anti-LFA-1 antibodies. Thus, although the aggregation required LFA-1, the ability of cells lacking LFA-1 to form aggregates with LFA-1-containing cells demonstrated that the LFA-1 binding partner was not LFA-1, but rather a previously undiscovered cell adhesion molecule. Figure 1 shows the mechanism of cell adhesion. B. Intercellular adhesion molecule 1 (ICAM-1); The new intercellular adhesion molecule ICAM-1 was first identified and partially characterized according to the method of R. Rothlein et al., (J. Immunol., 137:1270-1274 ; (1986)) , which reference is incorporated herein by reference. For detection of the ICAM-1 molecule, monoclonal antibodies were prepared from spleen cells of mice immunized with cells from individuals genetically lacking LFA-1 expression. Resulting antibodies were examined for their ability to inhibit the aggregation of LFA-1 expressing cells (Figure 2). In more detail, mice were immunized with EBV-transformed B cells from LAD patients that do not express the LFA-1 antigen. The spleen cells from these animals were then removed, fused with myeloma cells, and allowed to become hybridoma cells that produced monoclonal antibodies. EBV-transformed B cells from normal individuals expressing LFA-1 were then incubated in the presence of the monoclonal antibody from the hybridoma cell to identify any monoclonal antibody that could inhibit the phorbol ester-mediated, LFA-1-dependent, spontaneous aggregation of the EBV-transformed B cells. Since the hybridoma cells were derived from cells that had never encountered the LFA-1 antigen, no monoclonal antibody to LFA-1 was formed. Consequently, any antibody found to inhibit aggregation must be able to bind to an antigen which, although not LFA-1, participates in the LFA-1 adhesion process. Although any method of obtaining such monoclonal antibodies may be used, it is preferred to obtain ICAM-1 binding monoclonal antibodies by immunization of Balb/C mice using the methods and programs described by R. Rothlein et al. , (J. Immunol., 137:1270-1274 (1986)) with Epstein-Barr virus-transformed peripheral blood mononuclear cells from individuals deficient in LFA-1. Such cells are described by T.A. Springer et al., (J. Exper Med., 160:1901-1918 (1984)). ;In a preferred method for the formation and detection of antibodies that can bind to ICAM-1, mice are immunized with either EBV-transformed B cells that express both ICAM-1 and LFA-1, or more preferably with TNF-activated endothelial cells that express ICAM-1 but not LFA-1. In a most preferred method for the formation of hybridoma cells which form anti-ICAM-1 antibodies, a Balb/C mouse was sequentially immunized with JY cells and with differentiated U937 cells (ATCC CRL-1593). The spleen cells from such animals are removed, fused with myeloma cells and allowed to develop into antibody-producing hybridoma cells. The antibodies are tested for their ability to inhibit the LFA-1-dependent, phorbol ester-induced aggregation of an EBV-transformed cell line, such as JY cells, which express both the LFA-1 receptor and ICAM-1. As shown by R. Rothlein et al., (J. Immunol., 137:1270-1274 (1987)), antibodies which can. inhibit such aggregation, with regard to its ability to inhibit the phorbol ester-induced aggregation of a cell line, such as SKW3; (M. Dustin et al., J. Exper Med., 165:672-692 (1987)), if ability to spontaneously aggregate in the presence of a phorbol ester is inhibited by antibody that can bind LFA-1, but is not inhibited by anti-ICAM-1 antibodies. Antibodies which can inhibit the phorbol ester-induced aggregation of cells such as JY cells, but which cannot inhibit the phorbol ester-induced aggregation of cells such as SKW3 cells, are likely to be anti-ICAM-1 antibodies. Alternatively, antibodies that can bind to ICAM-1 can be identified by screening for antibodies that can inhibit the LFA-1-dependent aggregation of LFA-expressing cells (such as JY cells) but cannot bind to LFA-expressing cells -1, but little or no ICAM-1 (such as normal granulocytes), or that can bind to cells expressing ICAM-1 but not LFA-1 (such as TNF-activated endothelial cells). Another option is to immunoprecipitate from cells expressing ICAM-1, LFA-1, or both, using antibodies that inhibit the LFA-1-dependent aggregation of cells, such as JY cells, and by SDS-PAGE or an equivalent method determines some molecular character property of the molecule precipitated with the antibody. If the characteristic is the same as that of ICAM-1, the antibody can be assumed to be an anti-ICAM-1 antibody. Using monoclonal antibodies prepared as described above, the ICAM-1 cell surface molecule was purified and characterized. ICAM-1 was purified from human cells or tissues using monoclonal antibody affinity chromatography. In such a method, a monoclonal antibody that can react with ICAM-1 is coupled to an inert column matrix. Any method of performing such coupling may be used; however, it is preferred to use the method according to H.C. Oettgen et al., J. Biol. Chem., 259:12034 (1984)). When a cell lysate is passed through the matrix, the ICAM-1 molecules present are adsorbed and retained by the matrix. By changing the pH or the ion concentration in the column, the bound ICAM-1 molecules can be eluted from the column. Although any suitable matrix can be used, it is preferred to use Sepharose (Pharmacia) as matrix material. The formation of column matrices and their use in protein purification is well known in the art. In a manner understood by those of ordinary skill in the art, the above-described assays can be used to identify compounds that can impair or inhibit the degree or extent of cell adhesion. ;ICAM-1 is a cell surface glycoprotein that is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, certain other epithelial cells and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T-lymphocyte blasts and nucleated B cells and dendritic cells in tonsils, lymph nodes and the Peyer's patches. ICAM-1 is highly expressed on vascular endothelial cells in T-cell areas of lymph nodes and tonsils showing reactive hyperplasia. ICAM-1 is expressed in small amounts on lymphocytes in peripheral blood. Phorbol ester-stimulated differentiation of some myelomonocytic cell lines greatly increases ICAM-1 expression. Thus, ICAM-1 is preferentially expressed at sites of inflammation and is not usually expressed by resting cells. ICAM-1 expression on skin fibroblasts is increased three- to fourfold by either interleukin 1 or gamma interferon at levels of 10 U/ml over a period of 4 or 10 hours, respectively. The induction is dependent on protein and mRNA synthesis and is reversible. ;ICAM-1 shows molecular weight heterogeneity in different cell types with a molecular weight of 97 kd on fibroblasts, 114 kd on the myelomonocytic cell line U937, and 90 kd on the lymphoblastoid B cell JY. ICAM-1 biosynthesis has been found to involve an intracellular precursor of ca. 73 kd. The non-N-glycosylated form resulting from tunicamycin treatment (which inhibits glycosylation) has a molecular weight of 55 kd. ;ICAM-1 isolated from phorbol ester-stimulated U937 cells or from fibroblast cells gives an identical major product with a molecular weight of 60 kd after chemical deglycosylation. Monoclonal ICAM-1 antibodies interact with the adhesion of phytohemagglutinin blasts to cell lines lacking LFA-1. Pretreatment of fibroblasts, but not lymphocytes, with monoclonal antibodies capable of binding ICAM-1 inhibits lymphocyte-fibroblast adhesion. Pretreatment of lymphocytes, but not fibroblasts, with antibodies to LFA-1 has also been found to inhibit lymphocyte-fibroblast adhesion. ;ICAM-1 is thus the binding ligand for the CD-18 complex on leukocytes. It can be applied to fibroblasts and endothelial cells in vitro by inflammatory mediators such as IL-1, gamma interferon and tumor necrosis factor within a time frame consistent with the infiltration of lymphocytes into inflammatory lesions in vivo (M.L. Dustin et al., J. Immunol, 137:245-254 (1986); J.S. Prober et al., J. Immunol, 137:1893-1896. Furthermore, ICAM-1 is expressed on non-hematopoietic cells such as vascular endothelial cells, thymic epithelial cells, other epithelial cells and fibroblasts, and on hematopoietic cells such as tissue macrophages, mitogen-stimulated T-lymphocyte blasts and nuclear center B cells and dendritic cells in tonsils, lymph nodes and Peyer's patches (M.L. Dustin et al., J. Immunol, 137:245-254 (1986)).ICAM-1 is expressed on keratinocytes in benign inflammatory lesions such as allergic eczema, lichen planus, exanthema, urticaria and blistering diseases Allergic skin reactions produced by application of a hapten to the skin to which the patient is allergic showed o saw a strong ICAM-1 expression on the keratinocytes. On the other hand, toxic spots on the skin did not show ICAM-1 expression on the keratinocytes. ICAM-1 is present on keratinocytes from biopsies of skin lesions from various dermatological disorders, and ICAM-1 expression is induced on allergic patch test lesions, whereas keratinocytes from toxic patch test lesions did not express ICAM-1. ;ICAM-1 is therefore a cell substrate to which lymphocytes can attach, so that the lymphocytes can migrate to sites of inflammation and/or perform various effector functions that contribute to this inflammation. Such functions include the formation of antibody, lysis of virally infected target cells, etc. The term "inflammation" as used herein is intended to include reactions in the specific or non-specific defense system. As used herein, the term "specific defense system" is intended to denote the component of the immune system that responds to the presence of specific antigens. Inflammation is said to be the result of a response from the specific defense system if the inflammation is caused by, mediated by or associated with a response from the specific defense system. Examples of inflammation resulting from a response of the specific defense system include the response to antigens such as rubella virus, autoimmune diseases, delayed-type hypersensitivity response, mediated by T cells (such as would be seen in individuals who gives a "positive" test in the Mantaux test), psoriasis, etc. ;A "non-specific defense system reaction" is a response mediated by leukocytes that do not have immunological memory. Such cells include granulocytes and macrophages. As used herein, an inflammation is said to be the result of a response of the non-specific defense system if the inflammation is caused by, mediated by, or associated with a response of the non-specific defense system. Examples of inflammation that is, at least in part, a result of a reaction of the non-specific defense system include inflammation associated with conditions such as: asthma; adult respiratory distress syndrome (ARDS) or multiple organ injury syndromes secondary to sepsis or trauma; reperfusion injury to the myocardium or other tissues; acute glomerulonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other inflammatory disorders of the central nervous system; thermal damage; hemodialysis, - leukapheresis; ulcerative colitis; Chron's disease; necrotizing enterocolitis; granulocyte transfusion-associated syndromes; and cytokine-mediated toxicity. According to the present invention, functional ICAM-1 derivatives, and in particular such derivatives comprising fragments or mutant variants of ICAM-1 which have regions 1, 2 and 3, can be used for in vitro diagnosis of such reactions in the non-specific defense system. More preferred for such diagnosis are ICAM-1 fragments containing region 2 of ICAM-1. C. Cloning of the ICAM-1 gene; Any of many different methods can be used to clone the ICAM-1 gene. One such method involves analyzing a shuttle vector library of cDNA inserts (derived from an ICAM-1 expressing cell) for the presence of an insert containing the ICAM-1 gene. Such an analysis can be performed by cells being transfected with the vector and then analyzed for ICAM-1 expression. The preferred method for cloning this gene involves determining the amino acid sequence of the ICAM-1 molecule. To accomplish this task, ICAM-1 protein can be purified and analyzed by automated sequence analyzers. Alternatively, the molecule can be fragmented such as with cyanogen bromide or with proteases such as papain, chymotrypsin or trypsin (Y. Oike et al., J. Biol. Chem., 257:9751-9758 (1982); C. Liu et al. , Int. J. Pept. Protein Res., 21:209-215 (1983)). Although it is possible to determine the entire amino acid sequence of ICAM-1, it is preferred to determine the sequence of peptide fragments in the molecule. If the peptides have a length of more than 10 amino acids, the sequence information is usually sufficient to clone a gene such as the gene for ICAM-1. The sequence of amino acid residues in a peptide is designated herein either by the use of their commonly used 3-letter designations or by their single-letter designations. A listing of these 3-letter and 1-letter designations can be found in textbooks such as Biochemistry, A. Lehninger, Worth Publishers, New York, NY (1970). When such a sequence is listed vertically, the amino-terminal residue is meant to be at the top of the list, and the carboxy-terminal residue in the peptide is meant to be at the bottom of the list. In horizontal entry, similarly, the amino-terminal residue is intended to be at the left-hand end, while the carboxy-terminal residue is intended to be at the right-hand end. The residues of amino acids in a peptide can be separated by hyphens. Such hyphens are only intended to make the presentation of a sequence easier. As a purely illustrative example, the amino acid sequence denoted: ;-Gly-Ala-Ser-Phe- ; indicates that an Ala residue is bound to the carboxy group of Gly, and that a Ser residue is bound to the carboxy group of the Ala residue and to the amino group on a Phe residue. The designation further indicates that the amino acid sequence contains the tetrapeptide Gly-Ala-Ser-Phe. The designation is not intended to limit the amino acid sequence of this one tetrapeptide, but is intended to include (1) the tetrapeptide with one or more amino acid residues attached to either its amino or carboxy end, (2) the tetrapeptide with one or more amino acid residues attached to both its amino and its carboxy terminus, (3) the tetrapeptide without any additional amino acid residues. ;Once one or more suitable peptide fragments have been sequenced, the DNA sequences that can encode them are examined. Because the genetic code is degenerate, more than one codon can be used to code for a particular amino acid {J.D. Watson, I: Molecular Biology of the Gene, 3rd ed., W.A. Benjamin, Inc., Menlo Park, Ca (1977), pp. 356-357). The peptide fragments are analyzed for the identification of sequences of amino acids that can be encoded by oligonucleotides with the lowest degree of degeneracy. This is preferably carried out by identifying sequences containing amino acids coded for by only a single codon. Although such amino acid sequences may occasionally be encoded by only a single oligonucleotide, often the amino acid sequence may be encoded by any of a set of similar oligonucleotides. It is significant that although all the elements in the set contain oligonucleotides that can code for the peptide fragment, and thus potentially contain the same nucleotide sequence as the gene that codes for the peptide fragment, only one element in the set contains a nucleotide sequence that is identical to the nucleotide sequence in this gene. Because this element is contained within the kit and can hybridize to DNA even in the presence of the other elements of the kit, it is possible to use the non-fractionated set of oligonucleotides in the same way as one would use a single oligonucleotide to clone the gene that codes for the peptide. In a manner exactly analogous to that described above, one can use an oligonucleotide (or set of oligonucleotides) having a nucleotide sequence that is complementary to the oligonucleotide sequence or set of sequences that can code for the peptide fragment. ;A suitable oligonucleotide, or set of oligonucleotides, which may encode a fragment of the ICAM-1 gene (or which is complementary to such an oligonucleotide, or set of oligonucleotides) is identified (using the method described above), synthesized and is hybridized by aids which are well known in the field, against a DNA, or more preferably, a cDNA preparation originating from human cells which can express ICAM-1 gene sequences. Techniques for nucleic acid hybridization are described by R. Maniatis et al., in: Molecular Cloning, a Laboratory Manual, Coldspring Harbor, NY (1982), and by B.D. Haymes et al., I. Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington, DC; (1985), which references are incorporated herein by reference. The source of the DNA or cDNA used will preferably have been enriched with respect to ICAM-1 sequences. Such enrichment is most easily obtained by cDNA obtained by extraction of RNA from cells grown under conditions that induce ICAM-1 synthesis (such as U937 grown in the presence of phorbol esters, etc.). Techniques such as, or similar to, those described above have successfully enabled the cloning of genes for human aldehyde dehydrogenases (L.C. Hsu et al., proe. Nat. Acad. Sei. USA, 82:3771-3775 ( 1985), fibronectin (S. Suzuki et al., Eur. Mol. Biol. Organ. J., 4:2519-2524 (1985)), the human estrogen receptor gene (P. Walter et al., Proe. Nat. Acad. Sci. USA, 82:7889-7893 (1985)), tissue type plasminogen activator (D. Pennica et al., Nature, 301:214-221 (1983)) and human placental alkaline phosphatase complementary DNA (W. Kam et al., Proe. Nat. Acad. Sci. USA, 82:8715-8719 (1985)).; In a preferred alternative method to cloning the ICAM-1 gene, a library of expression vectors is prepared by cloning DNA, or more preferably cDNA, from a cell capable of expressing ICAM-1, in an expression vector. The library is then screened for elements capable of expressing a protein which binds to anti-ICAM-1 antibody, and which has a nucleotide sequence capable of encoding for polypeptides having the same amino acid sequence as ICAM-1 or fragments of ICAM-1. The cloned ICAM-1 gene, obtained by the methods described above, can be operably linked to an expression vector and introduced into bacterial or eukaryotic cells to produce ICAM-1 protein. Techniques for such manipulations are described by T. Maniatis et al., as above, and are well known in the art. D. Applications of assays of LFA-1-dependent aggregation; The above-described assay, which can measure LFA-1-dependent aggregation, can be used for the identification of agents that act as antagonists to inhibit the extent of LFA-1-dependent aggregation. Such antagonists can work by weakening the ability of LFA-1 or ICAM-1 to mediate aggregation. Thus, such agents include immunoglobulins such as an antibody that can bind to either LFA-1 or ICAM-1. In addition, non-immunoglobulin agents (ie, chemical) can be tested using the assay described above to determine whether they are antagonists of LFA-1 aggregation. ;E. Applications of antibodies that can bind to ICAM-1 receptor proteins; 1. Anti-inflammatory agents; Monoclonal antibodies to elements of the CD-18 complex inhibit many adhesion-dependent functions of leukocytes including binding to endothelium (D. Haskard et al., J. Immunol., 137:2901-2906 {1986)), homotypic adhesions CR. Rothlein et al., J. Exp. Med., 163:1132-1149 (1986)), antigen- and mitogen-induced proliferation of lymphocytes (D. Davignon et al., Proe. Nati. Acad. Sei. USA, 78 :4535-4539 ;(1981)), antibody formation (A. Fischer et al., J. Immunol., 136:3198-3203 (1986)), and effector functions of all leukocytes such as lytic activity of cytotoxic T cells (A.M. Krensky et al., J. Immuno., 132:2180-2182 (1984)), macrophages (G. Strassman et al., J. Immunol., 136:4328-4333 (1986)), .and all cells that comprise in antibody-dependent cell cytotoxicity reactions (S. Kohl et al., J. Immunol., 133:2972-2978; (1984)). In all of the above functions, the antibodies inhibit the leukocyte's ability to attach to the appropriate cell substrate, which in turn inhibits the final outcome. As discussed above, the binding of ICAM-1 molecules to the members of the LFA-1 family of molecules is of central importance in cellular adhesion. During the adhesion process, lymphocytes can constantly monitor an animal for the presence of foreign antigens. Although these processes are normally desirable, they are also the cause of organ transplant rejection, tissue transplant rejection and many autoimmune diseases. Consequently, any aid that could weaken or inhibit cellular adhesion would be highly desirable for recipients of organ transplants, tissue transplants, or for autoimmune patients. Monoclonal antibodies that can bind to ICAM-1 are very well suited as anti-inflammatory agents in a mammal. It is significant that such agents differ from conventional anti-inflammatory agents in that they can selectively inhibit adhesion and do not produce other side effects such as kidney toxicity found in the case of conventional agents. Monoclonal antibodies that can bind to ICAM-1 can therefore be used to prevent organ or tissue rejection, or to modify autoimmune responses without fear of such side effects in the mammal. ;It is significant that the use of monoclonal antibodies that can perceive ICAM-1 can make it possible to perform organ transplants even between individuals who have HLA incompatibility. ;2. Suppressors of delayed-type hypersensitivity reactions; Since ICAM-1 molecules are mostly expressed at sites of inflammation, such as those involved in delayed-type hypersensitivity reactions, antibodies (especially monoclonal antibodies) that can bind to ICAM-1 molecules have therapeutic potential by weakening or eliminating such reactions. This potential therapeutic application can be exploited in one of two ways. First, a composition containing a monoclonal antibody against ICAM-1 can be administered to a patient experiencing a delayed-type hypersensitivity reaction. E.g. such compositions can be administered to an individual who has been in contact with antigens such as poison ivy, poison oak, etc. In the second embodiment, the monoclonal antibody capable of binding to ICAM-1 is administered to a patient in association with an antigen for prevention of a subsequent inflammatory reaction. Thus, the additional administration of an antigen with an ICAM-1 binding monoclonal antibody may allow an individual to temporarily tolerate subsequent presentation of that antigen. ; 3. Therapy for Chronic Inflammatory Disease ; Since LAD patients lacking LFA-1 do not produce an inflammatory response, it is hypothesized that antagonism of LFA-1's natural ligand, ICAM-1, will also inhibit an inflammatory response. The ability of antibodies against ICAM-1 to inhibit inflammation provides the basis for their therapeutic use in the treatment of chronic inflammatory diseases and autoimmune diseases such as lupus erythematosus, autoimmune thyroiditis, experimental allergic encephalomyelitis (EAE), multiple sclerosis, some forms of diabetes Reynaud's syndrome , rheumatoid arthritis, etc. Such antibodies can also be used as therapy in the treatment of psoriasis. Generally, the monoclonal antibodies that can bind to ICAM-1 can be used in the treatment of such diseases that can usually be treated by steroid therapy. ; 4. Diagnostic and prognostic applications ;Since ICAM-1 is mostly expressed at sites of inflammation, monoclonal antibodies that can bind to ICAM-1 can be used as a means of imaging or visualizing the sites of infection and inflammation in a patient. In such use, the monoclonal antibodies are detectably labeled using radioisotopes, affinity markers (such as biotin, avidin, etc.), fluorescent markers, paramagnetic atoms, etc. Methods for carrying out such labeling are well known in the field. An overview of the clinical use of antibodies in diagnostic imaging of H.B. Grosman, Uroi. Clin. North Amerk., 13:465-474 (1986), E.C. Unger et al., Invest-Radiol., 20:693:700 (1985) and B.A. Khaw et al., Science, 209:295-297 (1980). ;The presence of inflammation can also be detected using binding ligands such as mRNA, cDNA or DNA, which bind to ICAM-1 gene sequences or to ICAM-1 mRNA sequences, in cells expressing ICAM-1. Techniques for performing such hybridization assays are described by T. Maniatis (as above). ;Detection of centers for such detectably labeled antibodies indicates a site of inflammation or tumor development. In one embodiment, this investigation of inflammation is performed by removing tissue samples or blood and incubating such samples in the presence of detectably labeled antibodies. In a preferred embodiment, this technique is performed in a non-invasive manner using magnetic imaging, fluorography, etc. Such a diagnostic test can be used to screen organ transplant recipients for early signs of potential tissue rejection. Such analyzes may also be performed in efforts to determine an individual's susceptibility to rheumatoid arthritis or other chronic inflammatory diseases. While the invention has now been generally described, it will be easier to understand by reference to the following examples which are given by way of illustration. ;EXAMPLE 1 ;Culturing of mammalian cells ;Typically, EBV-transformed cells and the hybridoma cells were maintained in RMPI 1640 culture medium, supplemented with 20 mM L-glutamine, 50 µg/ml gentamycin and 10% fetal bovine (or fetal calf) serum. The cells were cultured at 37°C in a humidified atmosphere of 5% CO 2 and 95% air.; To generate Epstein-Barr virus (EBV) transformants, T-cell-depleted peripheral blood mononuclear cells in a number of l0 <6>/ml in RPMI 164 0 medium supplemented with 20% fetal calf serum (FCS), and 50 µg/ml gentamycin incubated for 16 hours with EBV-containing supernatant from B95-8 cells (D.A. Thorley-Lawson et al ., J. Exper. Med., 146:495 (1977)). Cells in portions of 0.2 ml were placed in 10 microtiter wells. The medium was replaced with RPMI 1640 medium (supplemented with 20% fetal calf serum and 50 µg/ml gentamycin) until cell growth was observed. The cells grew in most wells and were expanded in the same medium. ;Phytohemagglutinin (PHA) blisters were created with IO <6> cells/ml in RPMI 1640 medium (supplemented with 20% fetal calf serum) containing a 1:800 dilution of PHA-P (Difco Laboratories, Inc., Detroit, MI). PHA lines were expanded with interleukin 2-(IL-2)-conditioned medium and pulsed weekly with PHA (D.A. Cantrell et al., J. Exper. Med., 158:1895 (1983)). The above method was described by T. Springer et al., J. Exper. Med., 160:1901-1918 (1984), which reference is incorporated herein by reference. Cells obtained by the above procedure are then examined with anti-LFA-1 antibodies to determine whether they express the LFA-1 antigen. Such antibodies are described by F. Sanchez-Madrid et al., J. Exper. Med., 158:1785 (1983). ;EXAMPLE 2 ;Analyses regarding cellular aggregation and adhesion ;For assessment of the extent of cellular adhesion, aggregation analyzes were used. Cell lines used in such assays were washed twice with RPMI 1640 medium containing 5 mM Hepes buffer (Sigma Chemical Co., St. Louis) and resuspended to a concentration of 2 x 10 <6> cells/ml. In flat-bottomed 96-well microtiter plates (No. 3596; Costar Cambridge, MA) were added 50 µl of appropriate monoclonal-antibody supernatant or 50 µl of complete medium with or without purified monoclonal antibodies, 50 µl of complete medium containing 200 ng/ ml of the phorbol ester-phorbol myristate acetate (PMA) and 100 µl of cells at a concentration of 2 x 10 <6> cells/ml in complete medium. This gave a final concentration of 50 ng/ml PMA and 2 x IO <5> cells/well. The cells aggregated spontaneously, and the degree of aggregation was noted at different time points. Scores ranged from 0 to 5+, where 0 indicated essentially no cells clustered; 1+ indicated that less than 10% of the cells were in aggregates; 2+ indicated that less than 50% of the cells were aggregated; 3+ indicated that up to 100% of cells were in small, loose clusters; 4+ indicated that up to 100% of the cells were aggregated into larger clusters; and 5+ indicated that 100% of the cells were in large, very compact aggregates. To achieve a more quantitative assessment of cell adhesion, reagents and cells were added to 5 ml polystyrene tubes in the same order as above. The tubes were placed in a rack on a rotary shaker at 37°C. After 1 hour at approx. 200 rpm (revolutions per minute), 10 µl of the cell suspension was placed in a hemocytometer, and the number of free cells was quantified. Percentage aggregation was determined by the following equation: The number of introduced cells in the above formula is the number of cells per ml in a control tube containing only cells and complete medium that had not been incubated. The number of free cells in the above equation is equal to the number of non-aggregated cells per ml from test tubes. The above methods are described by R. Rothlein et al., J. Exper. Med., 163:1132-1149; (1986) . ;EXAMPLE 3 ;LFA-1 dependent cell aggregation ;The qualitative aggregation assay described in Example 2 was performed using the Epstein-Barr transformed cell line JY. When PMA was added to the culture medium in the microtiter plates, aggregation of cells was observed. Time-lapse video recordings showed that the JY cells at the bottom of the microtiter wells were motile and showed active membrane ruffling and pseudopodia movement. Contact between the pseudopodia of neighboring cells often resulted in cell-cell adhesion. If the adhesion was long-lasting, the area of cell contact moved to the uropod. Contact could be maintained despite strong cell movements and jerking of the cells in opposite directions. The main difference between PMA-treated and untreated cells appeared to be in the stability of these contacts once formed. With PMA, cell clusters developed that grew in size as additional cells adhered to their periphery. As a second aid for measuring adhesion, the quantitative analysis described in example 2 was used. The cell suspensions were shaken at 200 rpm for 2 hours, transferred to a hemocytorneter, and cells not in aggregates were counted. In the absence of PMA, 42% (SD = 20%, N = 6) of JY cells were in aggregates after 2 h, whereas JY cells incubated under identical conditions with 50 ng/ml PMA had 87% (SD = 8%, N = 6) of the cells in aggregates. Kinetic investigations of the aggregation showed that PMA increased the amount and size of the aggregation at all the investigated time points (Figure 3). EXAMPLE 4 Inhibition of Aggregation of Cells Using Monoclonal Anti-LFA-1 Antibodies To examine the effects of monoclonal anti-LFA-1 antibodies on PMA-induced cell aggregation, such antibodies were added to cells incubated according to the qualitative aggregation assay of Example 2. The monoclonal antibodies were found to inhibit the formation of aggregates by cells either in the presence or absence of PMA. Both F(ab')2- and Fab The <1->fragments of monoclonal antibodies against the alpha chain in LFA-1 could inhibit cell aggregation. Although approximately 100% of cells formed aggregates in the absence of anti-LFA-1 antibody, less than 20% of cells were found to be in aggregates when antibody was added. The results of this experiment are described by R. Rothlein et al., (J. Exper. Med., 163:1132-1149 (1986)). ;EXAMPLE 5 ;Cell aggregation requires the LFA-1 receptor EBV-transformed lymphoblastoid cells were prepared from patients in the manner described in Example 1. Such cells were probed for monoclonal antibodies capable of recognizing LFA-1, and the cells were found to be deficient on the LFA-1. The qualitative aggregation assay described in Example 2 was performed using the LFA-1 deficient cells described above. Such cells did not spontaneously aggregate, even in the presence of PMA. ;EXAMPLE 6 ;Detection of ICAM-1 ;The LFA-1-deficient cells according to Example 5 were labeled with carboxyfluorescein diacetate (M. Patarroyo et al., Cell. Immunol., 63:237-248 (1981)}. The labeled cells were mixed at a ratio of 1:10 with autologous or JY cells, and the percentage of fluorescein-labeled cells in aggregates was determined according to the method of R. Rothlein et al., J. Exper. Med., 163:1132- 149 (1986). The LFA-1-deficient cells were found to be able to co-aggregate with LFA-1-expressing cells (Figure 4). ;To determine whether LFA-1 was only important in the formation of aggregates, or in maintaining them, antibodies capable of binding to LFA-1 were added to the formed aggregates described above. The addition of antibody was found to greatly disrupt the formed aggregation. Time-lapse video recording confirmed that addition of the monoclonal antibodies to formed aggregates began to cause tearing within 2 hours (table 1). After addition of monoclonal antibodies against LFA-1, pseudopodia movements and changes in the shape of individual cells in aggregates continued unchanged. Individual cells were gradually detached from the periphery of the aggregate; after 8 hours the majority of cells were dispersed. According to time-lapse video recording, the disruption of formed aggregates by monoclonal LFA-1 antibodies appeared equivalent to the aggregation process in the absence of monoclonal LFA-1 antibody when going back in time. ;EXAMPLE 7 ;Requirement of divalent ions for LFA-1-dependent aggregation LFA-1-dependent adhesions between cytotoxic T cells and target substances require the presence of magnesium (E. Martz, J. Cell. Biol., 84:584-598 ( 1980)). PMA-induced JY cell aggregation was examined for dependence on divalent cations. JY cells did not aggregate (using the assay of Example 2) in medium free of calcium or magnesium ions. Addition of divalent magnesium supported aggregation at concentrations as low as 0.3 mM. Addition of calcium ions alone had little effect. However, calcium ions were found to enhance the ability of magnesium ions to support PMA-induced aggregation. When 1.25 mM calcium ions were added to the medium, magnesium ion concentrations as low as 0.02 millimolar were found to support aggregation. These data show that the LFA-1-dependent aggregation of cells requires magnesium ions, and that calcium ions, although insufficient by themselves, can act synergistically with magnesium ions so that aggregation is made possible. ;EXAMPLE 8 ;Isolation of hybridoma cells capable of expressing monoclonal anti-ICAM-1 antibodies ;Monoclonal antibodies capable of binding to ICAM-1 were isolated according to the method of R. Rothlein et al., J. Immunol., 137: 1270-1274 (1986), which reference is incorporated herein by reference. Thus, 3 BALB/C mice were immunized intraperitoneally with EBV-transformed mononuclear cells from peripheral blood, from an LFA-1-deficient individual (T.A. Springer et al., J. Exper. Med., 160:1901 ;(1984)) . About. IO <7> cells in 1 ml of RPMI 1640 medium were used for each immunization. The immunization doses were administered 45, 29 and 4 days before spleen cells were removed from the mouse to generate the desired hybridoma cell lines. On day 3 before removal of the spleen cells, the mice received additional IO <7> cells in 0.15 ml medium (intravenous). Isolated spleen cells from the animals described above were fused with P3X73Ag8.653 myeloma cells in a 4:1 ratio according to the protocol of G. Galfre et al., Nature, 266:550 (1977). Aliquots of the resulting hybridoma cells were introduced into 96-well microtiter plates. The hybridoma supernatants were screened for inhibition of aggregation, and one inhibitory hybridoma (among over 600 wells screened) was cloned and subcloned by limiting dilution. This subclone was designated RR1/1.1.1 (hereinafter referred to as "RR1/1"). ;Monoclonal antibody RR1/1 was consistently found to inhibit PMA-stimulated aggregation of the LFA-1-expressing cell line JY. The RR1/1 monoclonal antibody inhibited aggregation similarly to, or slightly less than, some monoclonal antibodies against the LFA-1 alpha or beta subunits. In contrast, aggregation was not inhibited by control monoclonal antibody against HLA, which is abundantly expressed on JY cells. The antigen that was bound by monoclonal antibody RR1/1 is defined as the intercellular adhesion molecule 1 (ICAM-1). ;EXAMPLE 9 ;Use of anti-ICAM-1 monoclonal antibodies ;to characterize the ICAM-1 molecule ;To determine the nature of ICAM-1, and in particular to determine whether ICAM-1 was distinct from LFA-1, cell proteins were immunoprecipitated using monoclonal antibody RR1/1. The immunoprecipitation was performed according to the method of R. Rothlein et al., (J Immunol., 137:1270-1274 (1986)). JY cells were lysed with 5 x IO <7> cells/ml in 1% Triton X-100, 0.14 mM NaCl, 10 mM tris, pH 8.0, with freshly added 1 mM phenylmethylsulfonyl fluoride, 0.2 units per ml trypsin inhibitor aprotinin (lysis buffer) for 20 minutes at 4°C. The lysates were centrifuged at 10,000 x g for 10 minutes and clarified with 50 µl of a 50% suspension of CNBr-activated, glycine-stopped Sepharose CL-4B for 1 hour at 4°C. 1 ml of lysate was immunoprecipitated with 20 µl of a 50% suspension of monoclonal antibody RR1/1 coupled to Sepharose CL-4B (1 mg/ml) overnight at 4°C (T.A. Springer et al., J. Exper. Med ., 160:1901; (1984)). Sepharose-bound monoclonal antibody was prepared using DNBr activation of Sepharose CL-4B in carbonate buffer according to the method of S. March et al., (Anal. Biochem., 60:149 (1974)). Washed immunoprecipitates were subjected to SDS-PAGE and silver staining according to the method of J.H. Morrissey, Anal. Biochem., 117:307; (1981). ;After elution of proteins with SDS sample buffer (M.K. Ho et al., J. Biol. Chem., 258:636 (1983)) at 100°C, the samples were divided into two halves and subjected to electrophoresis (SDS-8% PAGE ) under reducing (Figure 5A) or non-reducing conditions (Figure 5B). Bands with molecular weight of 50 kd and 25 kd corresponded to the heavy and light chains of immunoglobulins from the Sepharose with the monoclonal antibody (Figure 5A, lane 3). Variable amounts of other bands in the 25-50 kd weight range were also observed, but were not seen in washes from hairy leukemia cells, which yielded only one band at molecular weight 90 kd. The 177 kd alpha subunit and the 95 kd beta subunit of LFA-1 were found to migrate differently than ICAM-1 under both reducing (Figure 5A, lane 2) and non-reducing (Figure 5B, lane 2) conditions. To determine the effect of monoclonal antibody RR1/1 on PHA lymphoblast aggregation, the quantitative aggregation assay described in Example 2 was used. Thus, T cell blast cells were stimulated for 4 days with PHA, washed thoroughly and then cultured for 6 days in the presence of IL-2 conditioned medium. PHA was found to be internalized during this 6 day culture and did not contribute to the aggregation assay. In three different assays with different T-cell blast preparations, monoclonal ICAM-1 antibodies inhibited aggregation consistently (Table 2). ;Monoclonal LFA-1 antibodies were consistently more inhibitory than monoclonal ICAM-1 antibodies, while monoclonal HLA-A, B and LFA-3 antibodies were without effect. These results show that among the investigated monoclonal antibodies, only those that could bind to LFA-1 or ICAM-1 could inhibit cell adhesion. ;EXAMPLE 10 ;Preparation of monoclonal antibody against ICAM-1 ;Immunization ;A Balb/C mouse was immunized intraperitoneally (i.p.) with 0.5 ml 2 x 10 <7> JY cells in RPMI medium 103 days and 24 days before fusion. On days 4 and 3 before fusion, mice were immunized i.p. with IO <7> cells of PMA-differentiated U937 cells in 0.5 ml RPMI medium. ;Differentiation of U937 cells ;U937 cells (ATCC CRL-1593) were differentiated by incubation with 5 x 10 <5>/ml in RPMI with 10% fetal bovine serum, 1% glutamine and 50 tig/ml gentamycin (complete medium) containing 2 ng/ml phorbol-12-myristate acetate (PMA) in a sterile polypropylene container. On the third day of this incubation, half of the medium volume was removed and replaced with fresh complete medium containing PMA. On day 4, the cells were removed, washed and prepared for immunization. ;Fusion ;Spleen cells from the immunized mice were fused with P3x63 Ag8-653 myeloma cells in a ratio of 4:1 according to Galfre et al., (Nature, 266:550 (1977)). After fusion, the cells were plated in flat-bottomed 96-well microtiter plates with IO <5 >spleen cells/well. ;Selection with respect to anti-ICAM-1-positive cells ;After one week, 50 µl of supernatant was examined by the qualitative aggregation analysis according to example 2 using both JY and SKW-3 as aggregating cell lines. Cells from supernatants that inhibited JY cell aggregation but not SKW-3 were selected and cloned 2 times using limiting dilution. This experiment resulted in the identification and cloning of three separate hybridoma lines that produced monoclonal anti-ICAM-1 antibodies. The antibodies produced by these hybridoma lines were IgG2a, IgG2b and IgM respectively. The hybridoma cell line that produced the IgG2a anti-ICAM-1 antibody was designated R6'5'D6'E9 <1>B2. The antibody produced by the preferred hybridoma cell line was designated R6 <1>5 <1>D6 <1>E9'B2 (hereinafter referred to as "R6-5-D6". ;EXAMPLE 11 ;Expression and regulation of ICAM-1 ;For the measurement of ICAM-1 expression, a radioimmunoassay was developed. In this analysis, purified RRl/1 iodinated using iodogen to a specific activity of 10 nCi/ng. Endothelial cells were grown in 96-well plates and treated as described for each experiment. The plates were cooled at 4°C by placing in a cold room for 0 ,5-1 hour, not immediately on ice. The monolayers were washed 3 times with cold complete medium and then incubated for 30 minutes at 4°C with <125>I-RR1/1. The monolayers were then waxed 3 times with complete medium. The bound <125>I was released using 0.1 N NaOH and counted. The specific activity for <125>I-RR1/1 was adjusted using unlabeled RR1/1 to obtain a linear signal over the antigen density range found in this study. Non-specific binding was determined in the presence of a thousand-fold excess of unlabeled RR1/1 and was subtracted from the total binding to obtain specific binding. ;ICAM-1 expression, measured using the radioimmunoassay described above, is increased on human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) by IL-1, TNF, LPS and IFN-gamma (Table 3 ). Saphenous vein endothelial cells were used in this study to confirm the results from umbilical vein endothelial cells in cultured adult large vein endothelial cells. The basal expression of ICAM-1 is twofold higher on saphenous vein endothelial cells than on umbilical vein endothelial cells. Exposure of umbilical vein endothelial cells to recombinant IL-l-alpha, IL-l-beta and TNF-gamma increases ICAM-1 expression 10-20 fold. IL-1-alpha, TNF and LPS were the most potent inducers, and IL-1 was less potent on a weight basis and also at saturating concentrations of the response (Table 3). IL-1-beta at 100 ng/ml increased ICAM-1 expression 9-fold on HUVEC and 7.3-fold on HSVEC, the half-maximal increase occurring at 15 ng/ml. rTNF at 50 ng/ml increased ICAM-1 expression 16-fold on HUVEC and 11-fold on HSVEC with half-maximal effects at 0.5 ng/ml. Interferon-gamma produced a significant increase in ICAM-1 expression of 5.2-fold on HUVEC or 3.5-fold on HSVEC at 10,000 U/ml. The effect of LPS at 10 fxg/ml was in the same order of magnitude as the effect of rTNF. Pairwise combinations of these mediators resulted in additive or slightly less than additive effects on ICAM-1 expression (Table 3). Cross-titration of rTNF with rIL-l-beta or rlFN-gamma showed no synergism between these at suboptimal or optimal concentrations. Since LPS increased ICAM-1 expression on endothelial cells to levels sometimes found in culture media, the possibility that the basal ICAM expression could be due to LPS was investigated. When several serum aliquots were examined, it was found that serum with low endotoxin content produced lower ICAM-1 basal expression by 25%. All results presented here were for endothelial cells cultured in low endotoxin serum. However, incorporation of the LPS-neutralizing antibiotic polymyxin B decreased by 10 n <g>/ ml ICAM-1 expression only by an additional 25% (Table 3). The increase in ICAM-1 expression upon treatment with IL-1 or TNF was not affected by the presence of 10 µg/ml polymyxin B, which is consistent with the low endotoxin levels in these preparations (Table 3). ;;Upregulation of ICAM-1 expression on HUVEC and HSVEC. HUVEC or HSVEC were seeded in 96-well plates at 1:3 from a confluent monolayer and allowed to grow to confluence. The cells were then treated with the indicated materials or media for 16 hours, and the RIA was performed according to the described procedure. All points were performed with four parallels. ;EXAMPLE 12 ;Kinetics of interleukin-1 and gamma interferon induction of ICAM-1 ;The kinetics of interleukin-1 and gamma interferon effects on ICAM-1 expression on skin fibroblasts were determined using 125 I goat antimouse IgG binding assay according to M.L. Dustin et al., (J. Immunol., 137:24 5-254 (1986); which reference is incorporated herein by reference). To perform this binding assay, human skin fibroblasts were cultured in a 96-well microtiter plate to a density of 2-8 x 104 cells/well (0.32 cm <2>). The cells were washed twice with RPMI 1640 medium supplemented as described in Example 1. The cells were additionally washed once with Hank's Balanced Salt Solution (HBSS), 10 mM HEPES, 0.05% NaN 3 and 10% heat-inactivated fetal bovine serum . Washing with this binding buffer was performed at 4°C. In each well, 50 μl of the binding buffer described above and 50 μl of the appropriate hybridoma supernatant with X63 and W6/32 were added as negative and positive control, respectively. After incubation for 30 minutes at 4°C with gentle agitation, the wells were washed twice with binding buffer, and the second antibody 125 I goat anti-mouse IgG was added at 50 nCi in 100 µl. The 125 I goat anti-mouse antibody was prepared using iodogen (Pierce) according to the method of P.J. Fraker et al., {Biochem. Biophys. Res. Commun., 80:849 (1978)). After 30 minutes at 4°C, the wells were washed twice with 200 µl of binding buffer, and the cell layer was solubilized by adding 100 µl of 0.1 N NaOH. This and a 100 µl wash were counted in a Beckman 5500 gamma counter. The specific counts per minute was calculated as [cpm with monoclonal antibody]-[cpm with X63]. All steps, including induction with specific reagents, were performed in four parallels. ;The effect of interleukin 1 with a half-life for ICAM-1 induction of 2 hours was faster than the effect of gamma interferon with a half-life of 3.75 hours (Figure 6). The time course for the return to resting levels of ICAM-1 was found to depend on the cell cycle or rate of cell growth. In quiescent cells, interleukin 1 and gamma interferon effects are stable for 2-3 days, while ICAM-1 expression in log-phase cultures is close to baseline 2 days after removal of these inducers. The dose-response curves for the induction of ICAM-1 by recombinant mouse and human interleukin 1, and for recombinant human gamma interferon, are shown in Figure 7. Gamma interferon and interleukin 1 were found to have the same concentration dependences with almost identical effects at 1 ng/ml. The recombinant human and mouse interleukin 1 also have similar curves, but are much less effective than preparations of human interleukin 1 in inducing ICAM-1 expression. ;Cycloheximide, an inhibitor of protein synthesis, and actinomycin D, an inhibitor of mRNA synthesis, abrogate the effects of both interleukin 1 and gamma interferon on ICAM-1 expression on fibroblasts (Table 4). Furthermore, tunicamycin, an inhibitor of N-linked glycosylation, only inhibited the interleukin 1 effect by 43%. These results demonstrate that protein and mRNA synthesis, but not N-linked glycosylation, is required for interleukin 1- and gamma interferon-stimulated increases in ICAM-1 expression. ;EXAMPLE 13 ;Tissue distribution of ICAM-1 ;Histogenetic studies were performed on frozen tissue from human organs to determine the distribution of ICAM-1 in the thymus, lymph nodes, intestines, skin, kidneys and liver. To carry out such an analysis, frozen tissue sections (4 µm thick) of normal human tissues were fixed in acetone for 10 minutes and stained with the monoclonal antibody RR1/1 by an immunoperoxidase technique using the avidin-biotin complex method (Vector Laboratories, Burlingame, CA) described by N. Cerf-Bensussan et al., (J. Immunol., 130:2615 (1983)). After incubation with the antibody, the sections were sequentially incubated with biotinylated horse anti-mouse IgG and avidin-biotinylated peroxidase complexes. The sections were finally dipped in a solution containing 3-amino-9-ethylcarbazole (Aldrich Chemical Co., Inc. Milwaukee WI) to produce a color reaction. The sections were then fixed in 4% formaldehyde for 5 minutes and counterstained with hematoxylin. Control included sections incubated with unrelated monoclonal antibodies instead of the RRl/1 antibody. ;ICAM-1 was found to have a distribution most similar to that of major histocompatibility complex (MHC) class II antigens. Most of the blood vessels (both small and large) in all tissues showed staining of endothelial cells with ICAM-1 antibody. Vascular-endothelial staining was more intense in interfollicular (paracortical) areas in lymph nodes, tonsils and Peyer's patches, compared to vessels in kidney, liver and normal skin. In the liver, staining was mostly restricted to sinusoidal-covering cells; the hepatocytes and endothelial cells lining most of the portal veins and arteries were not stained. In the thymus marrow, diffuse staining of large cells and a dendritic staining pattern were observed. In the cortex, the staining pattern was focal and mainly dendritic. Thymocytes were not stained. In the peripheral lymphoid tissue, the germinal center cells in the secondary lymphoid follicles were intensely stained. In some lymphoid follicles, the staining pattern was mostly dendritic, with no recognizable staining of the lymphocytes. Weak staining of cells in the cover zone was also observed. In addition, dendritic cells with cytoplasmic extensions ("middle finger" reticulum cells) and a small number of lymphocytes in the interfollicular or paracortical areas were stained with the ICAM-1 binding antibody. ;Cells resembling macrophages were stained in the lymph nodes and lamina propria of the small intestine. Fibroblast-like cells (spindle-shaped cells) and dendritic cells scattered in the stroma of most organs examined were stained with the ICAM-1-binding antibody. No staining could be seen in the long-glove/indeterminate cells of the epidermis. No staining was observed in smooth muscle tissue. The staining of epithelial cells was also seen in the mucous membranes of the tonsils. Although hepatocytes, bile duct epithelium, intestinal epithelial cells, and tubular epithelial cells of the kidney were not stained under most circumstances, sections of normal kidney tissue from a specimen of resected kidney with renal cell carcinoma showed staining of many proximal tubule cells for ICAM-1 . These tubule epithelial cells were also stained with an anti-HLA-DR binding antibody. ;In summary, ICAM-1 is expressed on non-hematopoietic cells such as vascular endothelial cells, and on hematopoietic cells such as tissue macrophages and mitogen-stimulated T-lymphocyte blasts. ICAM-1 was found to be expressed in small amounts on peripheral blood lymphocytes. ;EXAMPLE 14 ;Purification of ICAM-1 by Monoclonal Antibody Affinity Chromatography ;General Purification Scheme ;ICAM-1 was purified from human cells or tissues using monoclonal antibody affinity chromatography. Monoclonal antibody RR1/1, which was reactive with ICAM-1, was first purified and coupled to an inert column matrix. This antibody is described by R. Rothlein et al., J. Immunol., 137:1270-1274; (1986), and M.L. Dustin et al., J. Immunol., 137:245 (1986). ICAM-1 was solubilized from cell membranes by lysing the cells in a nonionic detergent, Triton X-100, at a near-neutral pH. The cell lysate containing solubilized ICAM-1 was then passed through pre-columns designed to remove materials that bind non-specifically to the column matrix material, and then through the column matrix with the monoclonal antibody, allowing ICAM-1 to bind to the antibody. The antibody column was then washed with a series of detergent wash buffers of increasing pH up to pH 11.0. During these washes, ICAM-1 remained bound to the antibody matrix, while non-binding and weakly binding contaminants were removed. The bound ICAM-1 was then specifically eluted from the column using a detergent buffer of pH 12.5. ;Purification of Monoclonal Antibody RRl/ 1 and Covalent Coupling to Sepharose CL-4B ;The monoclonal anti-ICAM-1 antibody RRl/1 was purified from the ascites fluid of hybridoma-bearing mice, or from hybridoma culture supernatants by standard ammonium sulfate precipitation techniques and protein A affinity chromatography (Ey et al., Immunochem., 15:429 (1978)). The purified IgG, or rat IgG (Sigma Chemical Co., St. Louis, MO) was covalently coupled to Sepharose CL-4B (Pharmacia, Upsala, Sweden) using a modification of the method of March et al., (Anal .Biochem., 60:149 (1974)). Briefly, Sepharose CL-4B was washed in distilled water, activated with 40 mg/ml CNBr in 5 M K2HPO4 {pH approx. 12) for 5 min, and then washed thoroughly with 0.1 mM HCl at 4°C. The filtered, activated Sepharose was resuspended with an equal volume of purified antibody (2-10 mg/ml in 0.1 M NaHCO 3 , 0.1 M NaCl). The suspension was incubated for 18 hours at 4°C with gentle end-to-end rotation. The supernatant was then checked for unbound antibody by absorbance at 280 nm, and remaining reactive sites on the activated Sepharose were saturated by addition of glycine to 0.05 M. The coupling efficiency was usually greater than 90%. ;Detergent solubilization of membranes prepared from human spleen ;All procedures were performed at 4°C. Frozen human spleen (200 g fragments) from patients with hairy cell leukemia was thawed on ice in 200 ml Tris-saline solution (50 mM Tris, 0.14 M NaCl, pH 7.4 at 4°C) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.2 U/ml aprotinin and 5 mM iodoacetamide. The tissue was cut into small pieces and homogenized at 4°C with a Tekmar machine homogenizer. The volume was then brought to 300 ml with Tris-saline solution, and 100 ml of 10% Tween 40 (polyoxyethylene sorbitan monopalmitate) in Tris-saline solution was added to achieve a final concentration of 2.5% Tween 40. For the preparation of membranes, the homogenate extracted using three strokes of a Teflon Dounce or more preferably Potter-Elvehjem homogenizer, and then centrifuged at 1000 x g for 15 minutes. The supernatant was retained and the precipitate re-extracted with 200 ml of 2.5% Tween 40 in Tris-saline. After centrifugation at 1000 x g for 15 minutes, the supernatants from both extractions were mixed and centrifuged at 150,000 x g for 1 hour to precipitate the membranes. The membranes were washed by resuspension in 200 ml Tris-saline solution, centrifuged at 150,000 x g for 1 hour. The membrane precipitate was resuspended in 200 ml of Tris-saline solution and was homogenized with a motorized homogenizer and Teflon pestle until the suspension was uniformly cloudy. The volume was then brought up to 900 ml with Tris-saline solution, and N-lauroylsarcosine was added to a final concentration of 1%. After stirring at 4°C for 30 minutes, insoluble material in the detergent lysate was removed by centrifugation at 150,000 x g for 1 hour. Triton X-100 was then added to the supernatant to a final concentration of 2%, and the lysate was stirred at 4°C for 1 hour. ;Detergent solubilization of JY-B-lymphoblastoid cells ;The EBV-transformed B-lymphoblastoid cell line JY was grown in RPMI-1640 containing 10% fetal calf serum (FCS) and 10 mM HEPES to an approximate density of 0.8- 1.0 x IO <6 >cells/ml. To increase the cell surface expression of ICAM-1, phorbol-12-myristate-13-acetate {PMA) was added at 25 ng/ml for 8-12 hours before harvesting the cells. Sodium vanadate (50 µM) was also added to the cultures during this time period. The cells were pelleted by centrifugation at 500 x g for 10 min and grown twice in Hank's Blanched Salt Solution (HBSS) by resuspension and centrifugation. The cells (approx. 5 g per 5 liter culture) were lysed in 50 ml lysis buffer (0.14 M NaCl, 50 mM Tris pH 8.0, 1% Triton X-100, 0.2 U/ml aprotinin, 1 mM PMSF, 50 (xM sodium vanadate) by stirring at 4°C for 30 min. Unlysed nuclei and insoluble cell debris were removed by centrifugation at 10,000 x g for 15 min, followed by centrifugation of the supernatant at 150,000 x g for 1 h, and filtration of the supernatant through Whatman 3 mm filter paper. ;Affinity chromatography of ICAM-1 for structural studies ;For large-scale purification of ICAM-1 for use in structural studies, a column of 10 ml RRl/1-Sepharose CL-4B was used (coupled with 2.5 mg antibody/ml gel), and two 10 ml precolumns of CNBr-activated glycine-stopped Sepharose CL-4B and rat IgG coupled to Sepharose CL-4B (2 mg/ml). The columns were connected in series and prewashed with 10 column volumes of lysis buffer, 10 column volumes of pH 12.5 buffer (50 mM triethylamine, 0.1% Triton X-100, pH 12.5 at 4°C), followed by equilibration with 10 column volumes of lysis buffer. One liter of the detergent lysate of human spleen was applied at a flow rate of 0.5-1.0 ml/minute. The two pre-columns were used to remove non-specific binding material from the lysate prior to passage through the RR1/1-Sepharose column. ;After loading, the column of RRl/1-Sepharose and bound ICAM-1 was washed sequentially at a flow rate of 1 ml/min with a minimum of 5 column volumes of each of the following: 1) lysis buffer, 2) 20 mM Tris, pH 8.0 /0.14 M NaCl/0.1% Triton X-100, 3) 20 mM glycine pH 10.0/0.1% Triton X-100 and 4) 50 mM triethylamine, pH 11.0/0.1% Triton X-100. All wash buffers contained 1 mM PMSF and 0.2 U/ml aprotinin. After washing, the remaining bound ICAM-1 was eluted with 5 column volumes of elution buffer (50 mM triethylamine/0.1% Triton X-100/pH 12.5 at 4°C) at a flow rate of 1 ml/3 minutes. The eluted ICAM-1 was collected in 1 ml fractions and immediately neutralized by the addition of 0.1 ml of 1 M Tris, pH 6.7. Fractions containing ICAM-1 were identified by SDS-polyacrylamide electrophoresis of 10 µl aliquots (J. Springer et al., Exp. Med., 160:1901 (1984)), followed by silver staining (J.H. Morrissey, Anal Biochem., 117:307 (1981)). Under these conditions, the main amount of ICAM-1 was eluted in approx. 1 column volume and had a purity of more than 90%, as judged by silver electropherograms (a small amount of IgG, eluted from the affinity matrix, was the main contaminant). The fractions containing ICAM-1 were mixed and concentrated approx. 20 times using Centricon 30 microconcentration devices (Amicon, Danvers, MA). The purified ICAM-1 was quantified by Lowry protein analysis of an ethanol-precipitated portion of the mixture: approx. 500 µg of pure ICAM-1 was prepared from the 200 g of human spleen. ;About. 200 µg of purified ICAM-1 was subjected to a second-step purification by preparative SDS-polyacrylamide gel electrophoresis. The band representing ICAM-1 was visualized by loading the gel in 1 M KCl. The region of the gel containing ICAM-1 was then excised and electroeluted according to the method of Hunkapiller et al., Meth. Enzymol., 91:227:236 (1983). The purified protein had a purity greater than 98%, as judged by SDS-PAGE and silver staining. ;Affinity purification of ICAM-1 for functional studies ;ICAM-1 for use in functional studies was purified from detergent lysates of JY cells as described above, but on a smaller scale (a 1 ml column of RRl/1-Sepharose), and with the following modifications. All solutions contained 50 nM sodium vanadate. After washing the column with pH 11.0 buffer containing 0.1% Triton X-100, the column was washed again with five column volumes of the same buffer containing 1% n-octyl-beta-D-glucopyranoside (octylglucoside) instead for 0.1% Triton X-100. The octylglucoside detergent replaces Triton X-100 bound to ICAM-1, and unlike Triton X-100 can be removed afterwards by dialysis. ICAM-1 was then eluted with buffer of pH 12.5 containing 1% octylglucoside instead of 0.1% Triton X-100, and was analyzed and concentrated as described above. ;EXAMPLE 15 ;Characteristics of purified ICAM-1 ;ICAM-1 purified from human spleen migrates in SDS-polyacrylamide gels as a broad band with Mr 72,000-91,000. ICAM-1 purified from JY cells also migrates as a broad band with Mr 76,500-97,000. These Mr are within the reported range for ICAM-1 immunoprecipitated from different cell sources: Mr=90,000 for JY cells, 114,000 for the myelomonocytic cell line U937, and 97,000 for fibroblasts (Dustin et al., J. Immunol., 137:245; (1986)). This wide range of Mr has been attributed to an extensive but variable degree of glycosylation. The non-glycosylated precursor has an Mr of 55,000 (Dustin et al.). The protein purified either from JY cells or human spleen retains its antigenic activity, as evidenced by its ability to rebind to the original affinity column, and by immunoprecipitation with RRl/1-Sepharose and SDS-polyacrylamide electrophoresis. ;For the production of peptide fragments of ICAM-1, approx. 200 µg reduced with 2 mM dithiothreitol/2% SDS, followed by alkylation with 5 mM iodoacetic acid. The protein was precipitated with ethanol, and redissolved in 0.1 M NH4CO3/0.1 mM CaCl2/0.1% zwittergent 3-14 (Calbiochem), and digested with 1 wt% trypsin at 37°C for 4 hours , followed by a further digestion with 1% trypsin for 12 hours at 37°C. The tryptic peptides were purified by reverse-phase HPLC using a 0.4 x 15 cm C4 column (Vydac). The peptides were eluted with a linear gradient from 0 to 60% acetonitrile in 0.1% trifluoroacetic acid. Selected peptides were subjected to sequence analysis on a gas-phase micro-equilibrator (Applied Biosystems). The sequence information obtained in this investigation is shown in Table 5. EXAMPLE 16 Cloning of the ICAM-1 gene The gene for ICAM-1 can be cloned using various methods. E.g. the amino acid sequence information obtained by sequencing the tryptic fragments of ICAM-1 (table 5) can be used for the identification of an oligonucleotide sequence that would correspond to the ICAM-1 gene. Alternatively, the ICAM-1 gene can be cloned using anti-ICAM-1 antibody to detect clones that produce ICAM-1. ;Cloning of the gene for ICAM-1 using oligonucleotide probes ;Using the genetic code (J.D. Watson, In: Molecular Biology of the Gene, 3rd ed., W.A. Benjamin, Inc., Menlo Park, CA (1977)) , one or more different oligonucleotides could be identified, each of which would encode the tryptic ICAM-1 peptides. The likelihood that a particular oligonucleotide will actually constitute the true ICAM-1 coding sequence can be assessed by considering abnormal base-pairing ratios and the frequency with which a particular codon is actually used (to code for a particular amino acid) in eukaryotic cells. Such "codon usage rules" are described by R. Latne et al., J. Molec. Biol., 183:12 (1985). By applying the "codon usage rules" according to Lathe, a single oligonucleotide, or a set of oligonucleotides, is identified that contains a theoretical "most likely" nucleotide sequence (i.e. the least abundant nucleotide sequence) that can code for the tryptic ICAM-1 peptide sequences . ;The oligonucleotide, or the set of oligonucleotides, which contains the theoretically "most likely" sequence that can code for the ICAM-1 fragments, is used for the identification of the sequence of a complementary oligonucleotide or set of oligonucleotides that can be hybridized to the "most likely" sequence , or set of sequences. An oligonucleotide containing such a complementary sequence can be used as a probe for the identification and isolation of the ICAM-1 gene (T. Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring Habo Press, Cold Spring Harbor, NY (1982) ;As described under part C above, it is possible to clone the ICAM-1 gene from eukaryotic DNA preparations that are assumed to contain this gene. For identification and cloning of the gene that codes for the ICAM-1 protein, a DNA library with respect to its ability to hybridize with the oligonucleotide probes described above, because it is likely that there will be only two copies of the gene for ICAM-1 in a normal diploid cell, and because it is possible that ICAM-1 -the gene may have large non-transcribed intervening sequences (introns) if cloning is not desired, it is preferred to isolate ICAM-1 coding sequences from a cDNA library prepared from the mRNA of an ICAM-1-producing cell, rather than from genomic DNA Suitable DNA or cDNA pre preparations are enzymatically cleaved, or randomly cut and ligated into recombinant vectors. The ability of these recombinant vectors to hybridize to the above described oligonucleotide probes is then measured. Procedures for hybridization are e.g. described in T. Maniatis Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982) or in B.T. Haymes et al., Nucleic Acid Hybridization a Practical Approach, IRL Press, Oxford, England (1985). Vectors found to be capable of such hybridization are then analyzed to determine the extent and nature of the ICAM-1 sequences they contain. Based on purely statistical considerations, a gene such as that encoding the ICAM-1 molecule could be unequivocally identified (via hybridization assay) using an oligonucleotide probe of only 18 nucleotides. Briefly, the actual identification of ICAM-1 peptide sequences thus enables the identification of a theoretical "most likely" DNA sequence, or a set of such sequences, which may code for such a peptide. By constructing an oligonucleotide that is complementary to this theoretical sequence (or by constructing a set of oligonucleotides that is complementary to the set of "most likely" oligonucleotides), one obtains a DNA molecule (or a set of DNA molecules) that can function as a probe for the identification and isolation of the ICAM-1 gene. Using the ICAM-1 peptide sequences in Table 5, the sequence of the "most likely" sequence of an oligonucleotide that could encode the AA and J peptides was determined (Tables 6 and 7, respectively). Oligonucleotides complementary to these sequences were synthesized and purified for use as probes for the isolation of ICAM-1 gene sequences. Appropriate size-selected cDNA libraries were generated from poly(A) <+->RNA for both PMA-induced HL-60 cells and from PS-stimulated umbilical vein endothelial cells. A size-selected cDNA library was prepared using poly(A) <+->RNA from PMA-induced HL-60 cells according to the method of U. Gubler et al., (Gene, 25:263-269 (1983)) and A. Corbi et al., (EMBO J. , 6:4023-4028 (1987)), which references are incorporated herein by reference. ;A size-selected cDNA library was prepared using poly(A) <+->RNA from umbilical vein endothelial cells stimulated for 4 h with PS 5 Mg/ml■ RNA was extracted by homogenizing the cells in 4 M guanidine isothiocyanate and the supernatant was subjected to ultracentrifugation through a CsCl gradient (J.M. Chirgwin et al ., Biochem., 18:5294-5299; (1979)). Poly(A) <+->RNA was isolated from the mixture of the total amount of RNA species using oligo-(dT)-cellulose chromatography (type 3, Collaborative Research) (H. Aviv et al., Proe. Nati. Acad. Sci. (USA), 69:1408-1412 (1972)). ;First strand cDNA was synthesized using 8 µg poly(A) <+->RNA, avian myeloblastosis virus reverse transcriptase (Life Sciences) and an oligo(dT) primer. The DNA-RNA hybrid was digested with RNase H (BRL), and the second strand was synthesized using DNA polymerase I (New England Biolabs). The product was methylated with Eco R1 methylase (New England Biolabs), blunt-ended to Eco Ri linkers (New England Biolabs), digested with Eco Ri, and size-selected on a low-melting point agarose gel. cDNA greater than 500 bp was ligated to XgtlO that had previously been Eco R1 digested and dephosphorylated (Stratagen). the product of the ligation was then packed (Stratågene gold). ;Umbilical vein endothelial cell and HL-60 cDNA libraries were then plated at 20,000 PFU/150 mm plate. Recombinant DNA was transferred in duplicate to nitrocellulose filters, denatured in 0.5 M NaOH/1.5 M NaCl, neutralized in 1 M Tris, pH 7.5/1.5 M NaCl and baked at 80°C for 2 h ( W.D. Benton et al., Science, 196:180-182 (1977)). The filters were prehybridized and hybridized in 5X SSC containing 5X Denhardf's solution, 50 mM NaPO 4 and 1 Mg/ml salmon sperm DNA. Prehybridization was performed at 45°C for 1 hour. ;Hybridization was performed using 32 bp (5'-TTGGGCTGGTCACAGGAGGTGGAGCAGGTGAC) or 47 bp (5'-GAGGTGTTCTC-AAACAGCTCCAGGCCCTGGGGCGCCAGGTCCAGCTC) antisense oligonucleotides based, as described above, on the ICAM-1 tryptic peptides J and AA, respectively ( tables 6 and 7) (R. Lathe, J. Molec. Biol., 183:1-12 (1985)). The oligonucleotides were end-labeled with r-( <32>P(ATP) using T4 polynucleotide kinase and conditions recommended by the manufacturer (New England Biolabs). After overnight hybridization, the filters were washed twice with 2X SSC/0.1% SDS for 30 minutes at 45°. Phage were isolated from those plaques that showed hybridization, and were purified by successive replating and reprobing. ;Cloning of the gene for ICAM-1 using anti-ICAM-1 antibody f ;The gene for ICAM-1 can alternatively be cloned using anti-ICAM-1 antibody. DNA, or more preferably cDNA, is extracted and purified from a cell capable of expressing ICAM-1. The purified cDNA is fragmented (by cutting, endonuclease digestion, etc.) to form a mixture of DNA or cDNA fragments. DNA or cDNA fragments from this mixture are then cloned into an expression vector to form a genomic library of expression vectors whose elements each contain a unique cloned DNA or cDNA fragment. ;EXAMPLE 17 ;Analysis of the cDNA clones ;Phage DNA from positive clones was digested with Eco Ri and examined by Southern analysis using a cDNA from one clone as probe. Maximum size cDNA inserts that cross-hybridized were subcloned into the Eco Ri site in plasmid vector pGEM 4Z (Promega). HL-60 subclones containing cDNA in both orientations were eliminated by exonuclease III digestion (S. Henikoff, Gene, 28:351-359 (1984)) according to the manufacturer's recommendations (Erase-a-Base, Promega). Progressively eliminated cDNA molecules were then cloned and subjected to dideoxynucleotide chain termination sequencing (F. Sanger et al., Proe. Nati. Acad. Sei (USA), 74:5463-5467 (1977)) according to the manufacturer's recommendations (Sequenase, U.S. Biochemical) . HL-60-CDNA-51 - and the coding regions were completely sequenced on both strands, and 3 The <1->region was sequenced approx. 70% on both chains. A representative endothelial cDNA was sequenced over the majority of its length by rapid cloning of 4 bp recognition restriction enzyme fragments. The cDNA sequence from one HL-60 and one endothelial cell cDNA was generated (Figure 8). The 3023 bp sequence contains a short 5' untranslated region and a 1.3 kb 3 <1->untranslated region with a consensus polyadenylation signal at position 2966. The longest open reading frame begins with the first ATG at position 58 and ends with a TGA termination triplet at position 1653. The identity between the translated amino acid sequence and sequences determined from 8 different tryptic peptides totaling 91 amino acids (underlined in Figure 8) confirmed that authentic ICAM-1 cDNA clones had been isolated. The amino acid sequences of tryptic ICAM-1 peptides are shown in Table 8. Hydrophobicity analysis (J. Kyte et al., J. Molec. Biol., 157:105-132 {1982)) suggests the presence of a 27-residue signal sequence. The determination of the +1-glutamine is consistent with our inability to obtain the N-terminal sequence for three different ICAM-1 protein preparations; glutamine can cyclize to pyroglutamic acid, resulting in a blocked N-terminus. The translated sequence from 1 to 453 is mainly hydrophilic, followed by a 24-residue hydrophobic putative transmembrane domain. The transmembrane field is immediately followed by several charged residues found in a 2 7-residue putative cytoplasmic field. The predicted size for the fully developed polypeptide chain is 55,219 daltons, in excellent agreement with the observed size of 55,000 for deglycosylated ICAM-1 (M.L. Dustin et al., J. Immunol., 137:245-254 (1986)). Eight N-linked glycosylation sites are predicted. Absence of asparagine in the tryptic peptide sequences in two of these sites confirms their glycosylation and their extracellular orientation. Assuming 2,500 daltons per N-linked carbohydrate with high mannose content, a size of 75,000 daltons is predicted for the ICAM-1 precursor, compared to the observed six of 73,000 daltons (M.L. Dustin et al., J. Immunol., 137:245-254; (1986)). After conversion of high-mannose to complex carbohydrate, the fully developed ICAM-1 glycoprotein is 76-114 kd, depending on the cell type (M.L. Dustin et al., J. Immunol., 137:245-254 (1986)). Thus, ICAM-1 is a highly glycosylated, but otherwise typical, integral membrane protein. ;EXAMPLE 18 ;ICAM-1 is an integrin-binding member of the immunoglobulin supergene family ;Alignment of ICAM-1 internal repeats was performed using the Microgenie protein alignment program (C. Queen et al., Nucl. Acid Res., 12 :581-599 (1984)) followed by inspection. Alignment of ICAM-1 with IgM, N-CAM and MAG was performed using the Microgenie and ALIGN program (M.O. Dayhoff et al., Meth. Enzymol., 91:525-545 (1983)). Four protein sequence databases, maintained by the National Biomedical Research Foundation, were examined for protein sequence similarities using the FASTP program of Williams and Pearson (D.J. Lipman et al., Science, 227:1435-1439; (1985)). ;Since ICAM-1 is a ligand of an integrin, it was unexpected that it would be a member of the immunoglobulin supergene family. However, inspection of the ICAM-1 sequence shows that it fulfills all criteria proposed for membership in the immunoglobulin supergene family. These criteria are discussed below. ;The entire extracellular domain of ICAM-1 is constructed of 5 homologous immunoglobulin-like regions which are shown aligned in Figure 9A. Fields 1-4 are respectively 88, 97, 99 and 99 residues long and thus have typical Ig field size; field 5 is truncated within 68 residues. Examination of the NBRF database using the FASTP program revealed significant homologies with members of the immunoglobulin supergene family including IgM and IgG C fields, variable T cell receptor of subunit field, and alpha-l-beta glycoprotein (Figure 9B-D). ;Using the above information, the amino acid sequence of ICAM-1 was compared to the amino acid sequences of other members of the immunoglobulin supergene family. ;Three types of Ig superfamily domains, V, Cl and C2 have been differentiated. Both V and C domains are constructed of 2 S layers bound together by the intradomain disulfide bond; V domains contain 9 antiparallel fi chains, while C domains have 7. Constant domains were divided into C1 and C2 sets based on characteristic residues shown in Figure 9A. The Cl set includes proteins involved in antigen recognition. The C2 set includes several Fc receptors and proteins involved in cell adhesion including CD2, LFA-3, MAG and NCAM. ICAM-1 fields were found to be most homogeneous with fields in the C2 set when placing ICAM-1 in this set; this is reflected by stronger similarity to conserved residues in C2 than Cl fields as shown for fi chains B-F in Figure 9. Moreover, ICAM-1 fields matched much better with S chains A and G in C2 fields than with these chains in V and Cl fields, which made possible good arrangements over the entire C2 area. Arrangements with C2 fields from NCAM, MAG and alpha-1-S-glycoprotein are shown in Figures 9B and 9C, - the identity was in the range from 28 to 33%. Arrays with a T-cell receptor Va 27% identity and IgM C-field 3-34% identity are also shown (Figures 9B, 9D). ;One of the most important characteristics of immunoglobulin fields are the disulfide-bonded cysteines that bind together the B- and F-fi chains that stabilize the 6-layer formation; in ICAM-1, the cysteines are conserved in all cases except in chain f in field 4, where a leucine amino acid is present that may face the layer formation and stabilize the contact as suggested for some other V and C2 set fields. The distance between the cysteine amino acids (43, 50, 52 and 37 residues) is as described for the C2 set. ;To examine the presence of intrachain disulfide bonds in ICAM-1, endothelial cell ICAM-1 was subjected to SDS- ;PAGE under reducing and non-reducing conditions. Endothelial cell ICAM-1 was used because it shows less glycosylation heterogeneity than JY or hair cell spleen ICAM-1 and allows greater sensitivity to changes in Mr. ICAM-1 was therefore purified from cultures of umbilical vein endothelial cells stimulated for 16 h with LPS (5 µg/ml), by immunoaffinity chromatography as described above. Acetone-precipitated ICAM-1 was resuspended in sample buffer (U.K. Laemmli, Nature, 227:680-685 (1970)) with 0.25% 2-mercaptoethanol or 25 mM iodoacetamide and brought to 100°C for 5 minutes. The samples were subjected to SDS-PAGE 4670 and silver staining 4613. Endothelial cell ICAM-1 had an apparent Mr of 100 kd under reducing conditions and 96 kd under non-reducing conditions, strongly suggesting the presence of intrachain disulfides in native ICAM-1. ;Using the primary sequence for secondary structure prediction (P.Y. Chou et al., Biochem., 13:211-245 ;(1974)) showed the 7 expected fi chains in each ICAM-1 field, labeled a-g in Figure 9A top, which exactly fulfilled the prediction of an immunoglobulin field, corresponding to the positions of chains A-H in immunoglobulins (Figure 9A, lower). Field 5 lacks the A and C chains, but since these form edges of the layers, the layers could still be formed, possibly replacing chain D with chain C, as suggested for some other C2 fields; and the characteristic disulfide bond between the B and F chains would be unaffected. Thus, all the criteria for field size, sequence homology, conserved cysteine amino acids forming the putative intrafield disulfide bond, presence of disulfide bonds and assumed 6-layer structure are met for incorporation of ICAM-1 into the immunoglobulin superfamily. ;ICAM1 was found to be most highly homologous to NCAM and MAG glycoproteins in the C2 set. This is of particular interest since both NCAM and MAG mediate cell-cell adhesion. NCAM is important in neuron-neuron and neuromuscular interactions (B.A. Cunningham et al., Science, 236:799-806 (1987)), while MAG is important in neuron-oligodendrocyte and oligodendrocyte-oligodendrocyte interactions during myelination (M. Poltorak et al., J. Cell Biol., 105:1893-1899 (1987)). The cell surface expression of NCAM and MAG is developmentally regulated during nervous system formation and myelination, respectively, in analogy to the regulated induction of ICAM-1 during inflammation (T.A. Springer et al., Ann. Rev. Immunol., 5:223-252 (1987)). ICAM-1, NACM (B.A. Cunningham et al., Science, 236:799-806(1987)) and MAG (J.L. Salzer et al., J. Cell. Biol., 104:957-965 (1987)) are similar in overall structure as well as homologous, since each is an integral membrane glycoprotein constructed of 5 C2 domains forming the N-terminal extracellular region, although an additional non-Ig-like sequence is present in NCAM between the last C2 field and the transmembrane field. ICAM-1 matches over its entire length including the transmembrane and cytoplasmic fields, with MAG with 21% identity, - the same percentage identity is found when comparing the 5 fields of ICAM-1 and NCAM-1. A schematic comparison of the secondary structures of ICAM-1 and MAG is shown in Figure 10. Field-by-field comparisons show that the level of homology between fields in ICAM-1 and NCAM molecules (respectively x + s.d. 21 + 2.8% and 18 .6 + 3.8%) is the same as the level of homology when comparing ICAM-1 fields and NCAM and MAG fields (20.4 + 3.7 and 21.9 + 2.7, respectively). Although there is evidence for alternative splicing in the C-terminal regions of NCAM (B.A. Cunningham et al., Science, 236:799-806 (1987); D. Barthels et al., EMBO J., 6:907-914; (1987)) and MAG (C. Lai et al., Proe. Nati. Acad. Sei. (USA), 84:4377-4341 (1987)), no evidence for this has been found in the sequencing of endothelial- or HL-60 ICAM-1 clones or by studies of the ICAM-1 protein backbone and precursor in many different cell types (M.L. Dustin et al., J. Immunol., 137:245-254 (1986)). ;ICAM-1 acts as a ligand for LFA-1 in lymphocyte interactions with a number of different cell types. Lymphocytes bind to ICAM-1 incorporated into artificial membrane bilayers, and this requires LFA-1 on the lymphocyte, demonstrating direct LFA-1 interaction with ICAM-1 (S.D. Marlin et al., Cell, 51:813-819 (1987) ). LFA-1 is a leukocyte integrin and has no immunoglobulin-like features. Leukocyte integrins comprise one integrin subfamily. The other two subfamilies mediate cell-matrix interactions and perceive the sequence RGD in their ligands which include fibronectin, vitronectin, collagen and fibrinogen (R.O. Hynes, Cell, 48:549-554 (1987); E. Ruoslathi et al., Science, 238: 491-497 (1987)). The leukocyte integrins are only expressed on leukocytes, are involved in cell-cell interactions, and the only known ligands are ICAM-1 and iC3b, a fragment of the complement component C3 that shows no immunoglobulin-like features and is perceived by Mac-1 (T.K. Kishimoto et al., I; Leukocyte Typing III, M. McMichael (ed.), Springer-Verlag, New York (1987); T. A. Springer et al., Ann. Rev. Immunol., 5:223-252 (1987); D.C. Anderson et al., Ann. Rev. Med., 38:175-194 (1987)). Based on sequence analysis, any peptides in the ICAM-1 sequence that are recognized by LFA-1 are shown in Table 9. ;ICAM-1 is the first example of a member of the immunoglobulin supergene family that binds to an integrin. Although both of these families play an important role in cell adhesion, interaction between them was not previously expected. In contrast, interactions within the immunoglobulin gene superfamily are quite common. It is very possible that further examples of interactions between the integrin and immunoglobulin families will be found. LFA-1 perceives a ligand distinct from ICAM-1 (T.A. Springer et al., Ann. Rev. Immunol., 5:223-252 {1987)), and the leukocyte integrin Mac-1 perceives a ligand distinct from C3bi at neutrophil-neutrophil -adhesion {D.C. Anderson et al., Ann. Fox. Med., 38:175-194 (1987)). Furthermore, purified MAG-containing vesicles bind to neurites which are MAG, and thus MAG must be able to interact heterophilically with a special receptor (M. Poltorak et al., J. Cell Biol., 105:1893-1899 (1987)). The role of NCAM in neutral-neutral and neutral-muscle cell interactions has been suggested to be due to homologous NCAM-NCAM interactions (B.A. Cunningham et al., Science, 236:799-806;(1987)). The important role of MAG in interactions between neighboring turn loops of Schwann cells enclosing axons during myelin sheath formation may be due to interaction with a special receptor, or homophilic MAG-MAG interaction. The homology with NCAM and the frequent occurrence of field-field interactions within the immunoglobulin supergene family raise the possibility that ICAM-1 could participate in homophilic interactions as well as heterophilic ICAM-1-LFA-1 interactions. However, binding of B lymphoblast cells co-expressing similar densities of LFA-1 and ICAM-1 to ICAM-1 in artificial or cellular monolayers can be completely inhibited by pretreatment of the B lymphoblast with LFA-1-MAb, while adhesion is unaffected of B-lymphoblast pretreatment with lCAM-1-MAb. Pretreatment of the monolayer with ICAM-1-MAb completely abrogates binding M.L. Dustin et al., J. Immunol., 137:245-254; (1986); S. D. Marlin et al., Cell, 51:813-819 (1987)). These findings demonstrate that if homophilic ICAM-1 interactions occur at all, they must be weaker than heterophilic interactions with LFA-1. ;The possibility that the leukocyte integrins perceive ligands in a fundamentally different way is consistent with the presence of a 180-residue sequence in their α-subunits that may be important in ligand binding and which is not present in the RGD-sensing integrins (A. Corbi et al., (EMBO J., 6:4023-4028 (1987)). Although Mac-1 has been suggested to perceive RGD sequence present in iC3b 5086, there is no RGD sequence in ICAM-1 ( Figure 8). This is consistent with the fact that the fibronectin peptide GRGDSP and the control peptide GRGESP do not inhibit ICAM-1-LFA-1 adhesion (S.D. Marlin et al., Cell, 51:813-819 (1987)).However, are related sequences such as PRGGS and RGEKE present in ICAM-1 in areas that are predicted to form loops between 6-chains a and b in field 2, and c and d in field 2 (figure 9), and may thus be available for perception . It is of interest that the homologous MAG molecule contains an RGD sequence between fields 1 and 2 (M. Poltorak et al., J Cell Biol., 105:1893-1899 (1987); J. L. Salzer et al., J. Cell. Biol., 104:957-965 (1987)). EXAMPLE 19 Southern and Northern Blots Southern blots were performed using 5 µg of genomic DNA extracted from three cell lines: BL2, a Burkitt lymphoma cell line (a gift from Dr. Gilbert Lenoir); JY and Er-LCL, EBV-transformed B lymphoblastoid cell lines. The DNA molecules were digested with 5 times the manufacturer's recommended amount of BAM H1 and Eco R1 endonucleases (New England Biolabs). After electrophoresis through a 0.8% agarose gel, the DNA molecules were transferred to a nylon membrane (Zeta Probe, BioRad). The filter was prehybridized and hybridized according to standard procedures using ICAM-1 cDNA from HL-60 labeled with α-(32P)d-XTP molecules by random labeling (Boehringer Mannheim). Northern blots were performed using 20 µg total RNA or 6 µg poly(A) <+->RNA. RNA was denatured and electrophoresed through a 1% agarose-formaldehyde gel and electro-transferred to Zeta-Probe. The filters were prehybridized and hybridized as described previously (D.E. Staunton et al., Embo J., 6:3695-3701 (1987)) using the HL-60 cDNA probe of <32->P-labeled oligonucleotide probes (described above). ;Southern blots using the 3 kb cDNA probe and genomic DNA digested with BAM H1 and Eco Ri showed single-dominant hybridization fragments of 20 and 8 kb, respectively, suggesting a single gene and suggesting that the majority of the coding information is present within 8 kb. In blots of three different cell lines, there is no evidence of restriction fragment polymorphism. ;EXAMPLE 20 ;Expression of the ICAM-1 gene ;An "expression vector" is a vector which (due to the presence of appropriate transcriptional and/or translational control sequences) can express a DNA (or cDNA) molecule that has been cloned into the vector, thereby forming a polypeptide or protein. Expression of the cloned sequences occurs when the expression vector is introduced into a suitable host cell. If a prokaryotic expression vector is used, the appropriate host cell will be any prokaryotic cell capable of expressing the cloned sequences. Similarly, if a eukaryotic expression vector is used, the appropriate host cell will be any eukaryotic cell capable of expressing the cloned sequences. It is significant that since eukaryotic DNA can contain intervening sequences, and since such sequences cannot be correctly processed in prokaryotic cells, it is preferred to use cDNA from a cell that can express ICAM-1 for the production of a prokaryotic genome expression vector library. Methods for preparing cDNA and for generating a genome library are described by T. Maniatis et al., (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1982)). ;The expression vector genomic library described above is used to create a bank of host cells (each of which contains an element of the library). The expression vector can be introduced into the host cell by any of many different means (ie, transformation, transfection, protoplast fusion, electroporation, etc.). The bank of expression vector-containing cells is clonally propagated and its elements analyzed individually (using an immunoassay) to determine whether they form a protein that can bind to anti-ICAM-1 antibody. ;The expression vectors for the cells that produce a protein that can be bound to anti-ICAM-1 antibody are then further analyzed to determine whether they express (and thus contain) the entire ICAM-1 gene, whether they express (and contain) only a fragment of the ICAM-1 gene, or whether they express (and contain) a gene whose product, although immunologically related to ICAM-1, is not ICAM-1. Although such analysis can be performed in any convenient manner, it is preferred to determine the nucleotide sequence of the DNA or cDNA fragment that had been cloned into the expression vector. Such nucleotide sequences are then examined to determine whether they may code for polypeptides with the same amino acid sequence as the tryptic digestion fragments of ICAM-1 (table 5). An expression vector containing a DNA or cDNA molecule encoding the ICAM-1 gene can thus be understood by: (i) the ability to direct the expression of a protein that can bind to anti-ICAM-1 antibody; and (ii) presence of a nucleotide sequence that can encode each of the tryptic fragments of ICAM-1. The cloned DNA molecule in such an expression vector can be removed from the expression vector and isolated in pure form. ;EXAMPLE 21 ;Functional activities of purified ICAM-1 ;In cells, ICAM-1 normally functions as a surface protein associated with the cell membrane. The function of purified ICAM-1 was therefore investigated after the molecule had been reconstituted in artificial lipid membranes (liposomes or vesicles) by dissolving the protein in detergent-solubilized lipids, followed by removal of the detergent by dialysis. ICAM-1, purified from JY cells and eluted in the detergent octylglucoside as described above, was reconstituted into vesicles, and the ICAM-1-containing vesicles were fused to coverslips or plastic culture wells to allow detection of cells that bound to the protein. ;Preparation of planar membranes and plastic-bound vesicles ;Vesicles were prepared by the method of Gay et al., (J. Immunol., 136:2026 (1986)). Briefly, egg phosphatidylcholine and cholesterol were dissolved in chloroform and mixed in a molar ratio of 7:2. The lipid mixture was dried to a thin film while rotating under a stream of nitrogen gas, and then lyophilized for 1 hour while removing all traces of chloroform. The lipid film was then dissolved in 1% octylglucoside/0.14 M NaCl/20 mM Tris (pH 7.2) to a final concentration of phosphatidylcholine of 0.1 mM. About. 10 µg of purified ICAM-1, or human glycophorin (Sigma Chemical Co., St. Louis, MO) as a control membrane glycoprotein, was added to each ml of dissolved lipids. The protein-lipid-detergent solution was then dialyzed at 4°C against 3 changes of 200 volumes of 20 mM Tris/0.14 M NaCl, pH 7.2, and one change of HBSS. Planar membranes were prepared by the method of Brian et al., Proe. Nati. Acad. Sci., 81:6159 (1984). Coverslips (diameter 11 mm) were boiled for 15 min in a 1:6 dilution of 7X detergent (Linbro), washed overnight in distilled water, immersed in 70% ethanol and air dried. An 80 ( xl drop of vesicle suspension containing either ICAM-1 or glucophorin was placed in the bottom of wells in a 24-well "duster" plate, and the prepared coverslips were carefully allowed to float on top. After 20-30 minute incubation at room temperature, the wells were filled with HBSS, and the coverslips were inverted so that the planar membrane would face up. The wells were then washed thoroughly with HBSS while removing unbound vesicles. The planar membrane surface was never exposed to air. ;During experiments with planar membrane fused to glass surfaces, vesicles containing ICAM-1 were found to bind directly to the plastic surface of multiwell tissue culture plates, retaining functional activity as evidenced by specific cell binding. Such vesicles are hereafter referred to as "plastic-bound vesicles" (PBV ), since the nature of the lipid vesicles bound to the plastic has not been determined. ;Plastic bound vesicles were prepared by adding 30 µl of vesicle suspension directly to the bottom of wells in 96-well tissue culture plates (Falcon), followed by incubation and washing as described for planar membranes. ;Cell Adhesion Assays ;Cell adhesion assays using planar membranes or plastic-bound vesicles were both performed in essentially the same manner, except that the cell numbers and volume fractions for PBV assays were reduced to 1/5 of that used in planar membrane assays. ;T lymphocytes from normal controls and a patient with leukocyte adhesion deficiency (LAD) whose cells do not express LFA-1 (D.C. Anderson et al., J. Infect. Dis., 152:668 (1985)) were prepared by culturing mononuclear cells from peripheral blood with 1 µl Concanavalin-A (Con-A) in RPMI-1640 plus 20% FCS with 5 x IO <5> cells/ml for 3 days. The cells were then washed twice with RPMI and once with 5 mM methyl-alpha-D-mannopyranoside to remove residual lecithin from the cell surface. The cells were cultured in RPMI/2 0% FCS containing 1 ng/ml recombinant IL-2, and were used between 10 and 22 days after starting the culture. ;For the detection of cell binding to planar membranes or PBV, Con-A blasts, the T-lymphoma SKW-3 and the EBV-transformed B-lymphoblastoid cell lines JY (LFA-1-positive) - and LFA-1-lacking lymphoblastoid- cell line (BBN) (from patient 1, T.A. Springer et al., J. Exper Med., 160:1901-1918 (1986)) labeled by incubation of 1 x 10 <7> cells in 1 ml RPMI-1640/10% FCS with 100 /iCi Na <51>CrO4 for 1 h at 37°C, followed by four washes with RPMI-1640 to remove unincorporated label. In monoclonal-antibody-blocking experiments, cells or plastic-bound vesicles were pretreated with 20 µg/ml of purified antibody in RPMI-1640/10% FCS at 4°C for 30 minutes, followed by four washes to remove unbound antibody. In experiments regarding the effects of divalent cations on cell attachment, the cells were washed once with Ca <2+->, Mg <2+->free HBSS plus 10% dialyzed FCS, and CaCl and MgCl were added to the indicated concentrations. In all experiments, cells and planar membranes or PBV were pre-equilibrated at the appropriate temperature (4°C, 22°C or 37°C) in the appropriate assay buffer. ;For measurement of cell-bound to purified ICAM-1, was <51>Cr-labeled cells (5 x IO <5> EBV transformants in flat-membrane assays; 1 x 10s EBV transformant or SKW-3 cells, 2 x IO <5> Con-A vesicles in PBV assay) centrifuged for 2 min at 25 x g on planar membranes or PBV, followed by incubation at 4°C, 22°C or 37°C for 1 h. After the incubation, unbound cells were removed by eight cycles of loading and aspiration with buffer pre-equilibrated to the appropriate temperature. Bound cells were quantified by solubilizing the well contents with 0.1 N NaOH/1% Triton X-100 and counting in a gamma counter. Percent cell binding was determined by dividing cpm from bound cells by input cell-associated cpm. In planar membrane assays, entered cpm was corrected for the ratio of coverslip surface area compared to culture well surface area. In these assays, EBV-transformed B-lymphoblastoid cells, SKW-3 T-lymphoma cells and Con-A-T lymphoblasts bound specifically to ICAM-1 in artificial membranes (Figures 11 and 12). The binding was specific since the cells bound very weakly to planar control membranes or vesicles containing equivalent amounts of another human cell surface glycoprotein, glycophorin. Furthermore, LFA-1-positive EBV transformants and Con-A blasts bound, whereas their LFA-1-negative counterparts did not bind to any significant extent, demonstrating that binding depends on the presence of LFA-1 on the cells. Both the specificity of the cell binding and the dependence on cellular LFA-1 were confirmed by monoclonal antibody blocking experiments (Figure 13). The binding of JY cells could be inhibited by 97% when the ICAM-1-containing PBVs were pretreated with a monoclonal anti-ICAM-1 antibody RR1/1. Pretreatment of the cells with the same antibody had little effect. Conversely, the monoclonal anti-LFA-1 antibody inhibited TS1/18 binding by 96%, but only when the cells, but not PBV, were pretreated. A control antibody TS2/9 that reacted with LFA-3 (another lymphocyte surface antigen) had no significant inhibitory effect when both cells or PBV were pretreated. This experiment shows that ICAM-1 itself in the artificial membranes, and not a minor contaminant, produces the observed cell adhesion, and that the adhesion is dependent on LFA-1 on the binding cell. ;The binding of cells to ICAM-1 in artificial membranes also showed two other characteristics of the LFA-1-dependent adhesion system: temperature dependence and a requirement for divalent cations. As shown in Figure 14, Con-A blasts bound to ICAM-1 in PBV most efficiently at 37°C, partially at 22°C and very poorly at 4°C. As shown in Figure 15, the binding was completely dependent on the presence of divalent cations. At physiological concentrations, Mg <2+> alone is sufficient for maximum cell binding, while Ca <2+> alone gave very low binding levels. However, Mg <2+> at 1/10 of the normal concentration, combined with Ca <2+>, synergistic and gave maximum binding. Briefly stated, the specificity of cell binding to purified ICAM-1 incorporated in artificial membranes, the specific inhibition with monoclonal antibodies and the temperature and divalent cation requirements show that ICAM-1 is a specific ligand for the LFA-1-dependent adhesion system. EXAMPLE 22 Expression of ICAM-1 and HLA-DR in allergic and toxic patch test reactions Skin biopsies from five normal individuals were examined for expression of ICAM-1 and HLA-DR. It was found that although the endothelial cells of some blood vessels normally expressed ICAM-1, there was no ICAM-1 expression on keratinocytes from normal skin. No staining for HLA-DR on any keratinocyte from normal skin biopsies was observed. The expression kinetics of ICAM-1 and class II antigens were then examined for cells in biopsies from allergic and toxic skin lesions. Half of the six subjects examined were found to have keratinocytes expressing ICAM-1 4 hours after application of the hapten (Table 10). There was an increase in the percentage of individuals expressing ICAM-1 on their keratinocytes with exposure time to the hapten, as well as an increase in color intensity, showing more ICAM-1 expression per keratinocyte up to 48 hours. Indeed, at this time, a certain proportion of keratocytes in all biopsies stained positive for ICAM-1. At 72 hours (24 hours after the hapten was removed), seven of the eight subjects still had ICAM-1 expressed on their keratinocytes, while for one subject the expression of ICAM-1 decreased between 48 and 72 hours. ;;Histologically, the staining pattern for ICAM-1 on keratinocytes from biopsies taken 4 hours after application of the hapten was usually in small clusters. After 48 hours, ICAM-1 was expressed on the surface of the majority of keratinocytes, with no difference being seen between the center and periphery of the lesion. The staining intensity decreased as the keratinocytes approached the stratum corneum. This was found for biopsies taken from both the center and the periphery of the lesions. Also at this time the patch test was positive (infiltration, erythema and vesicles). No difference in ICAM-1 expression was observed when different haptens were applied to susceptible individuals. In addition to keratinocytes, ICAM-1 was also expressed on some mononuclear cells and endothelial cells at the lesion site. ;The expression of HLA-DR on keratinocytes in the allergic skin lesions was less frequent than the expression of ICAM-1. None of the subjects examined had lesions with keratinocytes that stained positive for HLA-DR up to 24 hours after application of the hapten. Indeed, only four biopsy specimens had keratinocytes expressing HLA-DR, and no biopsy had keratinocytes positive for HLA-DR and not ICAM-1 (Table 10). In contrast to the allergic patch lesion, the toxic patch lesion induced with croton oil or sodium lauryl sulfate had keratinocytes showing no, if any, ICAM-1 on their surface at all time points examined (Table 11). Indeed, at 48 hours after patch application, which was the optimal time for ICAM-1 expression in the allergic patch subjects, only one of the 14 toxic patch subjects had keratinocytes expressing ICAM-1 in their lesions. Also in contrast to the allergic patch test biopsies, there was no HLA-DR expressed on keratinocytes in toxic patch test lesions. ;These data show that ICAM-1 is expressed in immune-based inflammation, and not in toxic-based inflammation, and thus the expression of ICAM-1 can be used to distinguish between immune-based and toxic-based inflammation, such as acute renal failure in kidney transplant patients, where it is difficult to determine whether the failure is due to rejection or renal toxicity of the immunosuppressive therapeutic agent. Kidney biopsy and assessment of upregulation of ICAM-1 expression will enable differentiation of immune-based rejection and non-immune-based toxicity reaction. ;EXAMPLE 23 ;Expression of ICAM-1 and HLA-DR in ;Benign Skin Diseases ;Cells from skin biopsies of lesions from patients with various types of inflammatory skin diseases were examined for their expression of ICAM-1 and HLA-DR. A certain proportion of keratinocytes in biopsies from allergic contact dermatitis, pemphigoid/pemphigus and lichen planus expressed ICAM-1. Lichen planus biopsies showed the most intense staining with a pattern similar to, or even stronger than, that seen in 48-hour allergic patch biopsies (Table 12). Consistent with results seen with the allergic patch test, the most intense ICAM-1 staining was seen at sites of heavy mononuclear cell infiltration. Furthermore, 8 of the 11 examined lichen planus biopsies were positive with regard to HLA-DR expression on keratinocytes. ;The expression of ICAM-1 on keratinocytes from skin biopsies from patients with exanthema and urticaria was less pronounced. Only four of the seven examined patients with these diseases had keratinocytes expressing ICAM-1 at the lesion site. HLA-DR expression was found in only one patient, and this was in association with ICAM-1. Endothelial cells and a certain proportion of the infiltrate of the mononuclear cells from all the examined benign inflammatory skin diseases expressed ICAM-1 to varying extents. ;EXAMPLE 24 ;Expression of ICAM-1 on keratinocytes in psoriatic skin lesions ;The expression of ICAM-1 in skin biopsies from 5 patients with psoriasis was examined before the start of, and periodically during, a course of PUVA treatment. Biopsies were taken from 5 patients with classic psoriasis confirmed by histology. Biopsies were taken sequentially before and during the indicated time for PUVA treatment. PUVA was given 3-4 times weekly. Biopsies were taken from the periphery of the psoriatic plaques in 5 patients, and in addition, biopsies were taken from clinically normal skin in 4 of the patients. ;Fresh skin biopsy specimens were frozen and stored in liquid nitrogen. Cryostat sections of 6 µm were air-dried overnight at room temperature, fixed in acetone for 10 minutes and either stained immediately or wrapped in aluminum foil and stored at -80°C until staining. ;The staining was carried out in the following manner. Sections were incubated with monoclonal antibodies and stained by a 3-step immunoperoxidase method (H. Stein et., Adv. Cancer Res., 42:67-147; (1984)), using a diaminobenzidine-H 2 O 2 substrate . Tonsils and lymph nodes were used as positive controls for anti-ICAM1 and HLA-DR staining. Tissues stained in the absence of primary antibody were negative controls. ;The monoclonal antibodies against HLA-DR were supplied by Becton Dickinson (Mountainview, California). The monoclonal anti-ICAM-1 antibody was R6-5-D6. Peroxidase-conjugated rabbit anti-mouse Ig and peroxidase-conjugated pig anti-rabbit Ig were supplied from DAKAPATTS, Copenhagen, Denmark. Diaminobenzidine tetrahydrochloride was supplied from Sigma (St. Louis, Mo.). The results of the investigation show that the endothelial cells in some blood vessels express ICAM-1 in both diseased and normal skin, but the color intensity and the number of blood vessels expressing ICAM-1 were increased in the psoriatic skin lesions. Furthermore, the expression pattern of ICAM-1 in keratinocytes in untreated psoriatic skin lesions from the 5 patients varied from staining of only small cell clusters to staining of many keratinocytes. During PUVA treatment, ICAM-1 expression for two of the patients (patients 2 and 3) showed a marked reduction that preceded, or was concurrent with, clinical remission (table 13). Patients 1, 4 and 5 had decreases and increases of ICAM-1 expression during PUVA treatment that corresponded to clinical remissions and relapses, respectively. There was no ICAM-1 expression on keratinocytes from normal skin before or after PUVA treatment. This shows that PUVA does not induce ICAM-1 on keratinocytes from normal skin. The observation that the density of the infiltrate of mononuclear cells corresponded to the amount of ICAM-1 expression on keratinocytes was remarkable. This applied to both a decreased number of mononuclear cells in lesions during PUVA treatment when ICAM-l expression also decreased, and an increased number of mononuclear cells during PUVA treatment when ICAM-l expression on keratinocytes was more predominant. Endothelial cells and skin mononuclear cells are also ICAM-1 positive. In clinically normal skin, ICAM-1 expression was restricted to endothelial cells without any labeling of keratinocytes. ;The expression of HLA-DR on keratinocytes was variable. There was no HLA-DR positive biopsy that was not also ICAM-1 positive. Briefly stated, these results show that before treatment ICAM-1 expression is pronounced on the keratinocytes and is consistent with a dense infiltrate of mononuclear cells. During PUVA treatment, a pronounced decrease in ICAM-1 staining is seen to parallel the clinical improvement. Histologically, the skin infiltrate also became smaller. When a clinical relapse was evident during treatment, the expression of ICAM-1 on the keratinocytes increased, as well as the density of the skin infiltrate. When a clinical remission was observed during treatment, there was a corresponding decrease in ICAM-1 staining on the keratinocytes as well as a decrease in the skin infiltrate. Thus, the expression of ICAM-1 on keratinocytes responded to the density of the infiltrate of mononuclear cells in the dermis. These data show that clinical response to PUVA treatment resulted in a pronounced decrease in ICAM-1 expression on keratinocytes in parallel with a more moderate decrease in the mononuclear cells. This shows that ICAM-1 expression on keratinocytes is responsible for starting and maintaining the skin infiltrate, and that PUA treatment down-regulates ICAM-1, which in turn reduces the skin infiltrate and the inflammatory response. The data also show that there was variable HLA-DR expression on keratinocytes during PUVA treatment. ;The expression of ICAM-1 on keratinocytes in psoriatic lesions correlates with the clinical severity of the lesion as well as with the size of the skin infiltrate. Thus, ICAM-1 plays a central role in psoriasis, and inhibition of its expression and/or inhibition of its interaction with the CD 18 complex on mononuclear cells would be an effective treatment of the disease. Furthermore, control of ICAM-1 expression on keratinocytes will be an effective tool for diagnosis and prognostication, as well as assessment of the course of the treatment of psoriasis. ;EXAMPLE 25 ;Expression of ICAM and HLA-DR in ;malignant skin diseases ;In contrast to lesions from benign skin conditions, the expression of ICAM-1 on keratinocytes from malignant skin lesions was much more variable (Table 14). Among the 23 cutaneous T-cell lymphoma studies, ICAM-1-positive keratinocytes were identified in only 14 cases. There was a tendency for keratinocytes from biopsies of mycosis fungoides lesions to lose their ICAM-1 expression with the progression of the disease to more advanced stages. However, ICAM-1 expression was observed on a varying proportion of the infiltrate of mononuclear cells from most of the cutaneous T-cell lymphoma lesions. Among the remaining lymphomas examined, 4 of 8 had keratinocytes expressing ICAM-1. Among the 29 examined patients with malignant skin diseases, 5 had keratinocytes expressing HLA-DR without expressing ICAM-1 (Table 14). EXAMPLE 26 Effect of anti-ICAM-1 antibodies on the proliferation of human peripheral blood mononuclear cells Human peripheral blood mononuclear cells are induced to proliferate by the presence and perception of antigens or mitogens. Certain molecules, such as the mitogen, concanavalin A or the T-cell-binding antibody 0KT3, cause a non-specific proliferation of mononuclear cells from peripheral blood. ;Human peripheral blood mononuclear cells are heterogeneous in that they consist of subpopulations of cells that can recognize specific antigens. When a peripheral blood mononuclear cell capable of recognizing a particularly specific antigen encounters the antigen, proliferation of this subpopulation of mononuclear cells is effected. Tetanus toxoid and keyhole limpet hemocyanin are examples of antigens that are recognized by subpopulations of peripheral mononuclear cells, but not recognized by all peripheral mononuclear cells in sensitized individuals. The ability of anti-ICAM-1 monoclonal antibody R6-5-D6 to inhibit proliferative responses of human peripheral blood mononuclear cells in systems known to require cell-cell adhesions was tested. Peripheral blood mononuclear cells were purified on Ficoll-Paque (Pharmacia) gradients according to the manufacturer's instructions. After collection of the interfacial layer, the cells were washed three times with RPMI 1640 medium and cultured in flat-bottomed 96-well microtiter plates at a concentration of 106 cells/ml in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine and gentamycin (50 ng/ml). ;Antigen, either the T-cell mitogen, concanavalin A (0.25 Aig/ml) ; the T-cell binding antibody, OKT3 (0.001 ng/ml); keyhole limpet hemocyanin (10 g/ml) or tetanus toxoid (1:100 dilution from the source) was added to cells cultured as described above either in the presence or absence of anti-ICAM antibody (R6-5- D6; final concentration 5 g/ml). Cells were cultured for 3.5 days (concanavalin A assay), 2.5 days (0KT3 assay), or 5.5 days ("keyhole limpet" hemocyanin and tetanus toxoid assay) before assays were terminated. 18 hours before the end of the analysis was 2.5 fiCi <3>H-thymidine added to the cultures. Cell proliferation was analyzed by measuring the incorporation of thymidine into DNA by the mononuclear cells from peripheral blood. Incorporated thymidine was collected and counted in a liquid scintillation counter (Merluzzi et al., J. Immunol., 139:166-168 (1987)). The results of these tests are shown in Figure 16 (concanavalin A test), Figure 17 (0KT3 test), Figure 18 (keyhole limpet hemocyanin test) and Figure 19 (tetanus toxoid test). It was found that anti-ICAM -1 antibody inhibits proliferative responses to the nonspecific T-cell mitogen, ConA; the nonspecific T-cell-associated antigen, OKT-3; and the specific antigens, keyhole limpet hemocyanin and tetanus toxoid, in mononuclear cells The inhibition by anti-ICAM-1 antibody was comparable to the inhibition by anti-LFA-1 antibody, suggesting that ICAM-1 is a functional ligand of LFA-1 and that antagonism of ICAM-1 will inhibit specific defense system responses. EXAMPLE 27 Effect of Anti-ICAM-1 Antibody on the Mixed Lymphocyte Reaction As discussed above, ICAM-1 is required for efficient cell-cell interactions during an immune response mediated by LFA-1-dependent cell adhesion. The induction of ICAM -1 during immune responses or inflammatory disease makes possible interaction of leukocytes between and with endothelial cells. ;When lymphocytes from two unrelated individuals are cultured in the presence of each other, blast transformation and cell proliferation of the lymphocytes are observed. This response, of one population of lymphocytes to the presence of another population of lymphocytes, is known as the mixed-lymphocyte reaction (MLR), and is analogous to the response of lymphocytes to the addition of mitogens (Immunology The Science of Self-Nonself Discrimination, (J. Klein, John Wiley & Sons, NY (1982), pp. 453-458). ;Experiments were conducted to determine the effect of monoclonal anti-ICAM antibodies on the human MLR. These experiments were performed as follows. Peripheral blood was drawn from normal, healthy donors by venipuncture. The blood was collected in heparinized tubes diluted 1:1 at room temperature with Puck's G (GIBCO) balanced salt solution (BSS). The blood mixture (20 ml) was layered on top of 15 ml of a Ficoll/hypaque density gradient (Pharmacia, density = 1.078, room temperature) and centrifuged at 1000 x g for 20 min. The interface layer was then collected and washed 3 times in Puck's G. Cells were counted on a hemacytometer and resuspended in RPMI 1640 animal culture medium (GIBCO) containing 0.5% gentamycin, 1 mM L-glutamine (GIBCO) and 5% heat-inactivated (56°C, 30 minutes) human AB serum (Flow Laboratories) (hereafter referred to as RPMI culture medium) . Mouse anti-ICAM-1 (R6-5-D6) was used in these experiments. All monoclonal antibodies (prepared from ascites by Jackson ImmunoResearch Laboratories, Boston, MA) were used as purified IgG preparations. ;Peripheral blood mononuclear cells (PBMC) were cultured in medium with 6.25 x 10 <5> cells/ml in round-bottomed microtiter plates of the Linbro type (no. 76-013-05). Stimulator cells from a separate donor were irradiated at 1000 R and cultured with the responder cells at the same concentration. The total volume per culture was 0.2 ml. Controls included responder cells alone as well as stimulator cells alone. The culture plates were incubated at 37°C in a 5% CO 2 atmosphere with humidified air for 5 days. The wells were pulsed with 0.5 µl of tritiated thymidine (3HT) (New England Nuclear) for the last 18 hours of cultivation. In some cases, a two-way MLR was performed. The protocol was the same, except that the other donor's cells were not inactivated by irradiation. ;The cells were harvested on glass fiber filters using an automatic multiple sample harvesting device (Skatron, Norway), and rinsing with water and methanol. Filters were oven dried and counted in Aquasol in a Beckman (LS-3801) liquid scintillation counter. Results are given as mean CPM + standard error for 6 individual cultures. Table 15 shows that purified monoclonal anti-lCAM-1 antibodies inhibited MLR in a dose-dependent manner with significant suppression apparent at 20 ng/ml. Purified mouse IgG had little or no suppressive effect. Suppression of MLR by the monoclonal anti-ICAM-1 antibody occurs when the antibody is added within the first 24 hours of culture (Table 16). Briefly stated, the ability of antibody against ICAM-1 to inhibit MLR shows that monoclonal ICAM-1 antibodies are therapeutically useful in acute graft rejection. Monoclonal ICAM-1 antibodies are also therapeutically useful in related immune-mediated disorders dependent on LFA-1/ICAM-1 regulated cell-cell interactions. ;The experiments described here show that the addition of monoclonal antibodies against ICAM-1 inhibits the mixed-lymphocyte reaction (MLR) when added during the first 24 hours of the reaction. Furthermore, ICAM-1 is upregulated on human peripheral blood monocytes during in vitro culture. Furthermore, it was found that ICAM-1 is not expressed on resting human lymphocytes or monocytes from peripheral blood. ICAM-1 is upregulated on monocytes in cultured cells alone or cells co-cultured with unrelated donor cells in a mixed lymphocyte reaction using standard flow cytometric assays. This upregulation of ICAM-1 on monocytes can be used as an indicator of inflammation, especially if ICAM-1 is expressed on healthy monocytes in individuals with acute or chronic inflammation. ;ICAM-1's specificity towards activated monocytes, and the ability of antibody against ICAM-1 to inhibit MLR, suggests that monoclonal ICAM-1 antibodies may have diagnostic and therapeutic potential in acute graft rejection and related immune-mediated disorders that require cell-cell interactions . ;EXAMPLE 28 ;Genetic construction and expression of truncated derivatives of ICAM-1 ;In its native state, ICAM-1 is a cell membrane-bound protein containing an extracellular region with 5 immunoglobulin-like domains, a transmembrane domain, and a cytoplasmic domain. It was desirable to construct functional derivatives of ICAM-1 that lacked the transmembrane field and/or the cytoplasmic field in that a soluble, secreted form of ICAM-1 could be formed. These functional derivatives were constructed by oligonucleotide-directed mutagenesis of the ICAM-1 gene, followed by expression in monkey cells after transfection with the mutant gene. Mutations in the ICAM-1 gene resulting in amino acid substitutions and/or truncated derivatives were generated by the method of T. Kunkel, (Proe. Nati. Acad. Sei.; (U.S.A), 82:488-492 (1985 )). The ICAM-1 cDNA prepared as described above was digested with restriction endonucleases Sal 1 and Kpn 1, and the resulting 1.8 kb DNA fragment was subcloned into the plasmid vector CDM8 (B. Seed et al., Proe. Nat. Acad, Sei (U.S.A.), 84:3365-3369 (1987)). A dut"young" strain of E. coli (BW313/P3) was then transformed with this construct, designated pCD1.8C. A single-chain uracil-containing template was removed from the transformants by infection with the helper phage R408 (StratageneR). Mutant ICAM-1 cDNA molecules were then generated by priming second-strand synthesis with an oligonucleotide having mismatched bases, and subsequent transformation of a young <+->host (MC1061/P3) with the resulting heteroduplex. Mutants were isolated by screening for newly formed endonuclease restriction sites introduced by the mutant oligonucleotide. The mutant ICAM-1 protein was expressed by transfection of Cos-7 cells with the mutant DNA in the eukaryotic expression vector CDM8 using standard DEAE-dextran procedures (R.F. Seiden et al., In: Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.) pages 9.2.1-9.2.6 (1987)). ;A truncated functional derivative of ICAM-1 lacking the transmembrane and cytoplasmic domains, but containing the extracellular region possessing all 5 immunoglobulin-like domains, was prepared. A 30 bp mutant oligonucleotide (CTC TCC CCC CGG TTC TAG ATT GTC ATC ATC) was used to transform the codons for the amino acids tyrosine (Y) and glutamic acid (E) in positions 452 and 453, respectively, into a phenylalanine (F) and a . translational stop codon (TAG). The mutant was isolated at its unique Xba 1 restriction site and was designated Y <4S2>E/F, TAG. ;For expression of the mutant protein, COS cells were transfected with three mutant subclones (No. 2, No. 7 and No. 8). Three days after transfection with the three mutant subclones, culture supernatants and cell lysate were analyzed by immunoprecipitation with monoclonal anti-ICAM-1 antibody RR1/1 and SDS-PAGE. ICAM-1 was precipitated from the culture supernatants of cells transfected with mutant subclones #2 and #8, but not from detergent lysates of these cells. The molecular weight for ICAM-1 found in the culture supernatant was reduced by approx. 6 kd relative to the membrane form of ICAM-1, which is consistent with the size predicted for the mutant DNA. Thus, this functional derivative of ICAM-1 is secreted as a soluble protein. In contrast, ICAM-1 was not immunoprecipitated from control culture supernatants from cells transfected with native ICAM-1, demonstrating that the membrane form of ICAM-1 is not diffused from Cos cells. Furthermore, no ICAM-1 was immunoprecipitated from any culture supernatants or cell lysates from "mock" transfected negative control cells. The truncated ICAM-1 secreted from transfected cells was purified by immunoaffinity chromatography with a specific ICAM-1 antibody (R6-5-D6) and examined for functional activity in a cell binding assay. After purification in the presence of the detergent octylglucoside, preparations containing native ICAM-1, or the truncated, secreted form, were diluted to a final concentration of 0.25% octylglucoside {a concentration below the critical micelle concentration of the detergent). These preparations of ICAM-1 were allowed to bind to the surfaces of 96-well plastic plates (Nunc), forming ICAM-1 bound to a solid phase. After washing out unbound material, approx. 75-80% and 83-88% of SKW-3 cells that had LFA-1 on their surface bound specifically to the native and the truncated forms of ICAM-1, respectively. These data demonstrate that the secreted, truncated, soluble functional ICAM-1 derivative retained both the immunological reactivity and the ability to mediate ICAM-1-dependent adhesion characteristic of native ICAM-1. ;A functional derivative of ICAM-1 lacking only the cytoplasmic domain was prepared by similar methods. A 25 bp oligonucleotide (TC AGC ACG TAC CTC TAG AAC CGC CA) was used to change the codon for amino acid 476 (Y) to a translational TAG stop codon. The mutant was designated Y <476>/TAG. Immunoprecipitation analysis and SDS-PAGE of Cos cells transfected with the mutant revealed a membrane-bound form of ICAM-1 with a molecular weight of approx. 3 kd less than native ICAM-1. Indirect immunofluorescence of the mutant-transfected Cos cells showed a punctate staining pattern similar to native ICAM-1 expressed on LPS-stimulated human endothelial cells. Finally, cells transfected with the mutant DNA specifically bound to purified LFA-1 on plastic surfaces in a manner similar to Cos cells transfected with native ICAM-1 DNA (Table 17). ;EXAMPLE 29 ;Mapping of functional ICAM-1 fields Investigations of ICAM-1 have shown that the molecule has seven fields. Five of these fields are extracellular (field 5 is closest to the cell surface, field 1 is furthest from the cell surface), one field is a transmembrane field, and one field is cytoplasmic (ie inside the cell). For determining which fields contribute to ICAM-1's ability to bind LFA-1, epitope mapping studies can be used. To carry out such studies, various deletion mutants are prepared and these are characterized with respect to their capacity to bind to LFA-1. Alternatively, the assays can be performed using anti-ICAM antibody known to interfere with the capacity of ICAM-1 to bind LFA-1. Examples of such suitable antibodies include RR1/1 (R. Rothlein et al., J. Immunol., 137:1270-1274 (1986)), R6.5 (T.A. Springer et al., U.S. Patent Application Serial No. 07/250,446 ), LB-2 (E.A. Clark et al., Leukocyte Typing I, A. Bernard et al., eds.), Springer-Verlag pp. 339-346 (1984)), or CL203 D.E. Staunton et al., Cell, 56:849-853 (1989)). Deletion mutants of ICAM-1 can be generated in any of a number of different ways. However, it is preferred to produce such mutants via site-directed mutagenesis, or by other recombinant means (such as by constructing ICAM-1-expressing gene sequences where sequences coding for particular protein regions have been deleted). Methods which can be used for the production of such mutants are well known in the field. Using such methods, three ICAM-1 deletion mutants were produced. The first mutant lacks amino acid residues F185-P284 (ie deletion of field 3). The second mutant lacks the amino acid residues P284-R451 (ie deletion of fields 4 and 5). The third mutant lacks the amino acid residues after Y4 76 (ie deletion of cytoplasmic fields). The results of such investigations show that fields 1, 2 and 3 are mainly involved in interactions between ICAM-1 and anti-ICAM-1 antibody and LFA-1. ;EXAMPLE 30 ;Effect of mutations in ICAM-1 on LFA binding ;The ability of ICAM-1 to interact with, and bind to, LFA-1 is mediated by ICAM-1 amino acid residues present in field 1 of ICAM-1 -molecule (figures 8, 9 and 10). However, such interactions are supported by contributions from amino acids present in fields 2 and 3 of ICAM-1. Among the preferred functional derivatives according to the present invention are thus soluble fragments of the ICAM-1 molecule containing fields 1, 2 and 3 of ICAM-1. More preferred are soluble fragments of the ICAM-1 molecule containing domains 1 and 2 of ICAM-1. Most preferred are soluble fragments of the ICAM-1 molecule containing field 1 of ICAM-1. Several amino acid residues in the first ICAM-1 field are involved in the interaction between ICAM-1 and LFA-1. Substitutions of these amino acids with other amino acids change the ability of ICAM-1 to bind LFA-1. These amino acid residues and substitutions are shown in Figure 25. Figure 25 shows the effects of such mutations on the ability of the resulting mutant ICAM-1 molecule to bind to LFA-1. In Figures 23-25, residues are described with reference to the one-letter code for amino acids, followed by the position of the residue in the ICAM-1 molecule. Thus, e.g. "E90" the glutamic acid residue at position 90 in ICAM-1. Likewise, "E90V" denotes the dipeptide consisting of the glutamic acid residue at position 90 and the valine residue at position 91. The substitution sequence is indicated to the right of the slash ( <ii>/ <ii>), the V4, R13, Q27, Q58 and D60S61 residues in ICAM-1 are involved in LFA-1 binding. ;Substitution of these amino acids changed the capacity of ICAM-1 to bind to LFA-1. E.g. replacement of V4 with G results in the formation of a mutant ICAM-1 molecule that is less able to bind to LFA-1 (Figure 25). Replacement of the R13 residue in ICAM-1 with E leads to the formation of a mutant molecule with substantially less capacity to bind LFA-1 (Figure 25). Replacement of the Q58 residue in ICAM-1 with H leads to the formation of a mutant molecule with a substantially normal capacity to bind LFA-1 (Figure 25). Replacement of the D60S residues in ICAM-1 with KL leads to the formation of a mutant molecule with substantially less capacity to bind LFA-1 (Figure 25). ;Glycosylation sites in the second field are also included in LFA-1 binding (figure 23). Replacement of N103 with K, or A155N with SV, results in the formation of a mutant ICAM-1 molecule that is essentially unable to bind LFA-1. In contrast, replacement of the glycosylation site N175 with A did not appear to substantially alter the capacity of the mutant ICAM-1 to bind LFA-1. Mutations in the third ICAM-1 domain did not alter ICAM-1-LFA-1 binding to any significant extent {Figure 24). ;EXAMPLE 31 ;Multimeric forms of ICAM-1 with increased ;biological half-life, affinity and clearance Chimeric molecules are constructed where fields 1 and 2 are attached to the hinge region of the immunoglobulin heavy chain. Preferred constructs attach the C-terminal end of ICAM-1 domain 2 to a segment of the immunoglobulin heavy chain gene just N-terminal to the hinge region, allowing for the segment flexibility afforded by the hinge region. ICAM-1 fields 1 and 2 will thus replace the Fab fragment in an antibody. Attachment to heavy chains in the IgG class and formation of animal cells will result in the formation of a chimeric molecule. Formation of molecules containing heavy chains derived from IgA or IgM will result in formation of higher multimericity molecules containing from 2 to 12 ICAM-1 molecules. Co-expression of the J-chain gene in the animal cells that form the chimeric ICAM-1 heavy chain molecules will enable the correct assembly of IgA and IgM multimers, resulting mainly in IgA molecules containing 4-6 ICAM-1 molecules , and in the case of IgM, which contains approx. 10 ICAM-1 molecules. These chimeric molecules may have several advantages. First, Ig molecules are designed to last a long time in the circulation, and this can improve the biological half-life. Furthermore, the multimeric nature of these engineered molecules will enable them to interact with greater avidity with rhinovirus as well as with cell surface LFA-1, depending on the diagnostic context. If chimeric IgG-H chain molecules are desired, it would be possible to mutate regions involved in attachment to complement as well as in interactions with Fc receptors. ;EXAMPLE 32 ;Generation of ICAM-1 mutants ;Olignonucleotide-directed mutagenesis ;The coding region of an ICAM-1 cDNA in a 1.8 kb Sal1-Kpnl fragment, was subcloned into the expression vector CDMB (B. Seed et al., Proe. Nat. Acad. Sci. (U.S.A.), 84:3365-3369 (1987)). Based on the method of T. Kunkel, (Proe. Nati. Acad. Sei. ;(U.S.A.), 82:488-492 (1985)) and modifications of D. Staunton et al., (D.E. Staunton et al., Cell, 52:925-933 ; (1988)), this construct (pCD1.8) was used to generate a single-chain uracil-containing template for use in oligonucleotide-directed mutagenesis. Briefly, E. coli strain XS127 was transformed with pCD1.8. Single colonies were grown in 1 ml Luria broth (LB) medium (Difco) with 13 µg/ml ampicillin and 8 µg/ml tetracycline until near saturation point was reached. 100 l! of the culture was infected with R408 helper phage (Strategene) at a multiplicity of infection (MOI) of 10, and 10 ml of LB medium with ampicillin and tetracycline was added for a 16 hour culture at 37°C. After centrifugation at 10,000 rpm for 1 minute, and 0.22 µm filtration of the supernatant, the phage suspension was used to infect E. coli BW313/P3, which was then plated on LG agar (Difco) plates supplemented with ampicillin and tetracycline. Colonies were picked, grown in 1 ml of LB medium with ampicillin and tetracycline until near saturation was reached, and infected with helper phage at an MOI of 10. The culture volume was then increased to 250 ml, and the cells were grown overnight. Single-stranded DNA was isolated by standard phage extraction. Mutant oligonucleotides were phosphorylated and used with the pCD1.8 template in a second-strand synthesis reaction (D.E. Staunton et al., Cell, 52:925-933 (1988)). ;Transfection ;COS cells were seeded in 10 cm tissue culture plates so that they would be 50% confluent after 16-24 hours. The COS cells were then washed once with TBS and incubated for 4 hours with 4 ml RPMI containing 10% Nu-sera (Collaborative) 5 µg/ml chloroquine, 3 µg mutant plasmid and 200 µg/ml DEAE-dextran sulfate. The cells were then washed with 10% DMSO/PBS, followed by PBS, and cultured for 16 hours in culture medium. The culture medium was replaced with fresh medium, and 48 hours after transfection (OS cells were suspended by trypsin/EDTA (Gibco) treatment and split into 2.10 cm plates as well as 24-well tissue culture plates for HRV binding. After 72 hours, the cells were harvested from 10 cm plates with 5 mM EDTA/HBSS and processed for adhesion to LFA-1 coated plastic and immunofluorescence. ;LFA-1 and HRV binding ;LFA-1 was purified from SKW-3 lysates by immunoaffinity chromatograph on TS2/4 LFA-1 mAb-Sepharose and eluted at pH 11.5 in the presence of 2 mM MgCl 2 and 1% octyl glucoside LFA-1 (10 µg per 200 µl per 6 cm plate) were bound to bacteriological petri dishes by diluting octyl glucoside to 0.1% in PBS (phosphate buffered saline) with 2 mM MgCl2 and incubating overnight at 4° C. The plates were blocked with 1% BSA (bovine serum albumin) and stored in PBS containing 2 mM MgCl 2 , 0.2% BSA, 0.025% azide and 50 Mg/µl gentamycin. <51>Cr-labeled COS cells in PBS containing 5% FCS (fetal calf serum), 2 mM MgCl2 and 0.025% azide (buffer) were incubated with or without 5 µg/ml RRl/1 and R6.5 in LFA- l-coated microtiter plates at 25°C for 1 hour. Non-adherent cells were removed by 3 washes with buffer. Adherent cells were eluted by addition of EDTA to 10 mM and the γ counted. ;Results ;Anti-ICAM-1 antibodies such as RR1/1, R6.5, LB-2 and CL203 have been identified. If these antibodies can inhibit ICAM-1 function, they must be able to bind to a special place in the ICAM-1 molecule which is also important for ICAM-1 function. By producing the above-described deletion mutants of ICAM-1, and determining the extent to which the anti-ICAM-1 antibody can bind to the deletion site, it is thus possible to determine whether the deleted fields are important for the function. ;ICAM-1 is a membrane protein, where the extracellular field is expected to consist of 5 Ig-like C fields. For the identification of fields involved in the binding of LFA-1, field 3 and fields 4 and 5 (carboxyl-terminal) were deleted by oligonucleotide-directed mutagenesis and examined functionally after expression in COS cells. Moreover, the entire cytoplasmic domain was obliterated to ascertain its potential impact on ICAM-1 interactions. As expected, the deletion of the cytoplasmic field, Y476/ <*>, no loss of RRl/1, R6.5, LB-2 and CL203 reactivity, whereas deletion of field 3, F185-R451, resulted in a decrease and loss of CL203 reactivity (Figure 20) . Thus, the CL203 epitope appears to be found in field 4, while RR1/1, R6.5 and LB-2 appear to be found in the two amino-terminal fields.

All 3 deletion mutants show wild-type levels of attachment to LFA-1 (Figure 21). Amino acid substitutions in predicted S-turns in domains 1, 2 and 3 were also generated and functionally tested after expression in COS cells. The R6.5 epitope was thus localized to the sequence E111GGA in field 2, and may also include E39 in field 1, while RRl/1 and LB-2 both depend on R13 in field 1 (Figure 22). Moreover, RR1/1 binding is reduced by mutations in the sequence D71GQS. Mutations that delete N-linked glycosylation sites at N103 and N165 result in decreased RR1/1, R6.5 and LB-2, LFA-1-HRV binding. These mutations appear to cause processing to form ICAM-1 dimers.

Other mutations in fields 2 or 3 did not result in altered LFA-1 adhesion (Figures 23 and 24). The amino acids in field 1, Ri3 and D60, are both involved in the binding of LFA-1 (figure 25).

Thus, it appears that LFA-1 and HRV binding is a function of the amino-terminal Ig-like domain of ICAM-1.

Figure 26 shows an arrangement of amino-terminal ICAM fields.

Claims (2)

1. Diagnostically applicable water-soluble, functional derivative of ICAM-1 which is a fragment of ICAM-1, characterized in that it lacks the cytoplasmic domain and the transmembrane domain of ICAM-1, and it contains domain 1 or domains 1 and 2, or domains 1, 2 and 3. 2. Use of the derivative according to claim 1, for in vitro diagnosis of the presence and location of a tumor cell expressing a member of the LFA-1 molecular family in an individual who is suspected of having such a cell, by a) incubating a tissue sample from the individual in the presence of a mixture containing a detectable, labeled, water-soluble, functional derivative according to claim 1, and b) detection of said binding ligand which is bound to a member of the LFA-1 molecular family present in the tissue sample.