JPH05505391A - Endothelium-derived IL-8 - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] This invention is part of U.S. Application No. 07/232,224 (filed August 15, 1988). It is a continuation.

The present invention relates to endothelial-derived interleukin-8 (IL-8). In the past “white” These polypeptides were identified as ``blood cell adhesion inhibitory factor'' (LAI).

Tide is involved in a process that inhibits leukocyte populations from adhering to cell substrates.

The present invention is also useful as an anti-inflammatory drug, and for treating damage to vascular endothelium and other tissues. The present invention relates to the use of endothelium-derived IL-8 as a therapeutic agent for clinical indicators occurring.

Description of related technology Foreign invaders such as bacteria, viruses, and other invasive parasites, skin or mucous Once permeated through the membrane, cellular defense mechanisms are immediately triggered.

Two of the phagocytic-type cells of this defense system are local and blood-producing mononuclear cells and polycystic cells. Morphonuclear leukocytes (PMNs) accumulate around the invader and begin phagocytosis. This defense system An excellent review of “Microbio” by Eisen, H.W. ", 3rd edition, pp. 290-295 and 381-418 (Harpe r and Row, Ph1ladelphia, PA (1980)). It is stated.

The presence of such foreign substances causes (1) dilation of surrounding blood vessels, (2) increased vascular permeability. Large, (3) extravasated hemorrhage (migration of mononuclear cells and PMNs through the vessel wall) ), triggering an inflammatory response characterized by

Accumulation of blood leukocytes at the site of inflammation and injury is due to the increase in blood against its vascular lining. Depends on localized adhesion of leukocytes. Localized adhesions are involved in various pathophysiological processes It is an essential element. White blood cells are responsible for properly defending the host against these invaders. In order to bind to the cell matrix, it must be able to bind to the cell matrix. Also, white blood cells They must also bind to endothelial cells so that they can move from the circulation to sites of ongoing inflammation. Must be. In addition, leukocytes present antigens so that a specific immune response can occur. Must bind to cells. Finally, virus-infected cells or tumor cells are The leukocytes must bind to the appropriate collar cells so that lysis of the cells can occur. stomach.

Leukocyte adhesion to vascular endothelium in areas of inflammation and injury has long been known. . It has also long been assumed that molecular changes in the blood vessel wall are involved in these endothelium-leukocyte interactions. It has been thought that this may be the case. However, the experimental evidence supporting this concept is It wasn't even close (Harlan).

J., Blood 65:513-525 (1985)).

Recent studies have shown that certain inflammatory cytokines, such as interleukins, 1 (IL-1) (e.g. Bevilacqua et al., J, CI in, I nvest.

76:2003-2011 (1985); Bevilacqua et al., “Le ukocyte Emigration and 1ts 5equelae” , S, Karger, AC, Ba5el and New York, Pages 79-93 (1986): D ■ nn et al.,’The Physiologic, MeLabolic, and I mmunologic Actions of 1interleukin-1” , Alan R, Li5s, Inc., New York, 45-5 9 pages (1985); Sch imer et al., J. Immunol. +36:649-654 (1986) ; Gamble et al., PNAS USA 82:8667-8671 (198 5); Pohlman et al., J. Immunol, 136:454g- 4553 (1986); Cave■ See Der et al., Fed, Proc, 46:113-117 (1987). ), tumor necrosis factor (TNF) (Gamble et al., P!IAs US A 82:8667-8671 (1985)), and Gram-negative bacterial endotoxins (reactive Popolysaccharide XL P S Schleimer et al. (1986); Poh lman et al., J. Immunol, 136:4548-4553 (198 8)) acts directly on the vascular endothelium in vitro, causing blood leukocytes and related Increases adhesion of endothelial cell surfaces to leukocyte cell lines (HL-60 and U937) It was shown that it would be a big deal.

Purified complement components, formyl-methionyl-leucyl-phenylalanine and Other chemotactic factors, such as leukotriene B4, also induce PMNs to cultured endothelial monolayers. can increase binding (e.g. Sm1th et al., EXI), Ce11Res, 122:169-177 (1979): Hoover et al., J. Ce1 l Sci, 45 near 3-W6 (1 980) ; Hoover et al., PNAS USA 81:2191-219 3 (1984): Zimmerman et al., Thr.

mb, Res, 35:203-217 (1984); Gimbrone et al., J, Cl1n, 1nvest, 74:1552-1555 (1984) ; Tonnesen et al., J, C11n, Invest, 74:15 81-1592 (1984); Ch≠Chichi B et al., Blood 65:473-479 (1985); Harlan et al. Lab, Invest, 52:141-150 (1985)) . However, the relative significance of endothelial versus leukocyte responses and the cellular mechanisms involved in their adhesion is still largely unknown.

Inhibition of leukocyte adhesion is potentially of central importance for therapeutic intervention in inflammatory disease processes have sex. Although leukocyte adhesion is usually desirable, organ transplant rejection, tissue transplant body rejection, allergic reactions, autoimmune entrapment 1, rheumatoid arthritis, vasculitis, Blood clots, respiratory distress syndrome (ARDS), glomerulonephritis, and others Immune and non-immune inflammation, including tissue- or organ-specific acute and chronic inflammation It is also associated with sexual disease processes. Furthermore, in situations of ischemia-reperfusion, leukocyte adhesion Microvascular occlusion, tissue damage and death can result. For some patients, Therefore, some means of reducing or inhibiting cell adhesion is highly desirable. Probably.

definition For brevity, the following abbreviations are used herein: CM conditioned medium (conditioned medium (also called Fundi/Yondo medium). FC8/fetal bovine serum. H.E. C. Human venous endothelial cells. T■S Transferrin/insulin/selenium. hm IL-1: Human mononuclear cell-derived interleukin 1゜r　I　L-1: Recombinant I L-1°rTNF: Recombinant tumor necrosis factor. LPS: Gram-negative bacterial endotoxin. L.A. I: leukocyte adhesion inhibitory factor. P, MN polymorphonuclear leukocytes. GM-GSF: Granulocyte-mono Nuclear cell colony stimulating factor. fMLP: N-formyl-methionyl-loin roof Phenylalanine. LTB: Leukotriene B. PMA phorbol 12-mi Restate 13-acetate.

Disclosure statement US Application No. 07/232,224 relates to leukocyte adhesion inhibitor LAI. after that studies have shown that LAI consists of a mixture of polypeptides, one of which is A 72-amino acid neutrophil-activating polypeptide secreted from T cells and mononuclear cells It was shown to be interleukin-8 (IL-8). therefore In this application, LAI is referred to as "endothelial-derived +L-8."

The documents listed below are relevant to the present invention and constitute a part of this specification.

Hoover et al., J. Ce1l Biol, 95:10^ (1982) and B. a5ford et al., Blood 68 Near 9a (1986), in unstimulated humans and Conditioned medium from bovine endothelial cells stimulates superoxide production by neutrophils. Discloses a decrease in lending.

Harlan et al., Blood 66:167-178 (1985), CDw1 Monoclonal antibodies against 8 complex (leukocyte surface differentiation antigen complex) Disclose that St (agonist) confers significant inhibition of polymorphonuclear leukocyte adhesion induced by There is.

Zimmerman et al., J. 1mmuno1.134:1866-1874 (1985) showed that endothelial cell prostaglandin intermediate metabolites contain several types of metabolites, including adhesion. is disclosed to inhibit leukocyte function in patients.

Kerr et al., J. Ceil Biol. 103:1760 (1986) , when polymorphonuclear hemocytes are exposed to unstimulated endothelial cells or medium conditioned with these cells, , leukocyte adhesion to unstimulated endothelial cells and tissue culture plastic was inhibited. Discloses that.

Cronstein et al., J, CI in, Invest, 78 Near 6O -770 (1986) Adenone and its analogue 2-chloroadenone However, N-formylmethionyl-leucyl on cultured human) 19 vein endothelial cells - discloses that phenylalanine-stimulated neutrophil adhesion is inhibited; There is.

Wheeler et al., Fed, Proc. 45:450 (1986) and Poster presentation by Theeler et al. ``International Symposium'', August 1986, Netherlands) on interleukin-1. disclose that treated endothelial cells produce inhibitors of leukocyte-endothelial adhesion. .

Wheeler et al., Fed, Proc. 46:758 (1987). Some features of the natural form of skin-derived leukocyte adhesion inhibitor are disclosed.

Varani Laboratory Investigation 59:2 92 (1988) showed that pretreatment of rat endothelial cells with TNF led to activated human disclose that their susceptibility to lethality by neutrophils is increased.

Summary of the invention The present invention relates to endothelial cells, specifically human cells treated with IL-1, TNF or LPS. Concerning factors produced by venous endothelial cells. These factors are responsible for hyperadhesion (site block PMN and mononuclear cell adhesion to endothelial surfaces (stimulated with caine) but , a non-cyclooxygenase-derived inhibitor that does not block lymphocyte adhesion. child The production of these inhibitory factors is not limited to human sacrificial vein endothelial cells, but is also expressed by Sv4° virus. Cell types include transformed human venous endothelial cells and human diploid dermal fibroblasts. included. These factors, called endothelium-derived IL-8, contain leukocyte adhesion inhibitory molecules. as well as as a drug to protect endothelial cells from neutrophil-mediated damage. It has many uses. Compositions of these factors and methods of their use are provided.

Substantially pure endothelial-derived IL-8 encompassed by the present invention comprises at least two [ ^la I L-8]? ? and [Ser　I　L-8]7. A unique named Consists of polypeptides. This substance is used in endothelial cultures activated with cytokines. inhibits both mononuclear cell and neutrophil adhesion to However, highly refined Endothelium-derived IL-8 and recombinant human 14Ala I L-817-v and Se r　I　L-81vy does not inhibit adhesion. Naturally derived from endothelium + L-8 The preparation includes one or more other leukocyte adhesion inhibitors active against blood mononuclear cells. It probably contains some activity.

The action of this endothelial-derived IL-8 (LAI) is directed selectively against leukocytes. It seems to be possible. However, this is a formyl-methionyl-leucyl-phenylar Ranin, leukotriene B4 or phorbol 12-myristate 13-a Lends global inhibition of leukocyte responsiveness to soluble inflammatory irritants such as acetate. do not have. Rather, this newly discovered inhibitory molecule may be at the center of inflammation and/or injury. appears to play a role in modulating leukocyte-tube wall interactions at the site.

Specifically, human endothelial cultures stimulated with cytokines and bacterial endotoxins The cultured human endothelial cells are endothelial-derived, a soluble natural product of activated endothelial cells. It produces IL-8. Its production involves de novo protein synthesis (new protein synthesis), but does not appear to depend on the cyclooxy/genase pathway.

Endothelial-derived IL-8 activity is stable under extreme temperature and pH conditions as well as under physiological conditions. It is fixed. This heat and acid stability and at concentrations that eliminate prostacyclin production As there is no effect of aspirin treatment, endothelial-derived IL = 8 and several types including adhesion. endothelial cell prostaglandin intermediate that has been shown to inhibit leukocyte function in metabolites (Zimmerman et al., J. Immunol, 1 34:1866-1874 (1985)).

No detectable endothelial-derived IL-8 activity was observed in conditioned media from untreated endothelial cultures. I can't stand it. It is possible to add PM to unstimulated endothelial cells or to media conditioned with these cells. Exposure to N inhibits the attachment of PMNs to unstimulated endothelial monolayers and tissue culture plastic. This contrasts with reports of inhibition of adhesion (Kerr et al., J, Ce1l B). iol, 103:971a (1986). Obtained from unstimulated human and orchid endothelium. Conditioned medium also reduces superoxide production by stimulated neutrophils (Ba srord et al., Blood 68 Near 9a (1986); Harlan et al., B 66:167-178 (1985)). In contrast, endothelial-derived + L-8 is an agonist for cytosolic calcium concentration and superoxide production. Leukocyte adhesion without total inhibition of leukocyte function, including changes induced by stimulation It is said to be selectively blocked.

The present inventors demonstrated that endothelial-derived [L-8] was expressed in cytokine-activated human [intravenous] We have also demonstrated that the tight binding of human PMN to skin cells can be reversed. That is, When endothelial-derived IL-8 is added to PMNs attached to activated endothelial cells, PMNs dissociate from the endothelial cell surface. The kinetics of this “dissociation” is rapid (1-5 minutes) and is potentially reversible.

Discriminative effects of endothelium-derived IL-8 on leukocyte adhesion (i.e., significantly inhibiting PMNs) (or does not inhibit mononuclear cell or lymphocyte adhesion) on hyperadhesive endothelial cell surfaces. Suggests that the action of endothelium-derived IL-8 is related to distinct leukocyte receptors that recognize are doing. Similarly, promyelocytic cell line HL-60 and mononuclear cell-like cell line U937 The lack of effect on this undifferentiated cell line may be due to a lack of endothelial-derived IL-8 in this undifferentiated cell line. This suggests that the surface structure or function of mature leukocytes is targeted.

Adhesive interactions between PMNs and endothelium involve the leukocyte surface differentiation antigen complex CDwlB ( LFA-1, Mac-1, p150.95) has been proven to be involved. (Gamble et al., PNAS USA 82:8667-8671 (1985 ); Pohlman et al., J. Immunol, 136:454g-455 3 (1986)). In the presence of concentrated endothelium-derived IL-8 (up to 5x relative concentration) and activated by fMLP, LTB, or PMA (see “Definitions” below) Adhesion of treated PMNs to control endothelial monolayers is not inhibited. In contrast, CDw1 Monoclonal antibodies against the 8-complex were used to detect agonist-induced PMN adhesion. (Harlan et al., Blood 66:167-178 (198 5)). Furthermore, PMN adhesion to serum-coated tissue culture plastic is endothelial-mediated. The endothelium-derived IL-8 of PMN adhesion is not altered by the presence of IL-8. The inhibition appears to be specific to "hyperadhesive" endothelial cell surfaces.

Endothelial-derived IL-8 protects against leukocyte-mediated damage to endothelial cells You can also do it. This protection may occur due to the leukocyte adhesion inhibitory activity of endothelium-derived IL-8. Ru. However, other mechanisms than adhesion-inhibiting activity may also be involved. Therefore, the present invention The method is not limited by any of the mechanisms disclosed.

Prior to the present invention, TL-8 was referred to as a pro-inflammatory chemoattractant.

Leukocyte adhesion inhibitory activity has never been considered to be attributable to IL-8. However, Thus, the present invention provides anti-inflammatory drug and/or leukocyte-mediated effects on vascular endothelium or other tissue. IL-8 and vascular endothelium as therapeutic agents for clinical indicators of tissue damage It includes the use of related peptides obtained from sources other than cells. in vivo The effective amount of TL-8 to be administered is determined by the in vitro assay described in more detail below. can be determined by in vitro) and in vivo bioassays.

The present invention relates to endothelial-derived IL-8 and functional derivatives thereof. Or the present invention Production and purification method of skin-derived IL-8, screening for endothelial-derived IL-8 Assays, Diagnostic and Therapeutic Uses of Endothelial-Derived IL-8, Endothelial-Derived IL-8 or Also related are compositions containing functional derivatives thereof.

Specifically, the present invention provides a leukocyte adhesion inhibitory factor that is substantially free of natural impurities. Includes endothelial-derived IL-8 or a functional derivative thereof. Furthermore, the present invention is capable of detecting The present invention relates to endothelium-derived IL-8 and derivatives thereof that are labeled such that they can be used.

The present invention also relates to a method for recovering endothelial-derived IL-8 in substantially pure form; The method involves, but is not limited to, the following operations: (a) harvesting conditioned medium from a cell source; (b) by ultrafiltration of M conditioned medium; An endothelium-derived IL-8-containing fraction is obtained.

(C) Endothelial-derived IL-8 is concentrated and desalted by ultrafiltration: (d) Endothelial-derived IL-8 is concentrated and desalted by ultrafiltration; Next, IL-8 is lyophilized and membrane-lipidized by acetone precipitation.

(e) Gel filtration of endothelial-derived TL-8 by HPLC chromatography.

(f) Endothelium-derived I L-8 is subjected to ion exchange chromatography: (g) Procedure The filtrate obtained in step (f) is recovered in substantially pure form.

The present invention includes endothelial-derived IL-8 obtained by the above method.

The present invention also provides a method for treating inflammation in a mammalian subject, and provides a method for treating inflammation in a mammalian subject. of the following group: endothelium-derived IL-8 in an amount sufficient to suppress the inflammation in the subject in need thereof. , fragments of endothelial-derived IL-8, chemical derivatives of endothelial-derived IL-8, endothelial-derived fL-8 8 variants, and analogs of endothelial-derived IL-8; Relating to a treatment method consisting of:

The present invention also provides compositions for protecting against leukocyte-mediated damage to endothelial cells. It also includes products and methods.

The present invention also provides a method for treating inflammation, wherein the anti-inflammatory agent is administered in a suitable carrier. It also includes methods of The present invention also provides the following groups: endothelial-derived IL-8, endothelial-derived I Fragments of L-8, chemical derivatives of endothelial IL-8, variants of endothelial IL-8, and and an analog of endothelium-derived IL-8. It also relates to a pharmaceutical composition for the treatment of inflammation in a mammalian subject comprising:

The present invention further relates to the presence of inflammation in a mammalian subject suspected of having inflammation and A method for diagnosing location, the method comprising: (a) binding to and identifying endothelial-derived IL-8; A composition containing a detectably labeled binding molecule (e.g., an antibody) is (b) detecting the binding molecule.

The present invention also relates to the presence and location of inflammation in mammalian subjects suspected of having inflammation. A method for diagnosing endothelial-derived IL-8, the method comprising: Biological samples obtained from elephants are subjected to a test that can bind to and identify endothelial-derived L-8. incubating in the presence of a binding molecule (e.g., antibody) that is releasably labeled; (b) detecting said binding molecule bound to said sample. the raw Physical samples include, for example, biopsy specimens, body fluids such as blood (serum or plasma), joint/ Pleural/peritoneal exudate, cerebral cerebrospinal fluid, or ocular fluid may be included.

Therefore, the present invention provides for the recovery of novel endothelial-derived IL-8 molecules and functional derivatives thereof. and purification methods, methods for diagnosing and treating inflammation in mammalian subjects, and endothelial-derived IL- 8 or a functional derivative thereof.

Understanding this process of leukocyte adhesion inhibition and the endothelium-derived TL-8 molecule itself will Its therapeutic use in the fields of rheumatology, organ transplantation, allergy and oncology. and/or facilitate the development of diagnostic uses. Furthermore, endothelial-derived IL- 8 for Adult Respiratory Disorder Syndrome, Septic Shock, Vasculitis, Cardiac and Vital Support. Ischemia-reperfusion injury of essential pond organs and leukemia to the vascular endothelium or pond tissue It may be useful in treating other inflammatory disease processes in which neutrophil-dependent damage occurs.

The present invention enables more effective diagnosis and treatment of inflammatory disease processes in the medical field. monitoring the time course and/or intensity of ongoing clinical episodes of inflammation It would be possible to use quantitative assays of endothelium-derived IL-8 to . Furthermore, the present invention focuses on the function of leukocytes in inflammation and leukocyte-endothelial cell interactions. This will enable the study of the mechanism of

■ Time indicated for indium-labeled PMN with hmIL-1 (5 U/ml) Added to pretreated HEC monolayer and measured the number of attached PMNs by standard 10 min monolayer adhesion. Determined by assay method. Black circles (“wash”) indicate that the conditioned medium was removed immediately before addition of PMN. and shows the wells where the monolayer was washed. Open circles (“unwashed”) indicate PMNs in conditioned medium. Direct addition wells are shown. in the presence (△) or absence (mu) of conditioned medium. Figure 3 shows PMN adhesion to untreated HEC at 1.4 and 8 hours.

Figure 2. Effect of various conditioned media on PMN adhesion to stimulated and unstimulated endothelial cells represents. Conditioned media were collected as follows: (1) with rlL-1β or rl Pretreated for 4 hours without L-1β, washed, and washed for 5 hours in RPMI+FC3. Collect lL-1 and pseudo-CM from HEC monolayers incubated for (CM), or (2) RP in a petri dish covered with ceratin without endothelial cells. MI+FC3 with rIL-1β (5u/ml) or rlL-1 Incubated for 9 hours without β (cell-free CM). For standard monolayer adhesion assays Next, Ill ln-labeled PMNs were transferred to non-incubated RPM [+FC3 ( fresh medium), mock, and IL-I CM cell-free CM or endothelial pseudo-CM and Diluted in IL-I CM (2x 10' cells/1) (see Example 5). and). Furthermore, immediately before the assay, rIL-β (5 U/ml) was added to a portion of the endothelial pseudo-CM. was added directly. Washed non-activated (black) or activated (hatched) (51j/ml r I L-β, 4 hours) PMN adhesion to HEC monolayer was evaluated. The value, Expressed as the mean ± 4 standard deviations of this representative experiment performed in triplicate. Same for 2 make-up exams I got similar results. (*p<o,ot IL-CM compared to pseudo-CM).

i13: Represents the IL-1 stimulation concentration dependence of LAI on human vascular endothelial production.

Fully grown HEC monolayers were incubated with various concentrations of hmI L-1 or rlL-1β for 4 hours. Pretreated, washed and incubated in RPMI FC3 for an additional 5 hours.

IL-I CM and pseudo-CM were collected and tested for LAI activity using a standard monolayer adhesion assay. Established. The percent inhibition of PMN adhesion was calculated as described in Example 7.

Each point represents the mean ± SEM of three separate experiments.

C104: IL-ICM concentration dependence of LAI-induced inhibition of PMN adhesion represent. IL-I CM was collected in serum-free TIS supplemented medium and Amicon (Ami con) Y　M　Filt through a 30 ultrafiltration membrane, and filter through an Amicon YM5 membrane with a 20 ( Concentrated to @concentration (volume/volume). Next, this concentrate was added to LAI endothelial-derived IL-8. Serially re-diluted in RPMI+FC3 for activity assay. Each point is measured 5 times separately. Represents the experimental mean ± SEM.

15. Attachment of different types of leukocytes to untreated and L-1 treated endothelial cell monolayers The effect of LAI on wear is shown. Isolated radiolabeled PMN, mononuclear cells, lymph spheres, or I(, L-60 cells) in pseudo CM (hatched) or IL-I CM (black). ), untreated (-) or IL-1 treated (+02.5 U/ml). T L-1β, 4 hours) Adhesion to HEC was evaluated using a 10 minute assay. The value , Table as mean ± SEM *P<0.01 sham CM vs. IL-I CM).

Figure 6: Hoffman derivative of PMN adhesion to HEC monolayer treated with I L-1 Represents interference cum microscopy. Isolated PMNs were subjected to pseudo-CM (A) or ILI-CM HEC (2,50/+nlr) resuspended in (B) and pretreated with IL-1 IL-1β, 4 hours) for 10 minutes and the monomer to remove non-adherent PMNs. The layer was washed. Photo B shows that the number of attached PMNs has decreased, and the surface of the HEC monolayer has decreased. Note that it does not appear to be well diffused over the surface. (Original magnification 400x) .

Figure 7. Represents agonist-induced transient Ca” rise and simulated membrane polarization in PMNs .

(A) P to 1.9 ml of HBS3 containing Ca” and Mg” (2IIIM) MN (2X10”/ml) was resuspended and disassembled at 37°C in a 5PEX fluorometer. C,-(5) (final concentration 1.5 μM). 100μl 20x concentration Pseudo-CM (dotted line) or IL-, ICM (solid line) was added. After 5 minutes, 10-' M fMLP (upper row), 10-'M LTB, (middle row), or 1100n/m l PMA (bottom row) was added and the increase in fluorescence was recorded. F = 10% full scale.

(B) Fura-2 loaded PMN (1x 10@/ml) with Ca” and Mg” The suspension was suspended in 1.9 ml of HBS3 containing 100 μl of 20x concentrated jL-ICM ( (upper row) or pseudo CM (lower row) were added. After 5 minutes, 10-'MfMLP was added, Changes in fluorescence emission (excitation 340 and 380) were recorded at 505 nm. cytoplasm Gry free calcium concentration (y-axis) was calculated from the ratio of emitted fluorescent signals (Gry Nkiewicz et al., J. Biol. Chell, 260:3440- 3450 (1985)).

Shows the fractionation of 1L-I CM by high-pressure liquid chromatography. IL- ICM was collected in serum-free TIS supplemented medium and filtered through an Amicon YM30 membrane. It was filtered, concentrated on a YM5 membrane, washed to remove salts, and lyophilized. part of it resuspended in 0.2M acetic acid, o 1M triethylamine (pH 3,9), and Applied to a Waters Proteinvite 125 gel filtration column. Flow rate 1.8 Fractions were collected at ml/min, dialyzed against PBS, and used for LAI activity assay (mesh bars). For this purpose, it was diluted 1:10 with RP MI + FCS. Undiluted in PBS The protein content in the image was determined (black circle). Similar results were obtained in three additional tests. .

[19: Represents LAI fractionation. Collection medium determined by fractionation (hourly) The appearance of LAI endothelium-derived IL-8 in the cells was time-dependent. HEC culture for 50 /ml Interloyl Beef-1 at time 0. Collect the medium at 1 hour and add fresh Replaced with fresh medium. Cytokines were added to the replacement medium until time 5 hours. this As shown in the figure, endothelial-derived IL-8 became evident in the culture medium in about 1 hour and Maximum production was reached in 5 hours and maintained for up to 9 hours. After 9 hours, observe a decrease in activity. It was done.

Cytokine activity from neutrophil-mediated damage by endothelial-derived LAI protection of the endothelial monolayer. After incubation with neutrophils (50·1) for 90 min. Micrograph of HEC monolayer. (a) Non-activated HEC monolayer, (b) cytokine activated HEC monolayer: Note significant monolayer shrinkage and cell loss, (C) Endothelium-derived LAI present Cytokine-activated HEC monolayer in the absence of damage evident in (b) Pay attention.

Description of preferred embodiments One aspect of the present invention is the production of a natural intercellular adhesion inhibitory factor called endothelial-derived IL-8. Regarding seeing. The present invention relates to substantially pure endothelial-derived IL-8. virtually pure Endothelial IL-8 includes a mixture of IL-8 molecules. This mixture Contains [AIaIL-8] and 7 as peptides, and [SerI] as a minor component. L-8], t. Generally [Ala IL-8E? ? is about this mixture It will constitute 70% to about 95%. [Serr L-8] tt to [A la I L-8]? ? Biochemical methods to separate rAla I L-s tt itself is co-pending U.S. Application No. 443131 (filed November 29, 1989). ) and are charged.

The term "functional derivative" refers to "fragments," "variants," and "analogs" of the subject polypeptide. or "chemical derivatives". Endothelium-derived TL-8 polypeptide "Fragment" of refers to a subset of a polypeptide.

A “variant” of an IL-8 polypeptide is one that is essentially similar to the entire molecule or a fragment thereof. Refers to naturally occurring molecules that are similar. Endothelium-derived ■What are “analogs” of L-8? , refers to a non-natural molecule that is essentially similar to the whole molecule or a fragment thereof. both When the amino acid sequences in the molecules are substantially the same, and when both molecules are similar organisms A molecule is said to be “essentially similar” to another when it has chemical activity. ”. Therefore, if two molecules have similar activities, even if their Even if one of the molecules contains additional amino acid residues that the other does not have, or Even if the amino acid sequences of the baits are not the same, they still have the same meaning as used in this specification. considered to be a variant.

As used herein, a molecule includes additional chemistry that is not normally part of the molecule. A molecule is considered to be a “chemical derivative” of another molecule when it contains a specific moiety. say. Such moieties improve the solubility, absorption, biological half-life, etc. of that molecule. It is possible. Alternatively, these moieties may reduce the toxicity of the molecule or It is also possible to eliminate or alleviate undesirable side effects. This kind of effect Examples of moieties that can mediate effects include “Rosington’s Pharmaceut ical 5 sciences'' (1980) and is obvious to those skilled in the art. Will.

Suitability for determining whether a compound is an endothelial-derived IL-8 functional derivative A useful screening method is, for example, biological Assay, specific neutralizing antibody (monoclonal or immunoassay using RIA or ELISA methods. Consists of fixed laws.

The endothelial-derived IL-8 disclosed herein is such that preparations containing it are If it does not contain substantially any substances normally associated with nature, it means that it does not contain substantially any natural impurities. However, as mentioned above, substantially pure endothelial-derived IL-8 is a　TL　-8], 7 and [Ser　I　L-8]7. From polypeptide It is a mixture of This mixture can be separated from natural contaminants by the method of the present invention. however, the two components are separated by this method or other methods known in the art. It is not possible.

Preferred purification methods for the purposes of this invention include immunoaffinity chromatography. Rabbit heterosera and mouse monoclonal for use in fie purification Preparation of antibodies, ion exchange chromatography using cation exchange columns such as Mono S Raffy, and preparative 5DS-PAGE followed by electroelution. illustration The procedures and materials involved in the preferred purification method are described below, including but not limited to: Not determined a) Endothelium-derived IL-8 was purified by ITs (Collaborative Research ('C).

11aborative Re5search, Bedford, MA supplement Added RPMT-1640 (M.A. ducts))).

b) Ultrafilter the medium in an Amicon stirred ultrafiltration vessel using membrane Y~1-30.

C) Concentrate the medium obtained in step (b) with YM-5 and perform Mi1)-Q purification to desalt it. Wash with water (Millipore), freeze and lyophilize. frozen Contaminants are removed by washing the dried pellets with 5 volumes of water-cooled acetone. Remove lipids.

d) The lyophilized material was dissolved in 0.2M acetic acid (Fischer Scientific). sher 5 scientific)) and 0.1M triethylamine (sig In Sigma Chemical Co. Reconstitute and apply to Protein-Vac 125 gel filtration column (Waters) .

e) The fraction obtained in (d) was transferred to 1011 IM T r i 5 (pH 7) (biolat (dialyzed into BioRad) and equilibrated in 10mM T r i 5 (pH 7). CM-Sephadex or SP-Sephadex (Pharmacia (Ph armacia)) and 20 minutes at 25°C. Both power chion exchange The endothelial-derived IL-8 activity is removed by a catalytic resin.

Buffer systems based on citric acid can also be used. Elution of activity was performed using a linear salt gradient. Performed by distribution (0.01M to IM NaCI). The fraction obtained by this procedure was were tested for IL-8 activity and subjected to SDS polyacrylamide gel electrophoresis. I can do it.

Purified endothelial-derived IL-8 fragments or organic synthesis or recombinant DNA techniques Derivatives thereof prepared by proteolytic hydrolysis are also encompassed by the present invention. Included. 'The present invention provides the ability to detect endothelial-derived IL-8 or functional inducers in a sample or subject. It also includes methods for detecting conductors. For example, endothelial-derived IL-8 or a functional derivative thereof Combine specific antibodies with appropriate ligands, e.g. radioisotopes, enzymes, fluorescent It can be detectably labeled with a label, a paramagnetic label or a free radical. Detectable By using such functionally labeled substances, the presence of inflammation can be detected. Ru. Methods for making such detectably labeled antibodies or functional derivatives thereof and detection methods are well known to those skilled in the art.

Detection of the collection location of such detectably labeled antibodies indicates the site of inflammation.

- In embodiments, a sample of tissue or blood is removed and such sample is detectable. This assay for inflammation by incubating in the presence of labeled antibodies carry out the experiment. In a preferred embodiment, for use in magnetic imaging, fluorofluorography, etc. Therefore, this technique is performed in a non-intrusive manner. For example, early stage of potential tissue rejection The use of such diagnostic tests in monitoring organ recipients for symptoms I can do it. In rheumatoid arthritis and other chronic inflammatory diseases, Such tests can also be performed to determine floor condition.

A biological sample of the subject suspected of containing endothelial-derived IL-8 was In the presence of a detectably labeled binding molecule (e.g. an antibody) that can identify L-8. an endothelial-derived method comprising incubating and detecting the binding molecules bound to the sample. By using assays for IL-8, antibodies or functional derivatives thereof detecting the presence and location of inflammation in a mammalian subject suspected of having inflammation or Can it be used to diagnose?

Therefore, in this aspect of the invention, the biological sample is treated with nitrocellulose or with cells. , even treated with cell particles or other solid supports capable of immobilizing soluble proteins. good. The support is then washed with an appropriate buffer before detectably labeled endothelial Next, treat with I L-8 specific antibody. This solid phase support is then washed again with buffer. Unbound antibody is removed by washing. Next, the amount of label on the bound antibody is can be detected by conventional means.

When carrying out the assay method of the present invention on a sample containing endothelial-derived IL-8, the The process consists of the following operations: (a) collecting a sample suspected of containing endothelial-derived IL-8; Immobilization of endothelium-derived IL-8 is achieved by contacting with a solid support: ( b) contacting the solid support with a detectably labeled endothelium-derived IL-8 specific antibody; Ru: (c) the detectably labeled endothelial-derived IL-8-specific antibody together with the support; Endothelium-derived IL-8-specific antibodies can sufficiently bind to immobilized endothelium-derived IL-8 Incubate for an hour.

(d) Separating the solid support from the incubation mixture obtained in step (C).

(e) Detecting the bound label and thereby detecting and quantifying endothelial-derived IL-8.

This aspect of the invention provides a method for obtaining endothelial-derived IL-8 or or a fragment thereof: (a) containing endothelium-derived IL-8; The suspected sample is tested for endothelial-derived IL-8-specific antibodies or antibodies that bind to endothelial-derived IL-8. contact with that fragment.

(b) Detecting whether a complex is formed.

Of course, specific concentrations of detectably labeled antibodies and endothelial-derived rL-8, The temperature and time of cuvagecon, as well as other assay conditions, are will vary depending on a variety of factors, including the concentration of the IL-8 source, the nature of the sample, etc. Will. Those skilled in the art can determine optimal functional assay conditions through routine experimentation. could be determined for each measurement.

Other operations such as washing, agitation, shaking, and filtration may not be performed routinely or in specific situations. may be added to this assay if necessary.

One way in which endothelial-derived IL-8-specific antibodies can be detectably labeled is by enzymatically labeling them. It is to combine with. When subsequently exposed to the enzyme's substrate, the enzyme e.g. Chemical substances that can be detected spectrophotometrically, fluorometrically, or visually will react with its substrate in such a way that a moiety is produced. Endothelium-derived IL-8 Enzymes that can be used to detectably label specific antibodies include malate dehyde logonase, staphyloconocal nuclease, δ-■- steroid isome enzyme, yeast alcohol dehydrogenase, α-glucerophosphate dehydrogenase Drogenase, Triosephosphate Imerase, Horseradish Peroxy Dase, alkaline phosphatase, albarginase, glucose oxidase -ase, β-galactonase, ribonuclease, urease, catalase, glycanase Lucose-vl-phosphate dehydrogenase, glucoamylase and These include, but are not limited to, cetylcholine esterase.

The endothelial-derived IL-8-specific antibody was measured using a gamma counter or a anti-schillation counter. radiation that can be measured using means or by audioradiography It can also be labeled with active isotopes. Particularly useful isotopes for the purposes of this invention are , 3H, "'I", 1'' 1%"S%14C1 and "Cr.

Endothelial-derived IL-8-specific antibodies can also be labeled with fluorescent compounds. fluorescently labeled When the identified antibody is exposed to light of the appropriate wavelength, its presence is detected due to the fluorescence of the dye. can. Fluorescein isothiocyanate, rhodamine, phycoevolaserine , phycocyanin, allophyphucyanin, O-phthalaldehyde and fluorescein Samin (flu.

roscamine) are some of the most commonly used fluorescent labeling compounds. be.

Endothelial-derived IL-8-specific antibodies can be expressed using antibodies such as lS'Eu or other lanthanides. It can also be detectably labeled using a fluorescent emitting metal.

Diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid Using metal chelating groups such as acid (EDTA), these metals can be isolated from endothelium-derived IL- 8 specific antibodies.

This can be achieved by conjugating endothelial-derived IL-8-specific antibodies with chemiluminescent compounds. Antibodies can also be detectably labeled. then occurs during the progress of a chemical reaction Chemiluminescently labeled endothelium-derived I by detecting the presence of luminescence Determine the presence of L-8 specific antibodies. Examples of particularly useful chemiluminescent labeling compounds include Minol, isoluminol, theromat ic acrizi with nium esters, imidazoles, acridinium salts and oxalate esters. be.

Similarly, a bioluminescent compound is used to label the endothelial-derived IL-8-specific antibody of the present invention. It's okay. Bioluminescence is a type of chemiluminescence found in biological systems; Catalytic proteins increase the efficiency of chemiluminescent reactions. Detects the presence of luminescence The presence of bioluminescent proteins is determined by emitting bioluminescent proteins. Heavy for labeling Key bioluminescent compounds include lumferin, le/ferase and aequorin (a equorin).・ For example, if the detectable label is a radioactive gamma emitter, the or, for example, if the label is fluorescent, by a fluorometer. Detection of endothelial-derived IL-8-specific antibodies can be accomplished using this method. In the case of enzyme labeling, the It can be detected by colorimetric methods using enzyme substrates. Similarly, the degree of enzymatic reaction of the substrate is determined. It can also be detected by visual comparison with a manufactured standard.

Endothelium-derived IL-8 is a soluble product of activated endothelial cells and therefore this Factors involved in the inflammatory process. The term “inflammation” refers to both specific and non-specific defense systems. This includes both reactions. A specific defense system response is a specific immunity against an antigen. It is a system reaction response. Examples of specific defense system reactions include reactions to antigens such as rubella virus. antibody responses and T-cell-mediated delayed-type hypersensitivity responses (e.g., mantle-mediated hypersensitivity responses). (Mantaux) (found in “positive” individuals in the test).

Nonspecific defense system responses are inflammatory responses mediated by leukocytes without immune memory capacity.

Such cells include granulocytes, macrophages, neutrophils, and the like. non-singular Examples of protective system reactions include the rapid swelling of a bee sting, the swelling induced at the site of a burn. Reddening and cellular infiltration, and collection of PMN leukocytes at the site of cell infection (e.g., cell infiltration) This includes pulmonary infiltration in bacterial pneumonia and pus formation in abscesses.

Rheumatoid arthritis, acute and chronic inflammation, post-ischemic (reperfusion) leukocyte-mediated tissue injury, acute leukocyte-mediated lung injury (e.g., Adult Respiratory Disorder Syndrome), and pond tissue characteristics. Diagnosis of inflammatory disease processes such as atypical or organ-specific acute inflammation (e.g., glomerulonephritis) It will be appreciated that the present invention is easily adapted for diagnosis, monitoring and therapy.

As will be clear to those skilled in the art, the therapeutic effects of endothelium-derived IL=8 derived IL-8 molecules or therapeutically active peptide fragments thereof. It can be obtained by

Also, added to the carrier to increase the activity of endothelium-derived IL-8. Endothelial-derived IL with additional amino acid residues added to promote binding of Exploiting the therapeutic benefits of endothelial-derived IL-8 by using -8 mutants or variants. It is also clear that it can be increased. The scope of the present invention is directed to mutant forms of endothelial-derived IL-8. (The key to the ability of the mutant endothelium-derived IL18 molecule to affect cell adhesion or some amino acid residues have been deleted or some amino acid residues have been changed. It is also considered to include endothelium-derived IL-8 molecules (including endothelial-derived IL-8 molecules).

According to known methods for producing pharmaceutically useful compositions, the endothelial-derived IL- 8. Mixing a polypeptide or a functional derivative thereof with a pharmaceutically acceptable carrier excipient. Formulate these substances and their functional derivatives by mixing them into compounds can do. Suitable excipients and other human proteins (e.g. human serum protein) For example, “Remington's Pharma Ceutical 5 Sciences'' (16th edition, edited by 0sol, A, Mack, Easton, PA (1980)). effective such compositions to form pharmaceutically acceptable compositions suitable for administration. A therapeutically effective amount of endothelial-derived IL-8 or a functional derivative reduces inflammation. amount, protective amount or amount that inhibits leukocyte adhesion) with an appropriate amount of carrier excipient. It will contain.

Preferred products of the invention are endothelial-derived IL-8 or its A sterile pharmaceutical composition for therapeutic use containing a functional derivative. this product for reconstitution and use by adding a suitable carrier or diluent. It may be in the form of a freeze-dried product or an aqueous solution.

Sterile diluents can be used to reconstitute the lyophilized product of the invention. , this diluent contains commonly known substances and and/or may contain substances required by government regulations. In this connection, sterile diluent buffering agents (e.g. sodium chloride) to provide physiologically acceptable p)l. saline, phosphate-buffered saline) and/or physiologically acceptable (or and/or) other materials that are safe for use may be included. Generally intravenous in humans Substances for injection are available to those in this field.

Regulations established by ``d and Drug Administration'' The rules should be followed.

The pharmaceutical compositions of the invention contain many of the substances mentioned above for reconstitution of lyophilized products. It may also be in the form of an aqueous solution containing it.

As mentioned above, the products of the invention are incorporated into pharmaceutical preparations used for therapeutic purposes. be able to. However, the term "pharmaceutical preparation" as used herein in its broadest sense refers to For therapeutic purposes as well as for reagents or diagnostic agents known in the art, or Preparations containing the protein composition of the invention are also used for tissue culture. shall be included. Pharmaceutical preparations intended for therapeutic use are defined as “pharmaceutically acceptable”. or “therapeutically effective amount” (i.e., necessary for preventive or therapeutic health standards). amount) of endothelial-derived IL-8. Use of pharmaceutical preparations as reagents or diagnostics When used as a drug withdrawal drug, it should contain reagent or diagnostic amounts of endothelium-derived IL-8. It is possible.

By definition, one unit of endothelial-derived IL-8 activity is Achieve 50% inhibition of PMN adhesion with PMNIIIi at 2xLO/cells/sl. . A 10-fold higher concentration of endothelial-derived IL-8 (10 U/ml) produced a maximal inhibition of 85-90%. lend harm.

Therapeutic and/or diagnostic doses administered in vivo are determined by the specific inflammatory condition involved. depending on the mammal's age, weight, height, sex, general medical condition, and and the type of concurrent treatment, if any. Generally, about LOU/ The recipient may be given an amount that provides an effective concentration of endothelial IL-8 activity greater than ml. Although desirable, higher or lower amounts can be administered. Endothelium-derived I L-8 The specific activity of certain preparations of or derivatives thereof was determined in vitro as described in the Examples section below. It can be determined by bioassay method. In this way, any endothelial-derived IL-8 or Dosages can be determined for body preparations.

Useful methods for administering the molecules of the invention include topically, subcutaneously, intravenously, intraarterially. , intraarticular, intraperitoneal, intrapleural or intraocular administration. Endothelium-derived IL-8 also If its functional derivative is administered by injection, continuous infusion or single It can be administered via multiple pores.

Effective molecules useful in the methods of the invention can be, for example, in the form of a sterile injectable suspension; to specific tissue sites with antibodies against inflammation-related cell surface structures such as ELAM-1. It can be used in the form of a mounting medium for inducing cqua et al., PNAS USA 84:9238-9242 (19g7); otran et al., J. Exp. Med, 164:661-666 (198 6)).

Additional pharmaceutical methods can also be used to control the duration of action. controlled The released preparations may complex or absorb endothelial-derived IL-8 or its functional derivatives. This can be achieved by using polymers for Control of delivery is appropriate Polymers (e.g. polyester, polyamino acids, polyvinyl, pyrrolidone, ethylene Vinyl acetate, methylcellulose, carboxymethylcellulose, or plastic Selecting appropriate concentrations of rotamine sulfate) and polymers and controlling release This can be achieved by selecting an uptake method to achieve this goal.

Another possibility is to control the duration of action with controlled release preparations. Useful methods include polyesters, polyamino acids, hydrogels, poly(lactic acid), or Endothelial-derived substances are found in particles of polymeric substances such as ethylene vinyl acetate and copolymers. The next step is to incorporate IL-8 or a functional derivative thereof.

Alternatively, endothelial-derived IL-8 or a functional derivative thereof can be incorporated into polymer particles. Instead, these substances can be processed, for example, by coacervation techniques or interfacial polymerization. Microcapsules prepared according to the method (e.g., hydroxymethyl cellulose or gelatin-microcapsules and poly(methyl methacrylate) microphones colloidal drug delivery systems (e.g., liposomes, albumin microspheres, macrocapsules); microemulsions, nanoparticles, and nanocapsules) or macroemulsions. It can also be encapsulated in a solution. Such technology is Remington's Disclosed in ``Pharmaceutical 5 Sciences'' (1980) ing.

Example Having now fully described the invention, it is not intended to be limiting unless otherwise indicated; By reference to some specific examples set forth herein for purposes of illustration: , the present invention can be understood more clearly.

Inflammatory substances and biochemical reagents The following reagents were obtained from the sources listed. IL-1 derived from human mononuclear cells (hm lL-1), human recombinant IL-1-β (rTLL-Lβ) and human recombinant Granulocyte-Mononuclear Cell Colony Stimulating Factor (GM-C3F): Genzyme Inc. Horated (Genzyme, Inc., Boston, MA). Big Human recombinant IL-1-α (rIL-1α) expressed in S. enterica Cistron Technology, Pine Brook , NJ). Gram-negative bacterial endotoxin (B4 in large intestine 11011): Diff Lab Latries (Difc.

Laboratories, Detroit, ML). affinity purification Human interferon comma (γ-IFN): interferon scie Interferon 5 Sciences, Inc., + New Brunswick, NJ). Trypsin (X [type)], Thrombin (derived from human plasma), N-formyl-methionyl-leucyl-phenyl Alanine (fMLP), phorbol, 12-myristate, 13-acetate-1- (PMA), gelatin-agarose and ammonium sulfate (class III): Sig Sigma Chemical Co., Ltd. Louis, MO). Leukotriene B, (LTB,): Biomol In Coholated (Bio@ol, Inc, , Phi 1adelph ia, PA).

Concanavalin A-Agarose: Vector Laboratories (VectorLa boratories, Burlingame, CA). Heparin-cepha Rose CL-6B and pepsin-agarose: Pharmacia , Uppsala, 5veden). Flavonobacterium heparinium Heparinase derived from Dr. Howard Bernstein (Masachinese Provided by University of Technology, Cambridge, MA). Recombinant T NF (rTNF), rIL-2 and rIL-1β are Biogen, Inc., Ca@bri dge, MA).

In the following examples, endothelial-derived IL-8 is sometimes referred to as LAI (leukocyte adhesion inhibitor). I will post it.

Example 1 cell culture Human writing vein endothelial cells (HEC) were isolated, cultured from 2 to 5 zone segments, and pooled. and 20% fetal bovine serum (FCS; Gibco Laboratories .

Grand Land, NY) and previously described antibiotics (Bevi Lacqua et al.

J, CI in Invest, 76:2003-2011 (1985) ) containing medium 199 (M199.M.

A, Primary culture using Bioproducts, Bethesda, VD) It was grown in cultivation. 081% gelatin (Bacto gelatin 0143-02; Dif co Laboratories, Detr.

Costar tissue culture flask (75 c m”; Co endothelial growth factor (50-1 00 It g/mL: Biomedical Technologies, Inc., Stoughton, MA) and porcine intestinal heparin (50-1 00It g/+sl; Sigma Cheg+1cal Co,, St, Lo Several strains were cultured sequentially using M199-20% FC3 supplemented with M. uis, M. (Distribution ratio 1:3).

For use in experiments, 1511Il Thera-anox (Thera+anox) ・Plastic coverslip (Miles 5 scientific, Na perville, le), fibronectin (18 g/cm”; Meloy Laboratories, Springfield, VA) or tissue culture dishes coated with 01% gelatin (Corning Glass W Orks, Corning, NY) or in microtiter wells. Blood from normal human volunteers was mixed with CCD buffer (1:9; 10 OIQM Sodium citrate, 130d glucose, pH 6.5). PM A suspension of N (>95%) was prepared by Bevilacqua et al., J. CI in, Prepared according to the method of Invest, 76:2003-2011 (1985). Ta. Mononuclear leukocytes were collected in lymphocyte isolation medium (Litton Bionetics, I Charleston, SC) and Recal e et al., J, 1mmuno1. Meth, 69 Near 1-77 (1984) fractionate into a mononuclear cell-enriched population (75-88%) according to the method of Lymphocyte suspension is achieved by mononuclear cell depletion on serum-coated plastiplates and vetri dishes. It was fractionated into a turbid solution (>95%). Promyeloid cell line HL-60 and mononuclear cell-like cells Strain U937 was cultured as described by Bevilacqua et al. (supra). So Each type of leukocyte was isolated using the technique described by Bevilacqua et al. (supra). Indium-oxine (Amersham Corp., Arli Radioactively labeled with Arlln Heights, IL).

Example 3 Preparation of medium I LL-1 conditioned medium ('IL-I CM) or pseudo-conditioned medium (pseudo-CM) To prepare, the following ingredients: (1) No additional protein: (2) 5βg/ml Transferrin, 5μg/ml insulin and 5βg/ml selenium (final concentration) containing limited non-serum additives (TIS; Co11abora tive Research, Cambr idge, MA); (3 ) 1% (V/V) fetal bovine serum (FC3, endotoxin <0, 027 ng/ ml; Hyclone Laboratories Inc.

Logan, UT); and (4) 10 mg/ml bovine serum albumin (B Contains S A, V fraction; Sigma Chemical Co, ); RPMI-1640 (RPM I; M, A, Bioproduct Full-grown HEC monolayers (100 mm dish) to T L-1 (2,5-5 U/ml hmlL-1 or rIL-β) or in the absence of IL-1.

After 4 hours, all dishes were treated with Ca' or Mg to remove the cytokine-containing medium. Hanks Buffered Salt Solution (HBSS; M, A, Biopr. oducts) in 5 ml and washed in RPMI alone or in TIS, F Incubated for an additional 5 hours in RPMI supplemented with C81 or BSA. . The medium is collected, centrifuged (400xg, 10 minutes), and frozen, usually at -80°C. Ta.

In some experiments, actinomycin D (5βg/ml; Sigma Chemical Co, ), acetylsalicylic acid (500 μM;'Fis her 5 scientific Co, Med'ford', MA), or indomethacin (5 μM; Sigma Chemicat' Co, ) was added to the endothelial culture 30 minutes before addition of rlL-1 and the medium was maintained throughout the pretreatment step. I left it inside.

Example 4 Enzymatic treatment of LAI LAI was enzymatically treated as described below: in RPM++TIS. The collected pseudo-CM and IL-I CM were transferred to an Amicon YM30 membrane (nominal exclusion) under N. Molecular weight removed: 30,000'; Am1con Corp, Danvers' , MA) and trypsin (1000U/a+l, pH 7, 37° C118 hours), thrombin (2U/l111.37℃, 2 hours), henodelina (1'5 U/+1.30°C, 2 hours) or pepsin-conjugated agarose ( 4500 U/ml, pH 4, 18 hours, 37°C).

by boiling 4 (15 min, 100 °C) or for pepsin-conjugated agarose. by centrifugation with removal of agarose beads in a Beckman microcentrifuge. Enzyme treatment was stopped. Trypsin and thrombin each act as a chromogen ‘Ji BAEE (N-α-benzoyl-thi-arginine ethyl ester; Sigma a　CheIIlical　Co,　) Determined by hydrolysis O and plasma coagulation activity Full activity was retained in the simulated CM and in the IL-I CM.

Example 5 Assay of LAI activity LAI activity was determined using a standard radiometric monolayer adhesion assay (Bevilacqua et al., J.).

See CI in, Invest, '76:2003-2011 (1985). The test was carried out using the following method. In short, the Thermanox cover slip, top or bottom Target monolayers consisting of fully grown HEC monolayers grown in titration wells were RPM containing β (25-5U/a+1))+FC3, or r　I　L- Pretreatment was carried out for 4 hours in RPMI+FC3 without 1β. +1 indium Labeled leukocyte suspension (2xlO' cells/+al) was transferred to IL-ICM, pseudo-CM , or diluted directly (final concentration) into fresh RP M T + F CS or TIS. degree = 2xlO'' cells/ml) L, added to the washed monolayer and incubated at 37°C under static conditions. Incubated for 10 minutes. HEC monolayer on Thermanox coverslip Standardized cleaning methods (see Bevilacqua et al., supra) or in the case of microtiter plates, seal, invert, and centrifuge. By (Bevilacqua et al., Proc, Natl, Ac ad, Sci, USA 84:923g-9242 (1987); Ch aro etc., a 65:473-479 (1985)), stop this assay. did.

1llInllIn labeled platinum attached, Gunn 75500 counter (Beckm an Instruments, Inc., Fullerton, CA ) was used to determine the radioactivity bound to the monolayer and the specific activity (ape/cell) of the leukocyte preparation. Determined from cells).

Percentage inhibition (%) was calculated as follows and expressed as standard error of the mean (SEM): did: Significance was tested with a two-tailed Stindent T-test.

Example 6 Cytokine-induced increased adhesion of endothelial cells to PMNs is shown in Figure 1. Furthermore, treatment of HEC monolayers with hmIL-1 induces a significant increase in polymorphonuclear leukocytes (PMNs). This suppressed the time-dependent increase in surface adhesion.

Our standard monolayer adhesion assay method (Bevilacqua et al., J, CI) in, Invest.

76:2003-2011 (1985)), the site Aspirate the in-containing medium and freshen each well immediately before adding radiolabeled leukocytes. Washed with medium. In this “cleaning” system, the IL-1 treated HEC monolayer PMN adhesion was significantly increased compared to untreated monolayers at 1 h after IL-1 and between 3 and 3 Maximum adhesion occurred at 4 hours and then gradually decreased. In contrast, removing the pretreatment medium If PMNs are added directly to the wells (“no-wash” system), especially at 3 o’clock After some time, PMN adhesion was significantly less. PMN connections to unstimulated HEC monolayers The wear was the same at all test time points for both washed and unwashed conditions.

Based on these observations, we determined that the Conditioned medium inhibits PMN-endothelial adhesion (“leukocyte adhesion inhibitory factor”, LAI) activity, whereas conditioned media obtained from unstimulated HEC monolayers do not contain this activity. I hypothesized that.

Example 7 LAIAI activity determination and metabolic evaluation Testing the hypothesis that stimulated endothelial cells contain inhibitors of PMN-endothelial adhesion Conditioned medium was collected from endothelial cell cultures grown in 1,000,111 dishes to . HECs were washed to remove culture medium and incubated with IL-1 or with IL-1. Incubate for 4 hours in RPMI+FC3 in the absence of L-1, at which point The HEC was washed again. Incubate for additional 5 hours in fresh RPMI+FCS After treatment, IL-I CM and pseudo-CM were collected. Super adhesive HEC single layer (2, PM for 4 hours pre-treated with 5-5U/a+lrT L-1β) N adhesion was significantly inhibited in the presence of IL-I CM compared to sham CM (7 2±6%, n=10, P<0.001) (Figure 2). rlL-1β (5 Mock CM supplemented with 100 μg/ml) had no inhibitory effect. in the absence of endothelial cells Even when incubated with a medium containing rlL-Lβ (5 U/ml), no inhibitory activity was produced. I didn't.

Acetylsalicylic acid (500 gM) treatment of HEC was constitutive PCI.

production and calcium ionophore-stimulating PCI, under conditions where production is reduced; did not alter LAI production (LAIAI activity 0-97% of untreated HEC, 3 experiments) ). Similar results were obtained with indomethacin pretreatment, which Arachidonic acid metabolism via the sigenase pathway is unnecessary for LAIAI activity generation It shows.

However, H Treatment of EC with actinomycin D (5 μg/ml) inhibits subsequent LAI production. was essentially blocked (~10% LAIAI activity from untreated HEC, 6 experiments) . Blood in protein-free RPMI medium (65 ± 4% LAI activity, 4 experiments) in TIS-supplemented medium without serum (58 ± 3% LAI activity, 10 experiments), and BSA. IL-I collected in supplemented medium (57 + 6% LAI activity, 5 experiments). etc., and AI activity was detected.

The above observation results indicate that LAIAI activity is produced by endothelial cells stimulated with cytokines. requires de novo protein synthesis and that LAIAI activity requires some from intermediate metabolites of exogenously added media components (e.g. serum factors or IL-1). This suggests that it is not possible.

In addition to human recombinant rL-1β and natural human mononuclear cell-derived IL-1, human Recombinant IL-1α (IOU/ml), human recombinant TNF (100U/ml) , and bacterial LPS (111 g/mI>), as can be seen in Table 1 below. induced the production of LAI activity by the EC monolayer.

Table 1 Stimulator mediator of LAI production by cultured human endothelial cells - PMN adhesion Inhibition rate (%) hm-I L-1 (5U/ml) 72±6r [L-1a (1 0U/ll+l) 58±7rlL-1β(2,5U/ml) 69±10(+ Polymyxin B) 66±7 (10 fevers) 7±13 LPS (1βg/a+1) 39±8 (+polymyxin B) 12±3 (10 fevers) 33±5 rTNF (200U/+1) 54±4 IFNγ (200U/ml) 1± 9GM-CSF (200ng/ml) -1±15I L-2 (500U/ml ) Treated with −9±9 ether for 4 hours, washed, and incubated in RPMI+FC8 for 5 hours. Incubated for a while. The conditioned medium was collected and washed without dilution. 1 treatment (5U rIL-1β, 4 hours) for standard monolayer cell contact on HEC monolayers. It was tested using the wear test method. Data are expressed as mean: SEM.

Polymyxin B sulfate (20 μg/ml) treatment neutralized the stimulatory effect of LPS, but , did not attenuate the effect of rlL-1β. In contrast, rlL-2 (100 U/ml ), rTF N-γ (200U/+il), and GM-CSF (200 (U/ml) did not stimulate LAI production. Further details are provided in Table 2 below. As shown, LAI activity in IL-I CM was ) and PM on HEC monolayers activated with LPS (1βg/ml, 4 hours) It also inhibited N adhesion. Similarly, TNF-conditioned medium and LPS-conditioned medium also contain IL-1 , TNF, and LPS inhibited PMN adhesion to activated endothelial monolayers.

The conditioned medium obtained from HEC stimulated with IL-1 was supplemented with TNF (200 U/ml, 4 Time: 54±4%, 5 experiments), LPS (1βg/31.4 hours; 56±7%, 3 experiment), or IL-1 (2,5 U/+L 4 hours; 68 ± 4.5 experiments). comparably inhibited PMN adhesion to sexualized HEC target monolayers (see Table 2 below). ). In contrast, IL-I CM was prepared using fresh human blood/* (4 ± 3% inhibition, 4 experiments) , complement-depleted human serum (10 = 4%, 3 experiments) or zymosan-activated human PMN contact on positive surface coated with serum (-8 ± 10%, 3 experiments) It did not interfere with the wear. These results suggest that PMN adhesion to non-activated endothelial cells In addition to its lack of effect on the "hyperadhesive" endothelial cell surface and leukocyte This suggests that it acts to selectively inhibit the interaction of

Table 2 Cytokine-activated HEC monolayers and endotoxin-activated HEC monolayers were assayed. LAI activity from tocaine-stimulated HEC and endotoxin-stimulated HEC IL-ICM 68±4 54±4 56±7 TNF CM 45±9 38± 9 36±8 LPSCM 39±8 31±7 12±3 Stimulated with cytokines As described in Example 3, HEC monolayers stimulated with endotoxin and HEC monolayers stimulated with Conditioned medium (CM) was collected.

The target HEC monolayer was treated with rlL-1β (5U/ml), TNF (200U/a+ 1) or pretreated with LPS (1 μg/l1l) for 4 hours and resuspended in test medium. Immediately before adding the labeled PMNs, the cells were washed. Quantitative analysis of PMN adhesion Layer adhesion assay method (Bevilacqua et al., J, C11n, Invest, 76:2003-2011 (1985)) (see Example 5). ). Data are expressed as mean SEM of three separate experiments.

The generation of LAI activity is IL-1 degree dependent, and Q, lU/m1rjL-1β It was found to have a threshold at 25-5 U/ml and a maximum at 25-5 U/ml (Figure 3). IL' - LAI appearance in ICM is time dependent with respect to inhibitory activity, collected hourly IL-1 was detectable in 1-2 hours (30 ± 6%, n = 5) in It reached its maximum after 5 to 6 hours (65±2%). After 9 hours, a decrease in activity was observed. (See Figure 9).

LAI activity passed through Amicon YM30 (nominal exclusion molecular weight -30000), but It was concentrated 10-20 times with Amicon YM5 (nominal molecular weight exclusion = 5000). P Inhibition of MN adhesion was maximally blocked by 5x concentration of IL-ICM (7x of PMN adhesion 5 ± 7% inhibition, n-5) (Figure 4), detectable at lower IL-I CM concentrations LAI activity is proportionally reduced. Evaluation of targets for LAI activity. Does LAI activity exert an inhibitory effect on leukocytes or on endothelial cells? to selectively target each type of cell to determine whether it exerts an inhibitory effect. Prepared. Activated HE Cl 1 l (5 U/ml r I L-1, 4 hours) After 30 min incubation with IL-I CM, fresh medium (1,5+]) No inhibition of PMN adhesion was observed when replacing with ．． 05.3 experiment).

In contrast, PMNs (3xlO' cells in 1.5 ml) were incubated with IL-I CM for 30 min. Pre-treated for 2 min, centrifuged (100xg, 2 min) and resuspended in an equal volume of fresh medium. However, when an activated HEC monolayer was added, PMN adhesion was significantly inhibited (25 ±3% inhibition, P<0.001.3 experiments).

Additionally, a fixed IL-1 stimulated HEC monolayer (2% paraformaldehyde (PB) After 226C for 5 minutes, store overnight at 4℃ in PMS + BSS A. The adhesion of non-immobilized PMH to (45 soil 9% inhibition, 2 experiments). In contrast, PMNs are simply fixed (2 % paraformaldehyde (in PBS, on ice for 5 min), the inhibitory effect of LAI decreased by more than 80%. PMN fixation itself is not processed or IL-1 processed H'EC did not change monolayer adhesion. These observed facts indicate that the inhibitory effect of LAI is the main cause. It has been demonstrated that the cells are directed against white blood cells.

As shown in Figure 5, LAI is associated with cytokine-activated and non-activated endothelium. It appears to differentially affect the adhesion of some blood leukocytes to the skin. I L-ICM consistently conferred significant inhibition of PMN adhesion to activated HEC monolayers. (76 ± 4% inhibition of cytokine-stimulated adhesion, P < 0.001.5 experiment) had no effect on PMN adhesion to unactivated monolayers. Similarly, active Mononuclear cell adhesion to cultured HEC monolayers was significantly inhibited (stimulated with cytokines). 55 ± 12% inhibition of adhesion, P < 0.01.5 experiments), relative to unactivated monolayers. No effect on relatively large adhesion was observed. In contrast, IL-ICM , Peripheral blood return to IL-1 activated HEC monolayer and non-activated HEC monolayer lymphocytes (-4 soil 6% inhibition, P>0.1.3 experiment), HL-80 cells (2 soil 6% inhibition) Harm, p>o.

1.4 experiments) or 0937 (1 soil 9% inhibition, p>o, t, 4 experiments) No significant changes were made.

Barriers of PMN adhesion to activated HEC monolayers in the presence and absence of LAI Ital microscopy revealed significant differences. Single layer surface in pseudo CM A large number of PMNs were firmly attached to the membrane (Fig. 6A). In contrast, in the presence of LAI, PM The extent of N adhesion was significantly reduced, which was consistent with the stationary radiometric assay. interesting Remarkably, although individual PMNs resisted shape changes, they did not spread over the monolayer surface. It appeared that there were no such cases (Fig. 6B).

Example 9 Assay of PMN activation Soluble stimulants (10-'M fMLP, 10-'LTB, 1100n/I PMN responsiveness to PMMA) was determined by membrane potential and cytosolic release. Evaluation was made by quantitatively measuring changes in calcium concentration.

Changes in membrane potential were performed using a 5PEX membrane with a cuvette compartment controlled at 37°C. Fluorescent cyanine probe diS using Fluorolog I+ Fluorescence Spectrophotometer C, -5 (Thitin et al., J. Biol. Chem, 255+18 74-1878 (198G); Lazzari et al., J., Biol, C. Het 261:9710-9713 (1936)). .

For measurement of Ca”-sensitive Fura-2 fluorescence, a suspension of PMH (107 cells/ ml) in HBSS without Ca" and Mg" to a final concentration of 1 μ's F ura-2/AM (preservation solution, lll11 dimethyl sulfoxide) and Both were incubated at 37°C for 10 minutes. Centrifuge with cold HBSS Cells were washed twice and stored at 10°C. Dilute the cells 5x with HBSS. , and further incubated at 37°C for 15 minutes. Just before the test, take a small amount of Be-1kuman. Pellet the cells in a centrifuge (model B) and add warm HBSS+Ca” and Mg After resuspending in 1.6+al and equilibrating for 5 minutes in a cuvette, the agonist ( agonist) was added.

Beam splitter, 2 excitation monochromators, and 2 wavelengths (340 Gm and 380 na+) to enable rapidly changing (30 Hz) excitation of Fura-2. S equipped with a dual mirror cut-off mechanism with a specialized optical arrangement to enable PEX (Edison, NJ) Fluorolog II (Model CM-1) Firefly Fluorescence measurements were performed with a light spectrophotometer. The excitation band width was set to 6.6r+m+. release The cell number, dye loading and Intracellular Ca” concentration independent of pigment fading can be calculated. 80 μM digitonin After equilibrating (maximum) intracellular and extracellular Ca, IMTRIS, 30oI By adding (minimum) IIMEGTA (pH 〉10゜0), the fluorescence signal was I put a scale on it.

The basal amount and maximum increase in intracellular Ca'' were determined by Grynkiewicz et al., J. Biol.

Chew, 260:3440-3450 (1985). I calculated it. Cohen et al., J, Cl1n, Invest, 61:108 1-1088 (1978).

(Margin below) Example 10 Effects of LAI on other PMN functions Stimulus-responsive signaling and supero-activation in response to soluble inflammatory stimulants Oxide production was also tested. Addition of IL-I CM or pseudo-CM or 10-'MfMLP, 10-'I LTB 4, or 100 ng/ml P did not inhibit MA-induced membrane depolarization (Figure 7A). Similarly, both IL-ICM and sham CM induce fMLP or There was no change in the increase in cytosolic calcium levels (Fig. 7B). 10 -'11fMLP or lOOng/ml Suction in response to PMA Peroxide production was also not inhibited by LAI (data not shown). . These results suggest that in PMNs, transduction of activation signals (membrane depolarization and (reflected in elevated cytosolic free calcium) and cellular responses (superoxygen This shows that LAI does not inhibit LAI.

biochemical characterization To facilitate preliminary biochemical characterization, serum-free transferrin/infusion Collecting LAI from IL-1 activated H,EC monolayers in insulin/selenium supplemented medium Ta. The glue, AI activity in these preparations is increased by boiling for 20 minutes, acidification (pH 2 or 4) Freeze for 18 hours (-80°C until 13 months) and remove protease inhibitors. Stable to various treatments, including storage for up to 14 days at 4°C without additives I understand. LAIAI activity sedimentation (250000xg, 45 minutes) , this indicates that it is not bound to membrane fragments but is soluble. is suggesting. LAIAI activated latin-agarose or concanavalin-A-a Not adsorbed by galose. IL filtered through a 30,000 molecular weight exclusion membrane -I CM, trypsin (1000 U/ml, pH 7,376C 118 hours) , thrombin (2 U/1.37°C, 2 hours), or hevarinase (15 U/ 1Ill, 30°C, 2 hours) had no effect on LA■ activity. . However, pepsin-conjugated agarose (4500 U/ml, pH 4, 37°C, 18 Treatment with time) significantly reduced LAI in untreated controls. LAl activity is Precipitate with 60-80% saturated ammonium sulfate, and all activity is recovered in this fraction. It was. These results demonstrate that the biological activity of LAI is dependent on protein components. It's proven.

Example 12 Purification of LAI by High Performance Liquid Chromatography Model 51.0 Solvent Pump 2 water system consisting of individual and programmable system controller gradient preparation device Liquid Chromatography Systems, Inc. (Milford, MA) HPLC was performed using a TEM.

Column effluent was measured at 280 nm using a Waters Model 481 absorbance detector. I monitored it. IL-I CM and pseudo CM in RPMI+TIS were converted to Amicon Y. Filter with M30 membrane, and filter the effluent with YM5 membrane (nominal exclusion molecular weight 5000) for 20~ Concentrate 40 times and dialyze against distilled water (Spectrapol 7, excluded molecular weight 100) 00; Spectrum Med.

Ind., Los Angeles, CA) L, lyophilized. Freeze drying The dried sample was diluted with 0.2M acetic acid, 01M triethylamine (H3,9) to approximately 1mg/sl. Water's Protein-Vac 125 gel filtration column with a concentration of (7.8mm x 30ca+). Using this column as an isocratic system, the flow rate is 1. It was run at 8 ml/min and fractions of 0.9 ml each were collected. Transfer the fractions to phosphate buffered saline ( 136mM NaCl, 0.3d KCl, 0.8mM NatHPO, 0,1 Dialysis was performed for 18 hours (4°C) against mM KH, PO, pH 7,4). minutes The fractions were diluted in RPMI+FC3 and assayed using a standard monolayer adhesion assay. Viola Bio-Rad Laboratories, Rich The amount of protein was determined using the following software (Mond, CA).

A sample of lyophilized IL-I CM was subjected to high pressure liquid chromatography as described above. Therefore, it was fractionated by Water's Protein-Pak 125 gel filtration. Fraction (0 , 8 ml) was dialyzed against phosphate buffered saline (pH 7.4) and RPMI+F Diluted 1.10 in C5 and assayed using a standard monolayer adhesion assay. In this system, LA It was separated from the maximum of I activity (12th fraction) or the maximum of the main protein (9th fraction) (Fig. 8). Continuation of LAI activity in maximum fraction until PMNMNOS 0% inhibition It was present at an effective concentration of 80 times as calculated by dilution. recovered activity and The yield of LAI, evaluated by serial dilution, was higher than 90%. In a preliminary experiment , LAI activity in the maximal fraction is stable to boiling and acidification, and completely destroyed by treatment (62 ± 10% LAI activity in untreated control). −8±6% LAI activity in pushin-treated samples).

Example 13 Endothelial protection test Human endothelial cells were cultured on gelatin-coated microtiter wells as previously reported. (Wheeler et al., J. C11n, Invest, 82 : 1211 (1988) and Lu5cinleas et al., J. Immun. ol. 142:2257 (1989)). Next this monolayer is cultured ( Washed twice with RPMI+1% FBS).

Add 100μ + medium +/-10 units/ml r h r L-1β to each well. , and incubated for 4 hours at 37°C. After IL-1 treatment, monolayers were incubated once with culture medium. Washed. Next, human blood PMN (2x 105 to 2xlO@/well) was added to the test 100 μm medium containing the substance (1-500 nM for rhI L-8) tested). This plate was then incubated at 37°C for 10-2 hours. did. After this incubation, the wells are filled with medium, sealed and inverted for 25 minutes. Centrifuged at 0g for 5 minutes. Next, remove the liquid from this well and add 2% paraforma Fixation was achieved by adding 100 μm of aldehyde for 15 minutes. After fixation, well with Light Geimsa staining (fright' 5-Ge1m5a 5tain). The degree of damage to PMNMNOS and endothelial monolayer was determined by staining and microscopic examination. It was determined. See Figure 10.

Although the invention has been described with respect to particular embodiments thereof, it is understood that further modifications are possible. It will be understood. This application generally describes any and all embodiments of the invention that are in accordance with the principles of the invention. Any modifications, uses or adaptations known or known in the art to which this invention pertains. Deviations from this disclosure as are customary and herein above and below. Deviations from the disclosure as may be applied to the essential features recited in the claims Any variations, uses or adaptations of the present invention are deemed to be covered.

Contact PMN/mm” Contact PMN/mm 2 rIL-1β concentration (U/mI) Figure 3 Relative concentration of endothelial cell conditioned medium treated with IL-1 Figure 4 Adhesion white blood cells/mm 2 FIG. 6A FIG.6B Figure 7 (A) Protein content (mg/ml) (Inhibition rate (%) of PMN care) time Figure 9 international search report -11-a---1 PCT/U59Q/Q6 e, An---^-C-11 @Death/IIguQ/1/＾4ku Ayu

Claims (19)

[Claims] 1. Contains substantially natural contaminants that can inhibit the adhesion of leukocytes to other cell surfaces. Endothelium-derived IL-8 polypeptide. 2. endothelial-derived polypeptides capable of inhibiting leukocyte adhesion to another cell surface The endothelial-derived IL-8 of claim 1, which comprises a fragment of IL-8. 3. The endothelium-derived IL-8 protein according to claim 1 or 2, wherein said molecule is detectably labeled. Ripeptide or fragment thereof. 4. If the polypeptide is labeled with radioisotopes, enzymes, fluorescent labels, paramagnetic labels, and Claim 3, wherein the label is detectably labeled with a label selected from the group consisting of leaving radical labels. Endothelial-derived IL-8 polypeptide. 5. An assay for determining the concentration of endothelial-derived IL-8 in a sample, in which the polypeptide is The endothelial-derived IL-8 polypeptide according to claim 4, which is used for. 6. Persons diagnosing the presence and location of inflammation in a mammalian subject suspected of having inflammation The law is (a) A detectably labeled binding molecule capable of identifying endothelial-derived IL-8 is added to the target. administered to; (b) detecting the binding molecule; A method consisting of things. 7. Persons diagnosing the presence and location of inflammation in a mammalian subject suspected of having inflammation The law is (a) identify detectably labeled endothelium-derived IL-8 in a sample of the subject's tissue; (b) incubating in the presence of a binding molecule capable of binding into said sample of said tissue; detecting said binding molecule that is 8. (a) harvesting a conditioned medium from a cell source; (b) ultrafiltering the conditioned medium; Obtaining an endothelium-derived IL-8-containing fraction by; (c) concentrating and desalting the endothelium-derived IL-8-containing fraction by ultrafiltration; (d) The endothelium-derived IL-8 oil-containing fraction is lyophilized and defatted by acetone precipitation. (e) The endothelium-derived I-8-containing fraction was purified by HPLC chromatography. Gel filtration; (f) subjecting the endothelium-derived IL-8-containing fraction to ion exchange chromatography; (g) recovering the filtrate obtained in step (f) in substantially pure form; A method for recovering endothelial-derived IL-8 in qualitatively pure form. 9. Endothelium-derived IL-8 produced by the method of claim 8. 10. A mammalian drug comprising an inflammation-reducing amount of IL-8 and a medically acceptable carrier. A pharmaceutical composition useful for treating inflammation in elephants. 11. 11. The pharmaceutical composition of claim 10, wherein said IL-8 is endothelial-derived IL-8. 12. A method of treating inflammation in a mammalian subject requiring such treatment. 11. A method comprising administering to said subject the pharmaceutical composition of claim 10. 13. 13. The method of claim 12, wherein said inflammation is part of an acute or chronic inflammatory disease process. 14. It is a method of protecting against damage to other cells mediated by white blood cells. hand, in the vicinity of the cell at an effective concentration sufficient to inhibit leukocyte-mediated damage. 8 or a derivative thereof; 15. 15. The method of claim 14, wherein said IL-8 is endothelial-derived IL-8. 16. It is a method of protecting against damage to other cells mediated by white blood cells. hand, IL-8 or a concentration effective to inhibit leukocyte-mediated damage to the cells. A method comprising: treating leukocytes with a derivative thereof. 17. 17. The method of claim 16, wherein said IL-8 is endothelial-derived IL-8. 18. 17. The method of claim 14 or 16, wherein said cells are endothelial cells. 19. 19. The method of claim 18, wherein the leukocytes are neutrophils.