WO1991008483A1 - Il-8 a derivation endotheliale - Google Patents

Il-8 a derivation endotheliale Download PDF

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Publication number
WO1991008483A1
WO1991008483A1 PCT/US1990/006580 US9006580W WO9108483A1 WO 1991008483 A1 WO1991008483 A1 WO 1991008483A1 US 9006580 W US9006580 W US 9006580W WO 9108483 A1 WO9108483 A1 WO 9108483A1
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Prior art keywords
endothelial
derived
adhesion
leukocyte
inflammation
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PCT/US1990/006580
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English (en)
Inventor
Michael A. Gimbrone, Jr.
Margaret Elyse Wheeler
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Brigham And Women's Hospital
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Priority to JP91500554A priority Critical patent/JPH05505391A/ja
Publication of WO1991008483A1 publication Critical patent/WO1991008483A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5421—IL-8
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is drawn to endothelial-derived interleukin-8 (IL-8). These polypeptides, previously identi ⁇ fied as “leukocyte adhesion inhibitor” (LAI), are involved in the process through which populations of leukocytes are inhibited from adhering to cellular substrates.
  • LAI leukocyte adhesion inhibitor
  • the invention additionally relates to the use of endothelial-derived IL-8 as anti-inflammatory agents and as therapeutics for clinical indications in which damage of vascular endothelium and other tissues occurs.
  • Leukocytes must be able to attach to cellular substrates in order to properly defend the host against these invaders. They must also attach to endothelial cells so that they can migrate from the circulation to sites of ongoing inflammation. Furthermore, they must attach to antigen-presenting cells so that a specific immune response can occur. Finally, they must attach to appropriate target cells so that lysis of virally-infected or tumor cells can occur.
  • TNF tumor necrosis factor
  • LPS Gram-negative bacterial endotoxin
  • HL-60 and U937 can act directly on vascular endothelium in vitro to increase the adhesiveness of the endothelial cell surface for blood leukocytes as well as the related leukocyte cell lines (HL-60 and U937).
  • che otactic factors such as purified complement components, formyl -methionyl -leucyl-phenylalanine, and leukotriene B can also augment the attachment of PMN to cultured endothelial monolayers (see, e.g.. Smith et al . , Exp. Cell Res. 122:169-177 (1979); Hoover et al., J. Cell Sci. 45:73-86 (1980); Hoover et al., PNAS USA 81:2191-2193 (1984); Zimmerman et al . , Thromb. Res. 35:203-217 (1984); Gimbrone et al., J. Clin. Invest.
  • leukocyte adhesion potentially is of central importance to therapeutic interventions in inflammatory disease processes.
  • leukocyte adhesion is normally desirable, it is also implicated in immune and non-immune inflammatory disease processes, including organ transplant rejection, tissue graft rejection, allergic reactions, autoimmune diseases, rheumatoid arthritis, vasculitis, septic shock, adult respiratory distress syndrome (ARDS), glomerulo- nephritis, and other tissue or organ specific forms of acute and chronic inflammation.
  • ARDS adult respiratory distress syndrome
  • leukocyte adhesion may produce microvascular occlusion, tissue injury and death.
  • any means capable of attenuating or inhibiting cellular adhesion would be highly desirable for certain patients.
  • CM conditioned media
  • FCS fetal calf serum
  • HEC human umbilical vein endothelial cell(s); TIS, transferrin/insulin/selenium
  • hmIL-1 human monocyte-derived interleukin 1
  • rIL-1 recombinant IL-1
  • rTNF recombinant tumor necrosis factor
  • LPS Gram-negative bacterial endotoxin
  • LAI leukocyte adhesion inhibitor
  • PMN polymorphonuclear leuko- cyte(s)
  • GM-GSF granulocyte-monocyte colony stimulating factor
  • fMLP N-formyl-methionyl-leucyl-phenylalanine
  • PMA phorbol 12-myristate 13-acetate.
  • LAI leukocyte adhesion inhibitor
  • Kerr et al., J. Cell Biol. 103:1760 (1986) disclose that exposure of polymorphonuclear leukocytes to unstimulated endothelial cells or medium conditioned by these cells inhibits adhesion of leukocytes to unstimulated endothelial onolayers and tissue culture plastic.
  • the present invention is drawn to factors produced by endothelial cells, in particular, human umbilical vein endothelial cells, treated with IL-1, TNF, or LPS. These factors are non-cyclooxygenase-derived inhibitors which act to block PMN and monocyte adhesion, but not lymphocyte adhesion, to the hyperadhesive (cytokine-stimulated) endothelial surface. Production of these inhibitory factors is not limited to human umbilical vein endothelial cells but includes cell types including SV40 virally transformed human umbilical vein endo- thelial cells and human diploid skin fibroblasts. These factors designated endothelial-derived IL-8 find use as leukocyte adhesion inhibitor molecules and as agents which protect endothelial cells from neutrophil mediated damage. The compositions of these factors and methods for their use are provided.
  • Substantially pure endothelial-derived IL-8 encompassed by the present invention, comprises a mixture of at least two unique polypeptides designated [Ala IL-8JI77 and [Ser IL-8]7£. This material inhibits both monocyte and neutrophil adhesion to cytokine activated endothelial cultures.
  • highly purified endothelial-derived IL-8 and recombinant human [Ala IL-8J7 and [Ser IL-8]7 do not inhibit adhesion. Therefore, natural preparations of endothelial-derived IL-8 may contain other leukocyte adhesion inhibiting activity, or activities, that are active on blood monocytes.
  • LAI endothelial-derived IL-8
  • endothelial-derived IL-8 which is a soluble, natural product of the activated endo- thelial cell. Its generation appears to require de novo protein synthesis but is not dependent on the cyclooxygenase pathway. Endothelial-derived IL-8 activity is stable under physiological conditions as well as extremes of temperature and pH.
  • endothelial- derived IL-8 can reverse the strong attachment of human PMN to cytokine-activated human umbilical vein endothelial cells. That is, when endothelial-derived IL-8 is added to PMN which are adherent to the activated endothelial cell, the PMN release from the endothelial cell surface. The kinetics of this "release” is rapid (1-5 minutes) and potentially reversible.
  • endothelial-derived IL-8 The differential effects of endothelial-derived IL-8 on leukocyte adhesion, that is, the significant inhibition of PMN, but not monocyte or lymphocyte adhesion, suggests that the action of endothelial-derived IL-8 may be related to distinct leukocyte receptors which recognize the hyperadhesive endo- thelial cell surface. Similarly, the lack of effect on the promyelocytic cell line HL-60 and the monocyte-like line U937 suggests that endothelial-derived IL-8 may be directed at functions or surface structures of the mature leukocyte which are deficient in this undifferentiated cell line.
  • Endothelial-derived IL-8 can also protect against leukocyte mediated damage to endothelial cells. This protec- tion may occur because of the leukocyte adhesion inhibiting activity of endothelial-derived IL-8. However, other mechan ⁇ isms separate from adhesion inhibiting activity may be involved. Therefore, the methods of the present invention are not bound by any disclosed mechanisms. Before the present invention IL-8 was designated as a proinfiammatory che oattractant. No leukocyte adhesion inhibiting activity had been attributed to IL-8.
  • the present invention encompasses the use of IL-8 and related peptides from sources other than vascular endothelial cells, as anti-inflammatory agents and/or as therapeutics for clinical indications in which leukocyte mediated damage of vascular endothelium or other tissues occurs.
  • Effective amounts of IL-8 to be administered in vivo can be determined by in vitro and in vivo bioassays as explained in more detail below.
  • the present invention relates to endothelial-derived IL-8 as well as to its functional derivatives.
  • the invention additionally pertains to processes for preparing and purifying endothelial-derived IL-8, screening assays for endothelial- derived IL-8, diagnostic and therapeutic uses of endothelial- derived IL-8, and compositions containing endothelial-derived IL-8 or its functional derivatives.
  • the invention includes the leukocyte adhesion inhibitor endothelial-derived IL-8 or its functional derivatives, which are substantially free of natural contami- nants.
  • the invention further pertains to endothelial-derived IL-8 and its derivatives which are detectably labeled.
  • the invention also includes a method for recovering endothelial-derived IL-8 in substantially pure form which includes, but is not limited to the following steps:
  • step (g) recovering the filtrate obtained in step (f) in substantially pure form.
  • the invention includes the endothelial-derived IL-8 obtained by the above methods.
  • the invention is also directed to a method of treating inflammation in a mammalian subject which comprises providing to said subject in need of such treatment an amount of an anti- inflammatory agent sufficient to suppress said inflammation; wherein said anti-inflammatory is selected from the group consisting of: endothelial-derived IL-8, a fragment of endothelial-derived IL-8, a chemical derivative of endothelial- derived IL-8, a variant of endothelial-derived IL-8, and an analogue of endothelial-derived IL-8.
  • the invention also encompasses compositions and methods for protecting against leukocyte mediated damage to endothelial cells.
  • the invention also includes the above method for treating inflammation wherein said anti-inflammatory agent is adminis ⁇ tered in a suitable carrier.
  • the invention also relates to a pharmaceutical composition for treating inflammation in a mammalian subject comprising administering an inflammation- reducing amount of an anti-inflammatory agent, wherein said agent is selected from the group consisting of: endothelial- derived IL-8, a fragment of endothelial-derived IL-8, a chemical derivative of endothelial-derived IL-8, a variant of endothelial-derived IL-8, and an analogue of endothelial- derived IL-8.
  • the invention is further directed to a method of diagnos ⁇ ing the presence and location of inflammation in a mammalian subject suspected of having an inflammation which comprises:
  • the invention also pertains to a method of diagnosing the presence and location of an inflammation in a mammalian subject suspected of having an inflammation which comprises: (a) incu ⁇ bating a biological sample from said subject suspected of containing endothelial-derived IL-8 in the presence of a detectably labeled binding molecule (e.g., antibody) capable of identifying endothelial-derived IL-8; and (b) detecting said binding molecule which is bound in said sample.
  • Said biologi ⁇ cal sample may include a biopsy specimen or body fluid such as blood (serum or plasma), joint/pleural/peritoneal exudate, cerebral spinal fluid, or ocular fluid, for example.
  • the invention therefore includes novel methods for recovering and purifying the new endothelial-derived IL-8 molecule and its functional derivatives, diagnosing and treating inflammation in a mammalian subject, and novel compositions containing endothelial-derived IL-8 or its functional derivatives.
  • An understanding of this process of leukocyte adhesion inhibition, and of the endothelial-derived IL-8 molecule itself, will aid in the development of its therapeutic and/or diagnostic uses in such fields as rheumatology, organ trans- plantation, allergy and oncology.
  • endothelial- derived IL-8 may be useful in the treatment of adult respira ⁇ tory distress syndrome, septic shock, vasculitis, ischemia- reperfusion injury in the heart and other vital organs, and other inflammatory disease processes in which leukocyte (neutrophil) dependent injury to vascular-endothelium or other tissues occurs.
  • This invention will enable those in the medical field to more effectively diagnose and treat inflammatory disease processes, and to utilize a quantitative assay for endothelial- derived IL-8 in order to monitor the time-course and/or intensity of on-going clinical episodes of inflammation.
  • the invention will further enable researchers to investigate the mechanisms of leukocyte-endothelial cell interactions and leukocyte function in inflammation.
  • FIG. 1 PMN adhesion to IL-1-treated endothelial monolayers is depicted.
  • Indium-labeled PMN were added to HEC monolayers pretreated with hmIL-1 (5 U/ml) for the times indicated, and the number of adherent PMN were determined in a standard 10-minute monolayer adhesion assay.
  • Closed circles (“washed”) indicate wells from which the conditioned medium was removed and the monolayers washed immediately prior to PMN addition.
  • Open circles (“unwashed”) indicate wells in which PMN were added directly into the conditioned medium.
  • PMN adhesion to untreated HEC in the presence (open triangles) or absence (closed triangles) of conditioned medium is indicated at 1, 4, and 8 h.
  • FIG. 1 Effect of various conditioned media on PMN adhesion to stimulated and unstimulated endothelial cells is depicted.
  • Conditioned media were collected as follows:
  • IL-1 CM and SHAM CM were collected from HEC monolayers pretreated with and without rIL-l for 4 h, washed, and incubated in RPMI+FCS for a subsequent 5 h (ENDOTHELIAL CM), or
  • FIG. 3 Concentration dependence on IL-1 stimulation of human vascular endothelial generation of LAI is depicted.
  • Confluent HEC monolayers were pretreated for 4 h with increas ⁇ ing concentrations of hmIL-1 or rIL-1J, washed, and incubated an additional 5 h in RPMI+FCS.
  • IL-1 CM and SHAM CM were collected and assayed for LAI activity in a standard monolayer adhesion assay. Percent inhibition of PMN adhesion was calculated as described in Example 7. Each point represents the mean + SEM of three separate experiments.
  • FIG. 4 Concentration dependence on IL-1 CM of LAI- induced inhibition of PMN adhesion is depicted.
  • IL-1 CM was collected in serum-free, TIS-supplemented medium, filtered through an Amicon YM 30 ultrafiltration membrane, and concen ⁇ trated on an Amicon YM 5 membrane to a 20X concentration (vol/vol).
  • the concentrate was then serially rediluted with RPMI+FCS for assay of LAI, endothelial-derived IL-8 activity. Each point represents mean ⁇ SEM of five separate experiments.
  • FIG. 5 Effect of LAI on adhesion of different types of leukocytes to untreated and IL-1-treated endothelial cell monolayers is depicted.
  • Isolated radiolabeled PMN, monocytes, lymphocytes, or HL-60 cells were resuspended in SHAM CM (striped bars) or IL-1 CM (solid bars) and adhesion to untreated (-) or IL-1-treated (+) (2.5 U/ml HL-10, 4 h) HEC was assessed in a 10-minute assay. Values are expressed as mean ⁇ SEM (*P ⁇ 0.01 IL-1 CM versus SHAM CM).
  • FIG. 6 Hoffman differential-interference contrast microscopy of PMN adhesion to IL-1-treated HEC monolayers is depicted. Isolated PMN were resuspended in SHAM CM (A) or IL-1 CM (B) and added to IL-1-pretreated HEC (2.5 U/ml HL-10, 4 h) for 10 min, and the monolayers washed to remove nonadherent PMN. Note that, in panel B, the adherent PMN are reduced in number and appear less well-spread on the surface of the HEC monolayer. (Original magnification, 400x.)
  • FIG. 7 Agonist-induced membrane depolarization and transient Ca ++ rise in PMN is depicted.
  • FIG. 8 Fractionation of IL-1 CM by high-pressure liquid chromatography is depicted.
  • IL-1CM was collected in serum-free, TIS-supplemented medium, filtered through an Amicon YM 30 membrane, concentrated on a YM 5 membrane, washed to remove salts, and lyophilized. Aliquots were resuspended in 0.2M acetic acid, 0.1M triethylamine (pH 3.9), and applied to a Waters Protein-PAK 125 gel filtration column. Fractions were collected at a flow rate of 1.8 ml/ in, dialyzed against PBS and diluted 1:10 with RPMI+FCS for assay of LAI activity (stippled bars). Protein content was determined in undiluted fractions in PBS (closed circles). Similar results were obtained in three additional experiments.
  • FIG. 9 Fractionated collection of LAI is depicted.
  • the appearance of LAI, endothelial-derived IL-8, in the collec ⁇ tion medium is time-dependent as determined by fractionated (hourly) collections.
  • the HEC cultures were treated at time zero with 5 U/ml interleukin-1. At one hour, the medium was collected and replaced with fresh medium. Cytokine was added to the replacement medium until hour 5.
  • endothelial-derived IL-8 was evident in the medium as early as 1 hour, reached peak production between 4 and 5 hours and was maintained until 9 hours. After 9 hours, a decline in activity was observed.
  • Figure 10 Fractionated collection of LAI is depicted.
  • the appearance of LAI, endothelial-derived IL-8, in the collec ⁇ tion medium is time-dependent as determined by fractionated (hourly) collections.
  • the HEC cultures were treated at time zero with 5 U/ml interleukin-1. At one hour, the medium was collected and replaced with fresh medium.
  • One aspect of the present invention relates to the discovery of a natural intercellular adhesion inhibitor, referred to as endothelial-derived IL-8.
  • the invention is directed toward substantially pure endothelial-derived IL-8.
  • Substantially pure endothelial IL-8 includes a mixture of IL-8 molecules.
  • the mixture comprises [Ala IL-8J 7 as the predomi ⁇ nant polypeptide with [Ser IL-8J72 as a minor component.
  • [Ala IL-8]77 will comprise from about 70% to about 95% of the mixture.
  • [Ala IL-8]77 and a biochemical method for its separation from [Ser IL-8] j 2 are described and claimed in copending U.S. application serial no.
  • a “fragment” of endothelial-derived IL-8 polypeptides is meant to refer to polypeptide subsets.
  • a “variant” of the polypeptides is meant to refer to a naturally occurring molecules substantially similar to either the entire molecules, or fragments thereof.
  • An “analogue” of endothelial-derived IL-8 is meant to refer to a non-natural molecule substantially similar to either the entire molecules or fragments thereof.
  • a molecule is said to be "substantially similar” to another molecule if the sequence of amino acids in both molecules is substantially the same, and if both molecules possess a similar biological activity. Thus, provided that two molecules possess a similar activity, they are considered variants as that term is used herein even if one of the molecules contains additional amino acid residues not found in the other, or if the sequence of amino acid residues is not identical .
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half-life, etc.
  • a suitable screening method for determining whether a given compound is an endothelial-derived IL-8 functional derivative comprises, for example, bioassays as described for PMN-endothelial cell adhesion as well as immunoassay, employing RIA or ELISA methodologies, based on the production of specific neutralizing antibodies (monoclonal or polyclonal) to natural endothelial-derived IL-8.
  • Endothelial-derived IL-8 disclosed herein is said to be "substantially free of natural contaminants" if preparations which contain it are substantially free of materials with which this product is normally and naturally found.
  • substantially pure endothelial-derived IL-8 is a mixture comprising [Ala IL-877 and [Ser IL-8372 polypeptides. While the methods of the present invention are able to separate this mixture from natural contaminants, the present methods, or others known in the art, are not able to separate the two components.
  • preferred methods of purification include generating rabbit heterosera and mouse monoclonal antibodies for use in immunoaffinity chromatography purification, ion-exchange chromatography on a cation exchange column such as Mono S, and preparative SDS-PAGE followed by electroelution.
  • a preferred method of purification includes the following steps and materials: a) endothelial-derived IL-8 is collected from cell source in RPMI-1640 (M.A. Bioproducts) supplemented with ITS (Collaborative Research, Bedford, MA).
  • b) Media is ultrafiltered on Amicon-stirred ultrafil ⁇ tration cell using membrane YM-30.
  • Media from step (b) is concentrated on YM-5, washed to desalt with Milli-Q purified water (Millipore), frozen and lyophilized. Contaminating lipid is removed by washing the lyophilized pellet with five volumes ice-cold acetone.
  • Lyophilized material is reconstituted in 0.2 M acetic acid (Fisher Scientific) and 0.1 M triethylamine (Sigma Chemical Co.) and applied to a Protein-Pak 125 gel filtration column (Waters).
  • Fractions from this procedure may be tested for endothelial-derived IL-8 activity and subjected to SDS polyaery1amide gel electrophoresis.
  • Also contemplated by the present invention are purified endothelial-derived IL-8 fragments or its derivatives manufac- tured using organic synthesis or recombinant DNA techniques, or by proteolysis.
  • the present invention also includes methods of detecting endothelial-derived IL-8 or functional derivatives in a sample or subject.
  • antibodies specific for endothelial- derived IL-8, or functional derivatives thereof may be detectably labeled with any appropriate ligand, for example, a radioisotope, an enzyme, a fluorescent label, a paramagnetic label, or a free radical.
  • ligand for example, a radioisotope, an enzyme, a fluorescent label, a paramagnetic label, or a free radical.
  • the presence of inflammation may be detected through the use of such detectably labeled materials.
  • Methods of making and detecting such detectably labeled antibodies or their functional derivatives are well known to those of ordinary skill in the art.
  • the detection of foci of such detectably labeled anti- bodies is indicative of a site of inflammation.
  • this examination for inflammation is accomplished by removing samples of tissue or blood and incubating such samples in the presence of detectably labeled antibodies.
  • this technique is accomplished in a non- invasive manner through the use of magnetic imaging, fluoro- graphy, etc.
  • a diagnostic test may be employed in monitoring organ transplant recipients for early signs of potential tissue rejection.
  • Such assays may also be conducted in efforts to determine a subject's clinical status in rheumatoid arthritis and other chronic inflammatory diseases.
  • antibodies, or their functional derivatives to detect or diagnose the presence and location of an inflammation in a mammalian subject suspected of having an inflammation by utilizing an assay for endothelial-derived IL- 8, comprising incubating a biological sample from said subject suspected of containing endothelial-derived IL-8 in the presence of a detectably labeled binding molecule (e.g., antibody) capable of identifying endothelial-derived IL-8, and detecting said binding molecule which is bound in a sample.
  • a detectably labeled binding molecule e.g., antibody
  • a biological sample may be treated with nitrocellulose, or other solid support which is capable of immobilizing cells, cell particles or soluble protein.
  • the support may then be washed with suitable buffers followed by treatment with the detectably labeled endothelial-derived IL-8-specific antibody.
  • the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on the antibody may then be detected by conventional means.
  • the process comprises: (a) contacting a sample suspected containing endothelial-derived IL-8 with a solid support to effect immobilization of endothelial-derived IL-8;
  • step (d) separating the solid phase support from the incubation mixture obtained in step (c);
  • This aspect of the invention relates to a method for detecting endothelial-derived IL-8 or fragment thereof in a sample comprising
  • endothelial-derived IL-8- specific antibody can be detectably labeled is by linking the same to an enzyme.
  • This enzyme when later exposed to its substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used to detectably label the endothelial-derived IL-8-specific antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
  • the endothelial-derived IL-8-specific antibody may also be labeled with a radioactive isotope which can be determined by such means as the use of a gamma counter or a scintillation counter or by audioradiography.
  • Isotopes which are particu- larly useful for the purpose of the present invention are: 3 H, 125 If 131 I ⁇ 35 S> 14 C , and 51 Cr .
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.
  • the endothelial-derived IL-8-specific antibody can also be detectably labeled using fluorescence emitting metals such as l- ⁇ Eu, or others of the lanthanide series. These metals can be attached to the endothelial-derived IL-8-specific antibody using such metal chelating groups as diethylenetriaminepenta- acetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepenta- acetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the endothelial-derived IL-8-specific antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged endothelial-derived IL-8-specific antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the endothel al-derived IL-8-specific antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Detection of the endothelial-derived IL-8-specific antibody may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorescent material.
  • the detection can be accomplished by colorimetric methods which employ a substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Endothelial-derived IL-8 is a soluble product of the activated endothelial cell and, as such, is involved in inflammatory processes.
  • the term "inflammation” is meant to include reactions of both the specific and non-specific defense systems.
  • a specific defense system reaction is a specific immune system reaction response to an antigen. Examples of a specific defense system reaction include the antibody response to antigens such as rubella virus, and delayed-type hypersen- sitivity response mediated by T-cells (as seen, for example, in individuals who test "positive" in the Mantaux test).
  • a non-specific defense system reaction is an inflammatory response mediated by leukocytes incapable of immunological memory. Such cells include granulocytes, macrophages, neutrophils, etc. Examples of a non-specific defense system reaction include the immediate swelling at the site of a bee sting, the reddening and cellular infiltrate induced at the site of a burn, and the collection of PMN leukocytes at sites of bacterial infection (e.g., pulmonary infiltrates in bacterial pneumonias, pus formation in abscesses).
  • the present invention will be easily adapted to the diagnosis, monitoring, and treatment of inflammatory disease processes such as rheumatoid arthritis, acute and chronic inflammation, post-ischemic (reperfusion) leukocyte-mediated tissue damage, acute leukocyte-mediated lung injury (e.g., Adult Respiratory Distress Syndrome), and other tissue- or organ-specific forms of acute inflammation (e.g., glomerulonephritis).
  • inflammatory disease processes such as rheumatoid arthritis, acute and chronic inflammation, post-ischemic (reperfusion) leukocyte-mediated tissue damage, acute leukocyte-mediated lung injury (e.g., Adult Respiratory Distress Syndrome), and other tissue- or organ-specific forms of acute inflammation (e.g., glomerulonephritis).
  • endothelial-derived IL-8 may be obtained by providing to a patient the entire endothelial- derived IL-8 molecules, or any therapeutically active peptide fragments thereof.
  • endothelial-derived IL-8 may be augmented through the use of endothelial-derived IL-8 mutants or variants possessing additional amino acid residues added to enhance its coupling to a carrier or to enhance the activity of endothelial-derived IL- 8.
  • the scope of the present invention is further intended to include mutant forms of endothelial-derived IL-8 (including endothelial-derived IL-8 molecules which lack certain amino acid residues, or which contain altered amino acid residues, so long as such mutant endothelial-derived IL-8 molecules exhibit the capacity to affect cellular adhesion).
  • endothelial-derived IL-8 polypeptides of the present invention and functional derivatives can be formulated accord- ing to known methods of preparing pharmaceutically useful compositions, whereby these materials or their functional derivatives are combined in a mixture with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their formulation including other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharma ⁇ ceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton, PA (1980)).
  • compositions suitable for effective administration will contain a therapeutically effective amount of endothelial-derived IL-8, or functional derivatives (an inflammation-reducing amount, protecting amount or leukocyte adhesion inhibiting amount), together with a suitable amount of carrier vehicle.
  • a preferred product of the invention is a sterile pharma ⁇ ceutical composition for therapeutic use containing endo ⁇ thelial-derived IL-8 or its functional derivatives, which is suitable for intravenous administration.
  • the product may be in lyophilized form to be reconstituted for use by addition of a suitable carrier, or diluent, or it may be in the form of an aqueous solution.
  • a sterile diluent which may contain materials generally recognized for approximating physiological conditions and/or as required by governmental regulation.
  • the sterile diluent may contain a buffering agent to obtain a physiologically acceptable pH, such as sodium chloride, saline, phosphate-buffered saline, and/or other substances which are physiologically acceptable and/or safe for use.
  • a physiologically acceptable pH such as sodium chloride, saline, phosphate-buffered saline, and/or other substances which are physiologically acceptable and/or safe for use.
  • the material for intravenous injection in humans should conform to regulations established by the Food and Drug Administration, which are available to those in the field.
  • the pharmaceutical composition may also be in the form of an aqueous solution containing many of the same substances as described above for the reconstitution of a lyophilized product.
  • the products of the invention may be incorporated into pharmaceutical preparations which may be used for therapeutic purposes.
  • pharmaceutical preparation is intended in a broader sense herein to include preparations containing a protein composition in accordance with this invention, used not only for therapeutic purposes but also for reagent or diagnostic purposes as known in the art, or for tissue culture.
  • the pharmaceutical preparation intended for therapeutic use should contain a "pharmaceutically accept- able” or “therapeutically effective amount” of endothelial- derived IL-8, i.e., that amount necessary for preventative or curative health measures. If the pharmaceutical preparation is to be employed as a reagent or diagnostic, then it should contain reagent or diagnostic amounts of endothelial-derived IL-8.
  • one unit of endothelial-derived IL-8 activity will produce 50% inhibition of PMN adhesion at a PMN concentration of 2 x 10 5 cells/ml in a standardized in vitro assay system.
  • a ten-fold higher concentration of endothelial- derived IL-8 (10 U/ml) produces a maximal inhibition of 85-90%.
  • the therapeutic and/or diagnostic dosage administered jjn vivo will be dependent upon the particular inflammatory condition involved and may be dependent upon the age, weight, height, sex, general medical condition, and kind of concurrent treatment, if any, of the mammalian subject. In general, it is desirable to provide the recipient with a dosage which is sufficient to obtain an effective concentration of greater than about 10 U/ml of endothelial IL-8 activity, although a lower or higher dosage may be administered.
  • the specific activity of a given preparation of endothelial-derived IL-8, or derivatives thereof can be determined by an in vitro bioassay, as set forth below in the Experimental Section. In this manner, the dosage for any preparation of endothelial-derived IL-8, or derivatives, may be determined.
  • Methods useful for administering the molecules of the present invention include topical, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, intrapleural , or intraocular.
  • the administration may be by continuous infusion, or by single or multiple boluses.
  • the effective molecule useful in the methods of the present invention may be employed in such forms as, for example, sterile suspensions for injection or encapsulated for targeting to specific tissue sites with antibodies directed to inflammation-related cell surface structures such as ELAM-1 (Bevilacqua et al., PNAS USA 84:9238-9242 (1987); Cotran et al., J. Exo. Med. 164:661-666 (1986)). Additional pharmaceutical methods may be employed to control the duration of action. Controlled release prepara ⁇ tions may be achieved through the use of polymers to complex or absorb endothelial-derived IL-8 or its functional derivatives.
  • the controlled delivery may be achieved by selecting appropri- ate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate) and the appro ⁇ priate concentration of macromolecules as well as the methods of incorporation, in order to control release.
  • appropri- ate macromolecules for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine, sulfate
  • Another possible method useful in controlling the duration of action by controlled release preparations is incorporation of the endothelial-derived IL-8 molecule or its functional derivatives into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid), or ethylene vinylacetate copolymers.
  • micro- capsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethyl- cellulose or gelatine-microcapsules and poly-(methylmethacy- late) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, micro- emulsions, nanoparticles, and nanocapsules), or in macroemul- sions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, micro- emulsions, nanoparticles, and nanocapsules
  • Human monocyte derived IL-1 (hmIL-1), human recombinant IL-1-J (rIL-lJ), and human recombinant granulocyte- monocyte colony stimulating factor (GM-CSF): Genzyme, Inc., Boston, MA; human recombinant IL-l- ⁇ (rlL-lct) expressed in J coli: Cistron Technology, Pine Brook, NJ; Gram-negative bacterial endotoxin (E.
  • coli 0111:B4 Difco Laboratories, Detroit, MI; affinity-purified human interferon gamma ( ⁇ -IFN): Interferon Sciences, Inc., New Brunswick, NJ; trypsin (Type XI), thrombin (from human plasma), N-formyl-methionyl-leucyl- phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), gelatin-agarose, and ammonium sulphate (Grade III): Sigma Chemical Co., St.
  • leukotriene B4 Biomol, Inc., Philadelphia, PA
  • concanavalin A-agarose Vector Laboratories, Burlingame, CA
  • heparin-sepharose CL-6B and pepsin-agarose Pharmacia, Uppsala, Sweden.
  • Heparinase from Flavobacterium heparinium was provided by Dr. Howard Bernstein (Massachusetts Institute of Technology, Cambridge, MA). Reco binant TNF (rTNF), rIL-2, and rIL-lj3 were provided by Biogen, Inc. (Cambridge, MA).
  • endothelial-derived IL-8 is sometimes referred to as LAI (leukocyte adhesion inhibitor).
  • HEC Human umbilical vein endothelial cells
  • HEC strains were plated (passage levels 2-5) on 15 mm Thermanox plastic coverslips (Miles Scientific, Naperville, IL), in microtiter wells, or 100 mm tissue-culture dishes (Corning Glass Works, Corning, NY) coated with either fibronectin (1 ⁇ g/c ⁇ , Meloy Laboratories, Springfield, VA) or 0.1% gelatin.
  • Each leukocyte type was radiolabeled with ⁇ Indium-oxine
  • IL-1-conditioned medium IL-1 CM
  • SHAM CM sham-conditioned medium
  • confluent HEC monolayers 100 mm dishes
  • IL-1 CM IL-1-conditioned medium
  • SHAM CM sham-conditioned medium
  • confluent HEC monolayers 100 mm dishes
  • IL-1 2.5-5 U/ml hmIL-1 or rIL-0
  • RPMI-1640 RPMI, M.A.
  • Bioproducts, Walkersville, MD which contained the following: (1) no additional protein; (2) a defined non-serum additive containing 5 ⁇ g/ml transferrin, 5 ⁇ g/ml insulin, and 5 ng/ml selenium (final concentration) (TIS; Collaborative Research, Cambridge, MA); (3) 1% (v/v) fetal calf serum (FCS, endotoxin ⁇ 0.027 ng/ml, Hyclone Laboratories Inc., Logan, UT); and (4) 10 mg/ml bovine serum albumin (BSA, Fraction V, Sigma Chemical Co.). After 4 h, all dishes were washed with 5 ml Hanks balanced salt solution (HBSS, M.A.
  • HBSS Hanks balanced salt solution
  • Bioproducts without Ca ++ or Mg ++ to remove the cytokine-containing medium and incubated for an additional 5 h in RPMI alone or supplemented with TIS, FCS, or BSA. Media were collected, centrifuged (400 x g, 10 min), and usually frozen at -80°C.
  • actinomycin D (5 ⁇ g/ml, Sigma Chemical Co.), acetylsalicylic acid (500 ⁇ M, Fisher Scientific Co., Medford, MA), or indomethacin (5 ⁇ M, Sigma Chemical Co.) were added to the endothelial cultures 30 min prior to the addition of rIL-1 and allowed to remain in the medium through ⁇ out the pretreatment phase.
  • LAI was treated enzymatically as follows: SHAM CM and IL- 1 CM collected in RPMI+TIS were filtered through an Amicon YM 30 membrane (nominal molecular weight cut-off 30,000; Amicon Corp., Danvers, MA) under N2 and treated with trypsin (1,000 U/ml, pH 7, 37 ⁇ C, 18 h), thrombin (2 U/ml, 37 ⁇ C, 2 h), heparinase (15 U/ml, 30 ⁇ C, 2 h), or pepsin-linked agarose (4,500 U/ml, pH 4, 18 h, 37"C).
  • Enzyme treatments were stopped by boiling (15 min, 100 * C) or, in the case of pepsin-linked agarose, by centrifugation in a Beckman Microfuge with removal of the agarose beads. Trypsin and thrombin retained full activity in both SHAM CM and IL-1 CM as determined by the hydrolysis of chromagenic substrate BAEE (N- ⁇ -benzoyl-L- arginine ethyl ester; Sigma Chemical Co.) and plasma clotting activity, respectively.
  • BAEE N- ⁇ -benzoyl-L- arginine ethyl ester
  • LAI activity was assayed in a standard radiometric monolayer adhesion assay (see Bevilacqua et al ., J. Clin. Invest. 76:2003-2011 (1985)).
  • target monolayers consisting of confluent HEC monolayers, grown on Thermanox covers!ips or in microtiter wells, were pretreated in RPMI+FCS with and without rIL-10 (2.5-5 U/ml, 4 h).
  • Indium-labeled leukocyte suspensions (2 x 10 7 cells/ml) were diluted directly in IL-1 CM, SHAM CM, or fresh RPMI+FCS or TIS
  • the number of adherent In-labeled leukocytes was determined from the monola er-bound radioactivity and the specific activity (cpm/cell) of the leukocyte preparation using a Gamma 5500 counter (Beckman Instruments, Inc., Fullerton, CA). Percent inhibition was calculated as:
  • conditioned media were collected from endothelial cell cultures grown in
  • Acetylsalicylic acid (500 ⁇ M) treatment of the HEC did not alter LAI production (90-97% of LAI activity from untreated HEC, three experiments) under conditions which ablated consti- tutive and calcium ionophore-stimulated PGI production. Similar results were obtained with indomethacin pretreatment, indicating that arachidonic acid metabolism via the cyclooxy- genase pathway was not required for generation of LAI activity. However, treatment of HEC with actinomycin D (5 ⁇ g/ml) for 30 minutes prior to stimulation with rIL-1 (2.5-5 U/ml, 4 h), essentially blocked subsequent LAI production (0-10% of LAI activity from untreated HEC,- 6 experiments).
  • Confluent monolayers were treated for 4 h in RPMI+FCS with the various mediators indicated, washed, and incubated 5 h in RPMI+FCS.
  • Conditioned media were collected and assayed (without dilution) in a standard monolayer cell adhesion assay against washed IL-1-treated (5 U rIL-10, 4 h) HEC monolayers. Data are presented as mean ⁇ SEM.
  • Polymyxin B sulfate (20 ⁇ g/ml) treatment neutralized the stimulatory effect of LPS but did not reduce the effect of rlL- l ⁇ .
  • rIL-2 100 U/ml
  • HFN- ⁇ 200 U/ml
  • GM- CSF 200 U/ml
  • the LAI activity in IL-1 CM also inhibited PMN adhesion to HEC monolayers activated with TNF (200 U/ml, 4 h) and LPS (1 ⁇ g/ml, 4 h) as detailed in Table Two, below.
  • TNF- and LPS- conditioned medium also inhibited PMN adhesion to IL-1, TNF-, and LPS-activated endothelial monolayers.
  • Conditioned medium from IL-1-stimulated HEC produced comparable inhibition of PMN adhesion to HEC target monolayers activated with TNF (200 U/ml, 4 h; 54 ⁇ 4%, 5 experiments), LPS (1 ⁇ g/ml, 4 h; 56 ⁇ 7%, 3 experiments), or IL-1 (2.5 U/ml, 4 h; 68 ⁇ 4, 5 experiments) (see Table Two, below).
  • IL-1 CM did not inhibit PMN adhesion to plastic surfaces coated with fresh human serum (4 ⁇ 3% inhibition, 4 experiments), decomple- mented human serum (-10 ⁇ 4%, 3 experiments) or zymosan-activated human serum (-8 ⁇ 10%, 3 experiments).
  • CM Conditioned media
  • LAI activity was found to be IL-1 concentra ⁇ tion-dependent with threshold at 0.1 U/ml rll- ⁇ and maximal at 2.5-5 U/ml (Figure 3).
  • the appearance of LAI in the IL-1 CM was time-dependent with inhibitory activity, in media collected at 1 h intervals, detectable at 1-2 h (30 ⁇ 6%, n - 5), reaching a maximum at 5-6 h (65 ⁇ 2%) post-IL-1. After 9 hours, a decline in activity was observed (see Figure 9).
  • each cell type was selectively pretreated.
  • activated HEC monolayers (5 U/ml rIL-1, 4 h) were incubated for 30 min with IL-1 CM (followed by exchange with fresh medium (1.5 ml)), no inhibi ⁇ tion of PMN adhesion was observed (5 ⁇ 8% inhibition, P>0.05, three experiments).
  • LAI activity appears to differentially affect the adhesion of certain blood leukocytes to cytokine-activated and unactivated endothelium.
  • IL-1 CM consistently produced a marked inhibition of PMN adhesion to activated HEC monolayers (76 ⁇ 4% inhibition of cytokine-stimu- lated adhesion, P ⁇ .001, 5 experiments), but had no effect on PMN adhesion to unactivated monolayers.
  • monocyte adhesion to activated HEC monolayers was significantly inhibited (55 ⁇ 12% inhibition of cytokine-stimulated adhesion, P ⁇ 0.01, 5 experiments), while no effect on the relatively extensive adhesion to unactivated monolayers was observed.
  • LTB4 100 ng/ml PMA was assessed by quantitative measurement of changes in membrane potential and cytosolic free calcium concentrations. Changes in membrane potential were evaluated with the fluorescent cyanine probe USC3-5 (see Whitin et al . , J. Biol. Chem. 255:1874-1878 (1980); Lazzari et al .. J. Biol. Chem. 261:9710-9713 (1986)) using a SPEX Fluorolog II spectrofluori- meter with the cuvette compartment controlled at 37 * C.
  • Fluorescence measurements were made in a SPEX (Edison, NJ) Fluorolog II (Model CM-1) spectrofluorimeter equipped with a beam splitter, two excitation monochromaters, and a dual-mirror chopping mechanism in a specialized optical configuration to allow rapid alternating (30 Hz) excitation of Fura-2 at two wavelengths, 340 nm and 380 nm. Excitation band widths were set at 6.6 nm. The ratio of emitted fluorescence signals (505 nm, 7.2 nM) permits the calculation of intracellular Ca ++ concentration which is independent of cell number, dye loading, and dye bleaching.
  • Fluorescence signals were calibrated using 80 ⁇ M digitonin to permit equilibration of intracellular and extracellular Ca ++ (maximum) followed by the addition of 1 M TRIS, 300 mM EGTA, pH > 10.0 (minimum).
  • the basal and maximum increase in intracellular Ca ++ were calculated as described in Grynkiewicz et al .. J. Biol. Chem. 260:3440-3450 (1985).
  • Superoxide generation was determined according to Cohen et al .. J. Clin. Invest. 61:1081-1088 (1978).
  • Stimulus-response signaling and superoxide production in response to soluble inflammatory stimuli were also examined.
  • LAI was collected from IL-1 activated HEC monolayers in serum- free, transferrin/insulin/selenium-supplemented medium. LAI activity in these preparations was found to be stable to a variety of treatments, including boiling for 20 min, acidifica ⁇ tion (pH 2 or 4) for 18 h, freezing (-80 ⁇ C, up to three months), and storage at 4'C for up to 14 days without the addition of protease inhibitors. LAI activity was non-sedi- mentable (250,000 x g, 45 min), suggesting that it is soluble, rather than associated with a membrane fragment. LAI activity was not adsorbed by gelatin-agarose or concanavalin-A-agarose.
  • HPLC HPLC was performed with a Waters Associates (Milford, MA) Liquid Chromatrography system composed of two model 510 solvent pumps and a programmable Systems Controller gradient maker. Column effluent was monitored at 280 nm with a Waters model 481 absorbance detector. IL-1 CM and SHAM CM in RPMI+TIS were filtered on an Amicon YM 30 membrane, the effluent concentrated 20-40x on a YM 5 membrane (nominal molecular weight cut-off 5,000), dialyzed against distilled H2O (Spectrapor 7, 10,000 molecular weight cut-off, Spectrum Med. Ind., Los Angeles, CA), and lyophilized.
  • Lyophilized samples were reconstituted in 0.2 M acetic acid, 0.1 M triethylamine (pH 3.9) at a concentration of approximately 1 mg/ml, and applied (250 ⁇ l sample) to a Waters Protein-PAK 125 gel filtration column (7.8 mm x 30 cm). The column was run as an isochratic system at a flow rate of 1.8 ml/ in and 0.9 ml fractions were collected. Fractions were dialyzed 18 h (4'C) against phosphate buffered saline (136 mM NaCl, 0.3 mM KC1, 0.8 mM Na 2 HP0 , 0.1 mM KH 2 P0 4 , pH 7.4). Fractions were diluted into RPMI+FCS and assayed in a standard monolayer adhesion assay. Protein determinations were per ⁇ formed using Bio-Rad protein reagent (Bio-Rad Laboratories, Richmond, CA) .

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Abstract

Cette invention concerne des facteurs d'inhibition d'adhérence intercellulaire produits par des cellules endothéliales activées par la cykotine. Ces facteurs, connus sous le nom d'IL-8 endothéliale sont utiles au diagnostic et au traitement d'inflammations et à la protection des cellules endothéliales contre les dommages dus aux neutrophiles.
PCT/US1990/006580 1989-11-29 1990-11-09 Il-8 a derivation endotheliale WO1991008483A1 (fr)

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EP0543476A1 (fr) * 1991-11-21 1993-05-26 Medical Research Council Utilisation de l'interleukine-8
US5346686A (en) * 1992-10-05 1994-09-13 Mallinckrodt Medical, Inc. Labelled interleukin-8 and medical uses thereof
WO1997021813A1 (fr) * 1995-12-12 1997-06-19 Schering Aktiengesellschaft Procede de purification de l'interleukine-8 humaine recombinee
US5665346A (en) * 1991-12-04 1997-09-09 Research Corporation Technologies, Inc. Human interleukin-8 analogs
US6989435B2 (en) 1997-09-11 2006-01-24 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7067117B1 (en) 1997-09-11 2006-06-27 Cambridge University Technical Services, Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response

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US4797277A (en) * 1987-09-22 1989-01-10 Pharmacia Ab Method for reperfusion therapy

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Blood, Vol. 66, No. 1, July 1985, HARLAN et al., "The Role of Neutrophil Membrane Glycoprotein GP-150 in Neutrophil Adherence to Endothelium In Vitro", pp. 167-178. See entire Abstract. *
Blood, Vol. 68, 1986, BASFORD et al., "Endothelial Cells Produce A Soluble Inhibitor of Superoxide Production by Neutrophils", p. 79a, See entire Abstract, No. 196. *
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See also references of EP0502897A4 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993009796A1 (fr) * 1991-11-21 1993-05-27 Medical Research Council Utilisation d'interleukine-8
US5624670A (en) * 1991-11-21 1997-04-29 Medical Research Council Use of interleukin-8 for inducing cervical ripening
EP0543476A1 (fr) * 1991-11-21 1993-05-26 Medical Research Council Utilisation de l'interleukine-8
US5665346A (en) * 1991-12-04 1997-09-09 Research Corporation Technologies, Inc. Human interleukin-8 analogs
US5346686A (en) * 1992-10-05 1994-09-13 Mallinckrodt Medical, Inc. Labelled interleukin-8 and medical uses thereof
AU714059B2 (en) * 1995-12-12 1999-12-16 Schering Aktiengesellschaft Process for the purification of recombinant human interleukin-8
WO1997021813A1 (fr) * 1995-12-12 1997-06-19 Schering Aktiengesellschaft Procede de purification de l'interleukine-8 humaine recombinee
US6114510A (en) * 1995-12-12 2000-09-05 Schering Aktiengesellschaft Process for the purification of recombinant human interleukin-8
US6989435B2 (en) 1997-09-11 2006-01-24 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7067117B1 (en) 1997-09-11 2006-06-27 Cambridge University Technical Services, Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7700087B2 (en) 1997-09-11 2010-04-20 Cambridge Enterprise Limited Compounds and methods to inhibit or augment an inflammatory response
US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
US7989466B2 (en) 1999-03-17 2011-08-02 Cambridge Enterprise Limited Methods to inhibit or augment an inflammatory response
US8481558B2 (en) 1999-03-17 2013-07-09 Cambridge Enterprise Limited Compounds to inhibit or augment an inflammatory response

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