WO1994029720A1

WO1994029720A1 - Leukocyte adhesion assay

Info

Publication number: WO1994029720A1
Application number: PCT/GB1994/001279
Authority: WO
Inventors: Robin Poston
Original assignee: Robin Poston
Priority date: 1993-06-15
Filing date: 1994-06-14
Publication date: 1994-12-22
Also published as: GB9312315D0; US5766838A; EP0704056A1; DE69428278D1; EP0704056B9; ATE205600T1; DE69428278T2; US6010845A; EP0704056B1; JPH09500722A

Abstract

The invention provides a method of assaying leukocyte binding to vascular tissue which comprises contacting a suspension of a monocyte-like cell line in a suitable medium with a human vascular tissue sample at a temperature of at least 10 °C, and quantitating the number of bound monocyte-like cells over a defined area of tissue sample. The assay allows agents which inhibit binding of monocytes to human vascular tissue to be identified and the invention also relates to the use of such agents in the therapy of atherosclerosis.

Description

LEUKOCYTE ADHESION ASSAY

The present invention relates to a leukocyte adhesion assay, more particularly a method of assaying leukocyte binding to vascular tissue.

Atherosclerosis is a disease of the arteries which results in approximately half of the total deaths in Western countries. The disease involves thickening of the inner part (intima) of the artery wall by infiltration of monocytes (a type of leukocyte normally present in the blood) . Following infiltration, the monocytes mature into large cells called macrophages . which is followed by the accumulation of cholesterol, first within the macrophages but later becoming more widespread. Smooth muscle cells also appear and proliferate, probably under the influence of growth factors released by the macrophages. All of these components contribute to the thickening of the artery wall reducing the passage of blood through the artery which may finally become blocked by a blood clot forming at the site.

In recent years much has been learned about the mechanisms by which monocytes and other types of leukocytes migrate from the blood into tissues suffering from inflammation. One such mechanism involves adhesion molecules present on the inner surface of the vessels at sites of inflammation. The inner surface of all blood vessels consists of thin delicate cells referred to as endothelial cells and these form protein adhesion molecules which are able to bind specifically to receptors on leukocytes. These receptors are themselves adhesion molecules and consist of either carbohydrates, proteins or sulphur compounds on the surface of the leukocyte. Leukocytes can also be bound by other cells and structures within tissues by a number of similar adhesion interactions.

Recent work has shown that certain adhesion molecules are present on the endothelial cells of the diseased parts of atherosclerotic arteries but not in the normal parts of these arteries (see Poston et a.1 , Am. J. Pathol., 140 (3) , 665-673 (1992)). One adhesion molecule (ICAM-1) also appears on smooth muscle cells within the thickened area but is not seen on these cells elsewhere. These observations suggest that atherosclerotic lesions may resemble foci of inflammation and that monocytes leave the blood under the influence of adhesion molecules entering and adding to the disease process.

Whatever the precise mechanism, it is clear that the adhesion of leukocytes, and particularly monocytes, to the arterial wall plays an important role in the development of atherosclerotic lesions. For this reason, there is a need for a method of assaying leukocyte binding to vascular tissue.

One method which has previously been used for studying the binding of leukocytes to tissue is the so-called Stamper- Woodruff assay. This assay was developed originally to study the mechanism of lymphocytes binding to lymph nodes (Stamper & Woodruff, J. Exp. Med. , 144 , 828-833 (1976)). A suspension of rat thoracic duct lymphocytes in RPMI medium was contacted at about 7°C with thin sections of rat or mouse lymph node tissue, bound cells were fixed and visualised and then identified under a light microscope.

The assay has subsequently been used in other applications and, for example, a modified version was used to investigate experimental autoimmune encephalomyelitis which is an inflammatory condition of the nervous system with similarities to multiple sclerosis (Yednock et al, Nature, 356 , 63-66 (1992)). A suspension of a human monocytic cell line (U937) in RPMI medium was contacted with thin sections of brain tissue from normal rats and rats suffering from experimental autoimmune encephalomyelitis, bound cells were fixed and visualised and then identified under a light microscope. The brain tissue was generally contacted with U937 cells on ice although there is reference to some experiments having been carried out at 25°C.

It has now been found that a version of the Stamper- Woodruff assay can be applied to provide a functional assay for leukocyte adhesion to vascular tissue, for example tissue derived from atherosclerotic lesions.

The present invention provides a method of assaying leukocyte binding to vascular tissue which comprises contacting a suspension of a monocyte-like cell line in a suitable medium with a human vascular tissue sample at a temperature of at least 10°C, and quantitating the number of bound cells over a defined area of tissue sample.

In principle, the method according to the invention can be applied to any vascular tissue although the method is particularly applicable to assaying the binding of monocytes to atherosclerotic human artery. Generally, the tissue will be prepared as a thin section by standard histological procedures for example forming a cryostat section. The thickness of the section is preferably up to about 30μm, more preferably up to about lOμm, most preferably about 5μm.

The section of tissue is mounted on a microscope slide. Preferably aminopropyl triethoxyεilane (A.PES) coated slides are used to improve adhesion of the sections, (see Maddox & Jenkins, J. Clin. Pathol. , 40 , 1256-1257 (1987)). Although a chemical fixation procedure can be applied if desired, the sections are preferably used in the assay without any previous chemical fixation procedure.

Any monocyte-like cell line can used in the method according to the invention. A monocyte-like cell line is a cell line which has adhesion properties similar to human monocytes so that it adheres to vascular tissue (for example arterial wall) in a similar manner to human monocytes. The adhesion properties of monocytes are, in turn, determined by the adhesion receptors on the surface of the cell.

The monocyte-like cell line is preferably a monoclonal cell line. One particularly preferred monocyte-like cell line is the U937 histiocytic lymphoma cell line according to Harris & Ralph, J. Leukocyte Biol., 12, 407-422 (1985) available to the public from ATCC number CRL 1593. The U937 cell line was first described by Sundstro and Nilsεon (Int. J. Cancer, 17. 565-577 (1976)). An alternative monocyte-like cell line is the THP-1 monocyte cell line available to the public from ATCC number TIB 202 (see Tsuchiya et al Int. J. Cancer, 2_6, 171 (1980)).

The above monoclonal monocyte like cell lines can be grown by standard methods in cell culture medium such as RPMI medium and will generally be used according to the invention in suspension in that or a similar medium. In the case of the U937 cell line, a preferred cell culture medium is RPMI medium containing 10% fetal calf serum and this medium can also be used for the assay but preferably containing lO M HEPES buffer. The monoclonal monocyte like cells are preferably activated in order to increase adhesion, for example by use of a phorbol ester. According to one embodiment: of the invention U937 cells can be activated by use of phorbol myristyl acetate, for example suspenεion in tissue culture medium containing 10 ng/ml phorbol myristyl acetate for 24-48 hours at 37°C.

Whilst monoclonal cell lines are preferred, normal human monocytes can also be used in the application of the method, according to the invention. Normal human onocyteε can be prepared from heparinised human blood by centrifuging on a Ficoll-Hypaque density gradient to isolate mononuclear leukocytes, followed by adhesion to plastic rissue culture flasks. Alternatively, monocytes can be isolated from blood in an elutriation apparatus. Furthermore, by use of monocytes derived from patients' blood, the asεay can also be employed to assess the adhesive properties of monocytes in patientε with atherosclerotic or other disease.

A suitable concentration of cellε for contacting with the vascular tissue sample is about 10⁶ to 10⁸ cellε per ml, preferably about 10⁷ cells per ml.

The εection of vaεcular tiεεue iε contacted with the monocyte-like cell suspenεion under conditionε and for a sufficient length of time which allow the cells to adhere to the vascular tissue where suitable adhesion molecules are present to bring about such adhesion. Preferably a section of vascular tissue on a microscope slide iε contacted with the cell εuεpenεion on a rotating table at a rotation εpeed of about 40 to 80 rpm, preferably about 60 rpm. The time of contact may be very short or may be up to about 1 hour or longer, preferably about 40 minutes.

As noted above, in the past the Stamper-Woodruff assay has generally been carried out at low temperature, i.e. around 4 to 7°C. Surprisingly, it has been found that the assay according to the present invention is inoperative at 4°C in that no binding takeε place at thiε temperature. Accordingly, the aεεay εhould be carried out at a temperature of at leaεt 10°C, for example about 15 to 45°C, preferably about 20 to 40^CC, more preferably about 37°C.

After contact with the suspension of monocyte-like cells, the vaεcular tiεsue sample may be treated by standard hiεtological techniques to bring the sample into a condition where the number of bound monocyte-like cellε may be quantitated. Thuε the sample may be washed for example using phosphate buffered saline at low temperature, for example 4°C, and then fixed for example using parafor aldehyde or glutaraldehyde. Finally, the samples are stained in a suitable manner, for example using haemotoxylin, and the binding of monocyte- like cells can then be observed under a microscope. Recognition can be facilitated by performing immuno- histochemical εtaining for monocyte associated antigens, e.g. CD15. Under the microscope bound cells can be distinguished from cells in the εection itεelf by being rounded and lying in a different focal plane. The number of bound monocyte-like cellε can be quantitated by counting the number of cells in a defined area of sample either manually or automatically. Automatic counting can be effected by the use of image analysis equipment programmed to recognise objects of the size and circularity of the adherent cells, for example the image analyser produced by Sight Systemε (Hove, U.K.).

The aεεay according to the present invention is valuable for a number of purposes. Thus the involvement of adhesion molecules in the entry of monocytes into atherosclerotic foci may be of profound significance as it appears to be a vital mechanism in this initial event in the generation of the disease. There is reason to suppose that once monocyte entry has started, it may be self-perpetuating, as factorε produced by the monocyte-derived macrophageε may elicit further formation of endothelial adheεion moleculeε. Aε well aε providing a meanε of inveεtigating the mechaniεm of monocyte entry, the method according to the invention has important uses in the development of therapeutic approaches to the treatment of atheroscleroεiε.

The aεεay can be uεed for the inveεtigation of inhibitorε of the adheεion proceeε. For example EDTA or EGTA remove divalent cations and thereby inhibit the interaction of monocytes with the endotheliu . The interaction may also be inhibited at least in part by peptides containing the RGD sequence, a property which is aεεociated with adhesion through the leukocyte adhesion moleculeε referred to as integrins. Adhesion may also be inhibited by certain carbohydrate binding proteins such aε the lectin, wheat germ agglutinin, and by a number of antibodies, in particular monoclonal antibodies, directed against various adhesion molecules. This suggests that carbohydrates may alεo have potential as binding inhibitors.

Agents that can inhibit the procesε of adhesion of monocytes to the inti a are candidateε for use as therapeutic agents against human atheroscleroεiε. The method according to the present invention can be used for screening posεible inhibitory agentε with the potential for the development of therapeutic approacheε againεt human atherosclerosis. Assay for adhesion of monocyte-like cells in the presence and the absence of a potential inhibitory agent will identify those agents which inhibit the adhesion procesε. Alternatively the cell suspension or the tissue section can be preincubated with a potential inhibitory agent.

Thus, according to another aεpect, the present invention provides a method of identifying agents which inhibit binding of monocyteε to human vaεcular tissue which comprises carrying out the assay defined above in the presence and in the absence of a potential inhibitory agent or with or without preincubation of the cell suεpenεion or the tissue with a potential inhibitory agent, and assesεing the effect of the agent on the extent of monocyte binding. The invention also provides agents for uεe in the therapy of atheroεcleroεis identified in this way.

A further application of the asεay according to the invention iε in conjunction with animal modelε for human atherosclerosis. Thus, use can be made of the assay to compare the effects of a range of agents on the binding of monocyte-like cells to human vaεcular tiεsue (particularly tissue from human atherosclerotic lesionε) with their effect on the binding to tiεεue (again particularly atheroεclerotic leεionε) from another species, for example the rabbit. Use of the assay according to the invention would indicate whether an agent found to be effective in inhibiting experimentally induced atherosclerosis in another specieε would be likely also to be effective in man.

In carrying out the asεay according to the invention as described above, binding of monocyteε iε observed to the intima as well aε the endotheliu . It iε likely that endothelial adheεion dependε on different adhesion reactions from intimal binding. In this event, the aεεay according to the invention can be made specific for endothelial binding, which may be critical to monocyte entry, by carrying out the assay in the presence of an inhibitor which selectively blocks intimal binding.

Resultε obtained using the assay according to the present invention have indicated for the first time the involvement of the monocyte cell surface molecule CD14 in atherosclerosis. In particular it has been found that an anti-CD14 antibody UCHM-1 (see below) gave strong inhibition of binding of U937 cellε to atherosclerotic lesions. This suggests a potential utility for agents which inhibit CD14 dependent monocyte adheεion in the treatment of atheroεcleroεiε. Suitable agentε include antibodieε against CD14 and other molecules, e.g. small chemical molecules, which inhibit CD14 dependent monocyte adhesion. It may be posεible to uεe rodent antibodieε againεt CD1 , for example UCHM-1 or OKM1, in a therapeutic context. UCHM-1 iε widely available commercially, for example from Sigma, and is deεcribed by Hogg et al , Immunology, 3., 753 (1984) . OKM1 iε also available to the public, for example from the ATCC, deposit number CRL 8026. Other anti-CD14 antibodies are available or can be derived using known methods. However, it is preferred to develop anti-CD14 antibodies which have lesε potential for eliciting a reaction from the human immune εyste using known techniques such as the production of chimeric or humanised (e.g. CDR- grafted) antibodieε. It iε more preferred in a therapeutic context to use small chemical molecules, for example moleculeε with a molecular weight up to about 1000.

According to another aεpect, the preεent invention provideε the uεe of an agent which inhibits CD14 dependent monocyte adhesion for the manufacture of a medicament for the treatment or prevention of atheroscleroεiε.

According to a further aspect, the present invention provides a method for the treatment or prevention of atheroscleroεiε which comprises administering to a patient an effective amount of an agent which inhibits CD14 dependent monocyte adhesion.

In some cases, it may be possible to administer an agent which inhibits CD14 dependent monocyte adhesion to a patient as the raw substance but the agent will generally be presented as a pharmaceutical composition. In this context a pharmaceutical composition comprises at least one agent which inhibits CD14 dependent monocyte adhesion (referred to herein as the "active ingredient") with one or more pharmaceutically acceptable carriers or diluents. The carrier(ε) or diluent(s) must be "acceptable" in the senεe of not having any deleteriouε effect on the patient and being compatible with other componentε of the formulation. The pharmaceutical composition may also contain other therapeutic ingredients having the same or a different therapeutic effect from the active ingredient, for example agents having an effect on the heart or circulation, such as anti-coagulants or anti- hypertensives.

In the case of small chemical moleculeε, the active ingredient may be formulated for administration by any suitable means provided that it is delivered to the circulation in such a manner that CD14 monocyte adheεion in the vicinity -of atherosclerotic plaque or at potential sites of atherosclerotic plaque formation can be inhibited. Examples of suitable forms of administration include oral, parenteral, rectal or intranasal, e.g. by inhalation.

A pharmaceutical composition for oral administration may take the form of, for example, tablets or capsules and may be prepared by processing the active ingredient in a conventional manner together with one or more pharmaceutically acceptable excipients. Tablets may be prepared by compreεεion or moulding in known manner and εuitable excipients include binding agents, fillers, lubricants, disintegrants and wetting agents. Tabletε or capsuleε may be coated in known manner, for example to provide εlow or controlled release of the active ingredient.

Liquid preparations for oral administration may take the form, for example, of solutionε, εyrupε or εuεpensions or may be presented as a dry product for re-constitution with water or another εuitable vehicle prior to use.

Compositions for parenteral administration include aqueous and non-aqueous sterile injection solutions which may be formulated in known manner. The formulations may be presented in unit-dose or multi-dose containers, for example, a pouleε or vialε, or may be stored in a lyophiliεed condition suitable for reconεtitution by addition of εterile liquid, for example water for injection.

Compositions for rectal administration may be presented in forms such as suppositorieε or retention enemas which may be formulated in known manner.

Compositions for intranasal administration may be formulated aε solutions for administration via a metered dose or unit device or as a powder including a εuitable carrier for administration using an appropriate delivery εyεtem.

Antibodieε which inhibit CD14 dependent monocyte adhesion will generally also be administered to patients in the form of a pharmaceutical composition which preferably includes, in addition to the antibody, a physiologically acceptable carrier or diluent, posεibly in admixture with one or more other agentε εuch aε other antibodies or drugs, such as antibiotics or agentε having an effect on the heart or circulation. Suitable carrierε include physiological saline and phoεphate buffered saline. Alternatively the antibody may be lyophilised and reconstituted before use by the addition of an aqueous buffered solution. Routes of administration of the antibody include intravenous, intramuscular, subcutaneous and intraperitoneal injection or delivery.

The method by which the agent which inhibits CD14 dependent adhesion is used in the treatment or prevention of atherosclerosis will depend on the nature of the agent. Small chemical molecules may be used prophylactically over long periods by subjects at risk of atherosclerosis. Antibodies carry more risk of an adverse reaction from the subject's immune system and are more suitable for short term therapy of patients at particular risk in εpecial circumstances, for example following heart transplantation. In all cases the precise dose to be administered will be at the discretion of the attendant physician but will depend on the nature of the agent and a number of other factors including the age and sex of the patient, the condition of the patient and the severity of the diεorder being treated.

The invention is described further with reference to the following Examples. In the examples, reference is made to the accompanying drawings in which:

FIG l showε U937 and control hybridoma cell binding to atheroεclerotic and normal tiεsue; FIG 2 showε the effect of RGD peptideε on U937 cell adhesion; and FIG 3 showε the effect of CD14 and control antibodies on U937 cell adhesion.

EXAMPLE 1 - ASSAY PROTOCOL

Samples of vascular tissue, such as atherosclerotic plague, of dimensions up to about 8mm, are snap frozen by immersion in isopentane cooled in liquid nitrogen. Sections of the tiεεue, 5μm thick are cut with a cryoεtat and mounted onto aminopropyltriethoxyεilane (APES) coated microεcope εlideε. A ring iε optionally drawn round the εection on the εlide uεing a felt-tip pen containing a hydrophobic compound (Dako Ltd., High ycombe, U.K.). The sections are used fresh in the assay, i.e. without chemical fixation and within one hour of cutting.

U937 cells (ATCC no. CRL 1593) are cultured in RPMI-1640 tissue culture medium with 10% fetal calf serum. They are harvested by centrifugation at 1000 rpm and resuspended in 10 ng/ml of phorbol myristyl acetate (PMA) for 24-48 hours at 37°C. This results in activation of the cells and increased adhesion. Studies can be done with or without the activation stage.

U937 cells are separated from the surface of the tissue culture flask by agitation, then centrifuged down, counted and adjusted to 10⁷ cells/ml in RPMI-1640 medium containing 10% fetal calf εerum and buffered with lOmM HEPES buffer.

200μl of the cell suspension iε placed on the εlide (within the marker ring if thiε iε uεed) and the εlide iε immediately placed on a rotating table turning at 60 rpm and maintained at 37°C. The section is incubated under these conditions for 40 minuteε. The εection iε then washed by gently dipping 5 times into a bath of ice cold phosphate buffered εaline. Adhered U937 cells are then fixed by incubating for 10 minutes in 4% paraformaldehyde on ice. The section is washed in water, εtained with Mayer's haematoxylin for 2 minutes, washed, dehydrated through alcohol and toluene baths and mounted in DPX mountant (BDH) in a routine histological manner (see for example Theory and Practice of Histological Techniques, Bancroft & Stevens, Churchill Livingstone, 1990) .

Binding of U937 cells can be observed under a microscope and bound cellε can be diεtinguished from cells in the εection itεelf by being rounded and lying in a different focal plane. Binding can be quantitated by counting cellε in multiple microscope high power fields over histologically defined areas of the specimen.

For assessment of the binding to atherosclerotic plaque, the number of cells binding to the intimal layer of plaques and control normal intimal areas of human artery wall can be counted. To quantitate endothelial adhesion, U937 cells contacting or overlying the endothelial cell layer can be assessed. If necessary, the layer can be recognised by the immunohistochemical staining of the section for von Willebrand factor after the fixation stage, (see Poston et al , Amer. J. Pathol., 140 , 665-673 (1992)).

EXAMPLE 2 - BINDING TO ATHEROSCLEROTIC PLAQUE

Sections of atheroεclerotic plaque were aεsesεed to define the nature and extent of leεionε and their subtype in the atherosclerotic spectrum. Lesions were subdivided into fatty streaks, fibro-fatty plaques, complex lesions and fibrouε leεionε. The method of aεεessment was by histology aided by immunohiεtochemiεtry with antibodies to macrophages, smooth muεcle cells, and endothelium. These procedures have been described previously (see Poston et al, Am. J. Pathol., 140 (3) , 665-673 (1992)). Staining of the endothelium with antibody to von-Willebrand factor is particularly useful in defining the integrity of the layer, and quantitation, as described below, was only performed in those areas that were de onstrably intact. Furthermore the same assessment defined areas that were histologically normal, and these were used as controls. The results described below were obtained with PMA stimulated U937 cells.

In the asεeεε ent of the binding of U937 cells to atherosclerotic plaque, the number of cellε binding was quantitated in two histological areas aε followε:

1) Endothelium:- A cell was counted as being adherent to the endothelium if the cell was over the endothelial layer or adherent to the luminal margin. The whole length of the intact endothelium over a plaque was counted, conεisting of 10-30 high power fields (HPF) , and results expressed either as cells per high power field (aε below) , or as cells per length of endothelium.

2) Intima:- The intimal area to be counted was defined as the layer extending from immediately beneath the endothelium to the internal elastic lamina. Ten random high power fields were chosen, and results expressed as mean value per HPF.

RESULTS

Thiε aεεay demonstrated a highly selective binding of U937 cellε to the endothelium and whole intimal layer of atherosclerotic plaque. Binding of U937 cells to the free border of the endothelium was often apparent. Much less was found in normal areas of artery. Some medial binding waε also seen in asεociation with the plaqueε. Levelε of binding varied conεiderably from one leεion to another, and were reproducible. Negligible binding occurred if the aεsay was performed at 4°C, compared to the usual 37°C.

In an aεεeεsment of 13 atherosclerotic arterieε (fibro- fatty plaqueε) and 6 normal controlε, 3.04 ± 2.51 cellε/HPF (mean ± SD) were bound to the endothelium of plagues, compared to 0.58 ± 0.42 over the endothelium of normal artery, p = 0.0075, Mann-Whitney U test.

In the intimal layer, 16.1 ± 10.2 cells/HPF were bound in the atheroεclerotic εpecimenε, compared to 1.3 ± 1.1 cellε/HPF in the normal arteries, p = 0.0025.

By contrast, a B lymphocyte/myeloma hybridoma cell line used as a control gave only 0.11 and 1.18 cellε/HPF bound in the atheroεclerotic endothelial and intimal regions respectively, and less in the normal areas. These resultε are εhown in Figure 1, in which the εhaded bars are the results of binding of U937 cells and the blocked bars are control (non-specific hybridoma) .

In the aεsessment of inhibitory εubεtanceε, endothelial and intimal binding behaved in a similar manner. EGTA and EDTA caused total inhibition of binding. This is compatible with the involvement of selectin or integrin adhesion molecules in the process, as both are known to be divalent cation dependent. Likewise wheat-germ agglutinin, which binds N-acetyl glucosamine, caused a marked decrease in binding. An involvement of sugar residueε iε therefore probable. The peptide GRGDS (Sigma) caused dose-related inhibition of adhesion (Figure 2) , again suggeεting the involvement of integrinε, which bind peptides containing the RGD sequence. In Figure 2 the circles show binding to the intima and the triangles binding to the endothelium. Antibodies to the &. and B₂ integrin chains (CD29 and CD18) gave inhibition, but an antibody to the endothelial adhesion molecule ICAM-1 had only a weak effect. An antibody UCHM-1 (20 μg/ml) to a monocyte cell surface molecule, CD14, gave strong inhibition of binding (Figure 3) , as compared to no blocking and to a control immunoglobulin (UPC10, 20 μg/ml) . In Figure 3 the blocked barε are no blocking, the cloεely εpaced shaded bars are control antibody and the wider spaced shaded bars are anti- CD14 antibody. UCHM-1 and UPC10 are both mouse immunoglobulins of the same class (IgG_2a) . The CD14 molecule has recently been found to mediate monocyte-endothelial adhesion (see Beekhuizen and Van Furth, J. Leukocyte Biol., 54, 363-378 (1993)). These experiments suggest that integrins, carbohydrateε, CD14, and poεεibly εelectins are involved in the adhesion reactionε identified.

Claims

CLAIMS :

1. A method of assaying leukocyte binding to vascular tissue which compriseε contacting a εuspension of a monocyte-like cell line in a εuitable medium with a human vaεcular tissue sample at a temperature of at leaεt 10°C, and quantitating the number of bound monocyte-like cellε over a defined area of tissue sample.

2. A method according to Claim 1 wherein the vascular tissue is derived from atherosclerotic artery.

3. A method as claimed in Claim 1 wherein the vascular tissue iε derived from atheroεclerotic human artery.

4. A method aε claimed in any of Claimε 1 to 3 wherein the monocyte-like cell line is contacted with the vascular tissue sample at a temperature of about 15 to 45°C.

5. A method according to any of Claims 1 to 4 wherein the vascular tissue is in the form of a cryostat εection.

6. A method according to any of Claims 1 to 5 wherein the monocyte-like cells are a monoclonal cell line.

7. A method according to any of Claims 1 to 5 wherein the monocyte-like cells are monocytes derived from the blood of a patient.

8. A method according to any of Claims 1 to 7 wherein the monocyte-like cellε are contacted with the vascular tissue sample in suspenεion in cell culture medium.

9. A method according to any of Claims 1 to 8 wherein the monocyte-like cells are contacted with the vascular tissue sample at a concentration in the medium of 10⁶ to 10⁸ cells per ml.

10. A method as claimed in Claim 9 wherein the concentration is about 10⁷ cells per ml.

11. A method according to any of Claims 1 to 10 wherein the vascular tissue sample is mounted on a microεcope slide.

12. A method as claimed in Claim 11 wherein the microεcope εlide is mounted on a rotating table at a rotation εpeed of about 40 to 8O rpm.

13. A method as claimed in Claim 12 wherein the rotation speed is about 60 rpm.

14. A method as claimed in any of Claims 1 to 13 wherein the contact time is up to 1 hour.

15. A method as claimed in Claim 14 wherein the contact time is about 40 minutes.

16. A method as claimed in any of Claims l to 15 wherein following contact with the vaεcular tissue sample the monocyte-like cells are fixed with paraformaldehyde or glutaraldehyde.

17. A method as claimed in any of Claimε 1 to 16 wherein the fixed sample is stained.

18. A method aε claimed in Claim 17 wherein the εample is stained with hae otoxylin.

19. A method as claimed in Claim 17 wherein the sample is stained by an immuno-histoche ical procedure.

20. A method as claimed in any of Claims 1 to 19 wherein the number of bound cells iε guantitated by manual counting under a microεcope.

21. A method according to any of Claims 1 to 20 for identifying agents which inhibit binding of monocytes to human vascular tissue which comprises carrying out the asεay in the preεence and in the abεence of a potential inhibitory agent, or with and without preincubation of the cell εuspension or the tiεεue with a potential inhibitory agent, and aεsessing the effect of the agent on the extent of monocyte binding.

22. An agent for uεe in the therapy of atheroεclerosis identified by a method according to Claim 21.

23. An agent according to Claim 22 which is a peptide, protein or carbohydrate.

24. An agent according to Claim 23 which is a peptide containing the RGD sequence.

25. An agent according to Claim 23 which is an antibody.

26. An agent according to Claim 23 which is an inhibitor of CD14 dependent monocyte adhesion.

27. The use of an agent which inhibits CD14 dependent monocyte adhesion for the manufacture of a medicament for the treatment or prevention of atherosclerosis.

28. Use according to claim 27 wherein the agent is a small chemical molecule.

29. Use according to claim 27 wherein the agent iε an anti- CD14 antibody.

30. A method for the treatment or prevention of atherosclerosis which compriεeε adminiεtering to a patient an effective amount of an agent which inhibitε CD14 dependent monocyte adhesion.

31. A method according to claim 30 wherein the agent is a small chemical molecule.

32. A method according to claim 30 wherein the agent is an anti-CD14 antibody.