Soluble Forms of Vascular Adhesion Molecules, E-Selectin, ICAM-1, and VCAM-1: Pathological Significance ANDREW J. H. GEARINGPC IAN HEMINGWAY,a ROD PIGCYIT," JEREMY HUGHES," ANDREW J. REES," AND =EVEN J. CASHMAN6 aBritkh B~achnologyLimited Watktppm Road Gnvky, Oxf3mi OX4 SLY United fiqpivn 6Dcpamncnt f Medicine R y l l%qpdua& Medkl School Hammevmtith Horpiatl DucaneRoad Limb W12 ONN United Kiqpivn INTRODUCTION The emigration of circulating leukocytes into tissues is a characteristic of the inflammatory response. The extravasation of leukocytes and some tumor cells is controlled by the expression of cell sudice adhesion molecules on both the circulating cells and on the vascular endothelium.' We have produced monoclonal antibodies that bind to cytokine-activated human umbilical vein endothelial cells, and which can block the adhesion of leukocytes to the endothelium.2 These antibodies have been used to expression clone the adhesion molecules ICAM-1, VCAM-1 and E-Selectin (ELAM-1) (see TABLE1). Functional studies using blocking monoclonal antibodies and mammalian cells stably transkcted with these adhesins have revealed their binding specificities. ICAM-1 will bind cells expressing the 62 integrins, or CD43, including lymphocytes, granulocytes, and m ~ n o c y t e s . ~ ~VCAM-1 ." will bind cells expressing the B1 integrin VLA4 (bla4), including lymphocytes, eosinophils, basophils, and m0nocytes.~-12 E-Selectin will bind granulocytes, monocytes and a subset of memory T cells (CLA+).l3-15 The identity of the physiological hgand(s) for E-Selectin on these cells is unclear; however, hcosylation and sialylation are thought to be important for binding. 16117 We have used monoclonal antibodies to investigte the expression ofadhesion molecules on endothelium and other tissues, in a range of inflammatory diseases. Reports Author to whom correspondence should be addressed; Tel.: (0865)748747; FAX: (0865) 717598.
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TABLE1. Properties of the Vascular Adhesion Molecules, ICAM-1,VCAM-I, and E-Selectin ICAM-1
property
Structure
immunoglobulin superfamily 5 Ig domains
VCAM-1
E-Selectin
immunoglobulin superfamily 6 and 7 Ig domains
selectinlLECCAM family 6 CRP domains
Expression
Broad
endothelid cells dendritic cells
endothelid cells
Induction
IL-1, TNF, IFN-y, LPS
IL1, TNF, IL4, LPS
IL1, TNF, LPS
Ligand
@2intcgrins, CD43
a401
? s k x , sLea
of an alternatively spliced soluble form of P-Selectin (GMP140),'* which is related to E-Selectin, prompted us to construct ELISAs capable of detecting soluble forms of either ICAM-1, VCAM-1, or E-Selectin. These ELISAs have been used to demonstrate the existence of soluble adhesins in culture supernatants of cytokine-activated endothelial cells, and in normal and pathological human sera. Assays for soluble forms of the adhesins may be useful diagnostic or prognostic tools.
EXPRESSION OF ESELECTIN, ICAM AND VCAM IN V N O A number of studies have investigated the distribution of E-Selectin, ICAM-1, and VCAM-1 on healthy and diseased tissues. ICAM-1 shows a very broad distribution. It is basally expressed on vascular endothelium and shows marked upregulation on most tissues in acute and chronic inflammatory diseases.4J-22 VCAM-1 has a more restricted distribution than ICAM-1, being expressed on vascular endothelium and on dendritic cells.22.23 E-Selectin is only expressed on vascular endothelium, predominantly on post-capillary venules.22,24,25 The relative expression of ICAM-1, VCAM-1 and E-Selectin on sections of inflamed human tonsil is illustrated in FIGURES1, 2, and 3. Expression of adhesion molecules on vascular endothelium can be observed in the proximity of cells expressing inflammatory cytokines such as interleukin (IL-1) and tumor necrosis factor (TNF),stimuli which, in vim, are known to cause upregulation of adhesins.26 Other studies have shown correlation between the time of expression of particular adhesins and the type of leukocyte infiltration. These results support the hypothesis that inflammatory stimuli, such as cytokines, induce the expression of adhesion molecules on the vascular endothelium, which control the emigration of leukocytes to the inflammatory site. Recent evidence has also implicated adhesion molecules in the metastasis of some tumors. Melanoma cells have been shown to adhere to endothelium via VCAM-1, and colonic carcinoma cells via E - S e l e ~ t i n . ~ ~ A critical role fbr these adhesins in acute and chronic inflammation is indicated by the results obtained in a number of in PiPo models using adhesion blockers. Most studies reported to date have used monoclonal antibodies which bind either to ICAM-1 or its p2 integrin lignds. These have shown therapeutic effects in models of graft rejection, graft versus host disease, asthma, and local or systemic reperfhion injury.28 Clinical trials with anti-LFA-1 (anti-lymphocyte function-associated antigen 1)have given
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FIGURE 1. Indirect immunopemxidase stained section of human tonsil with anti-ICAM-1 monoclonal antibody. Staining of cells within the germinal center (GC)and a small vessel (V). encouraging results in bone marrow transplantation. Anti-E-Selectin antibodies have proved effective in models of immune complex-mediated lung injury and asthma.29 In most if not all of these cases the protective effects are thought to be mediated by prevention of leukocyte infiltration into the tissue.
RECOMBINANT SOLUBLE ADHESINS
A number of groups have reported the construction of recombinant soluble brms of ICAM-1, VCAM-1 and E S e l e ~ t i n .Most ~ ~ . ~of~ these have been constructed tiom the full length cDNA by insertion of a stop codon immediately external to the transmembrane region. Secretion of these soluble adhesins has only been reported from mammalian expression systems. The soluble adhesins are all capable, when bound to a plastic surface, of binding appropriate target cells in a specific fashion. However, they are relatively ineffective as adhesion blockers in solution when compared to monoclonal antibodies. This probably reflects the multiple array display of adhesins on planar surfaces in cell-cell binding, a process which would be &cult to disrupt with a monomeric soluble adhesin. One approach to make effective adhesion-blockers has been used by Genentech who have constructed fusion proteins of Selectins and immunoglobulin. Their standard construct consists of the terminal lectin, epidermal
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FIGURE 2. Indirect immunopemxidase stained section of human tonsil with anti-VCAM-1 monoclonal antibody. The whole germinal center is visible. VCAM-1 is present on the dendritic cells of the germinal center (GC)and in the mantle mne (MZ).
gmwth fictor and two complement binding protein repeat domains of the selectin fused to the CH2 and CH3 domains of I@. The fusion protein forms a dimeric structure which has been demonstrated to block adhesion in vitro and in pipo.32 Some p r o p erties of the adhesins are displayed by soluble monomers. These include blockade of rhinovirus binding to ICAM-1 by soluble ICAM-1, and even by truncated forms consisting of the two N-terminal domains of ICAM-1. Soluble ESelectin has been shown to stimulate the activation of Mac-1 on neutrophils.33
NATURAL SOLUBLE ADHESINS There is no published evidence for naturally occurring soluble splice-variants of VCAM-1 or E-Selectin, nor of soluble material being found in culture supernatants or bodily fluids. It has recently been shown that a soluble material immunologically related to ICAM-1 is present in normal human serum, and is elevated in serum from patients with melanoma.34.35 We have developed two-site ELISAs which can detect recombinant soluble forms of ICAM-1, VCAM-1 and ESelectin. Standard preparations of the adhesins have been aliquoted and stored at -7OOC to establish a unitage. The assays were also found to
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FIGURE 3. Indirect immunoperoxidase stained section of human tonsil with anti-E-Selectin monoclonal antibody. Only the endothelial cells of small venules are stained 0.
detect adhesins solubdized with detergent from endothelial cell membranes. We were also surprised to find that soluble forms of ICAM-1 and VCAM-1 were released by cytokine-stimulated endothelial cells. E-Selectin was also detected but at very low levels. Samples of human serum from healthy volunteers were also found to contain immunoreactive ICAM-1, VCAM-1, and E-Selectin. In a sample of 62 individuals, mean levels of ICAM-1 were 56.5 U/ml,VCAM-1 54.7 U/mland of ErSelectin 10.8 U/ml. We also examined serum samples from groups of patients with Diabetes mellitus, impaired renal function (IRF), on chronic ambulatory peritoneal dialysis (CAPD), hemodialysis, and hypertension. The results in TABLE 2 clearly show that the mean levels of soluble ICAM-1, V W - 1 , and E-Selectin are elevated above control levels in each of the patient groups, except those with hypertension. Patients with impaired renal function or on chronic ambulatory peritoneal dialysis consistently gave elevations of all three adhesins, hernodialysis patients had high levels of E-Selectin and VCAM-1, and diabetics had elevated E-Selectin and ICAM-1.
BIOLOGICAL SIGNIFICANCE OF SOLUBLE ADHESINS The appearance of soluble fbrms of adhesins in serum begs the question ‘Why are they there?” Expression of adhesins on the sudace of the endothelium or circulating
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TABLE 2. Levels of Soluble Adhesins in Human Serum Sample Normal Diabetes IRF CAPD Haemodialysis Hypertension
ICAM-1
VCAM-1
E-Selectin
56.5 94.5 (0.0002)9/31 106.4 (0.0003)8/26 141.2 (0.0001) 11/19 88.7 (0.0002) 3/20 69.5 (0.5304)2/10
54.7 69.6 (0.0393)4/31 134.6 (0.OOOl) 17/26 134.2 (0.OOOl) 14/19 124.5 (0.0001) 16/20 73.9 (0.1363) 3/10
10.8 23.4 (0.0001) 14/31 17.9 (0.0144) 9/26 19.7 (0.0091)6/19 20.1 (0.0048) 6/20 15.2 (0.0829) 2/10
Values quoted are: mean Unitslml (p value compared to control group), number of individuals with levels greater than 2 standard deviations above the mean value for controldtotal number of individuals in the group.
cells is tightly regulated, both temporally and spatially, in order to avoid uncontrolled cell emigration.22-25Release of these adhesins from cultured endothelium requires cytokine treatment to upregulate expression, however the mechanism of release is unclear (Pigott, unpublished results). There is no evidence for alternativelyspliced soluble variants of these adhesins, which would suggest proteolytic cleavage fiom the cell s u d c e as a likely mechanism. The finding of detectable ICAM-1, VCAM-1, and E-Selectin in normal serum is surprising given the relative lack of E-Selectin, VCAM-1 and even ICAM-1 on normal tissue sections, but may reflect minor inflammatory insults which the body suffers on a daily basis. Release of cell s u d c e adhesins may simply be a mechanism for breaking adhesive interactions between cells, or may provide a means for clearing the cell s u d c e of adhesins to control adhesivity. The soluble forms may themselves have b i o l o g d functions. Recombinant soluble adhesins can partially block adhesion of leukocytes to surface bound adhesins; soluble forms at locally high concentrations may therefore also act to damp down adhesive interactions. Binding of soluble adhesins to their taxget cells may also deliver an activating stimulus, recombinant soluble ESeleain has been shown to be a neutrophil chemoattractant, and to activate the integrin CR3.j3 A further intriguing possibility is that as soluble ICAM-1 has been shown to inhibit rhinovirus infection of cells, release of ICAM-1 from the upper respiratory tract could have an antiviral action. The elevated levels of soluble adhesins in serum from patients with inflammatory diseases may provide some useful diagnostic or prognostic information. The broad tissue distribution of ICAM-1 would only be expected to give general information on the overall level of inflammation; VCAM-1 is more resmcted to endothelium and dendritic cell populations, however, and may have more particular uses. E-Selectin has only been described on endothelial cells; levels of &selectin may therebre provide a circulating sumgate b r measurement of endothelial damage or activation. Our results from diabetic patients, who frequently develop vascular pathology, would suggest that measurement of ESelectin levels could have prognostic utility. Prospective studies are needed to establish this point. REFERENCES 1. SPRINGER,T. A. 1990. Adhesion receptors of the immune system. Nature 346:425-434. 2. PI^, R , L. A. NEEDHAM,R M. EDWARDS, C. A. WALKER& C. POWER. 1991. Stmctural and hnctional studies of the activation antigen ELAM-1 using a panel of monoclonal antibodies. J. Immunol. 147: 130-135.
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3. SIMMONS, D., M. W. MAKGOBA & B. SEED.1988. ICAM an adhesion ligand of LFA-1 is homologous to the neural cell adhesion molecule NCAM. Nature 331: 624-627. 4. DUSTIN,M. L., R RDTHLEIN, A. K. BHAN,C. A. DINARELLO & T. A. SPRINGER. 1986. Induction by IL1 and Interferon y: Tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J. Immunol. 137: 245-254. 5. MARLIN, S. D. & T. A. SPRINGER.1987. Pu&ed ICAM-1 is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 51: 813-819. 6. DIAMOND, M.S., D. E. STAUNTON, S. D. MARLIN& T. A. SPRINGER. 1991. Binding of the integrin Mac-1 to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. Cell 65: 961-971. 7. ELICES, M. J., L. OSBORN,Y. TAKADA,C. CROUSE,S. LUHOWSKY, M. E. HEMLER & R R ~ B B1990. . VCAM-1 on activated endothelium interacts with the leucocyte integrin VLA-4 at a site distinct from the VLA4ibronectin binding site. Cell 60:577-584. 8. &BORN, L., C. HESSLON,R TIZARD, C. VASSALO,S. LUHOWSKY, G. CHI-ROW & R LDBB.1989. Direct expression cloning of VCAM-1, a cytokine-inducibleendothelial protein that binds to lymphocytes. Cell 5 9 1203-1211. 9. WAISH,G. M., J-J. MERMOD, A. HARTNBLL, A. B. KAY&A. J. WARDLAW. 1991. Human eosinophil, but not neutrophil adherence to IGl-stimulated human umbilical vein endothelial cells is or401 (VLA4) dependent. J. Immunol. 146: 3419-3423. 10. RICE, G. E., J. M. MUNRO& M. P. BEVILACQUA. 1990. Inducible cell adhesion molecule 110 (INCAM-110) is an endothelial meptor hr lymphocytes. J. Exp. Med. 171:1369-1374. 11. HESSION,C., R TIZARD,C. VASSNO, S. B. SCHIFPER,D. GOPP,P. MOY,G. CHI-ROSSO, S. LUHOWSKY, R R LDBB&L.OSBORN. 1991. Cloningofan alternate hrm ofVCAM-1. J. Biol. Chem. 266: 6682-6685. 12. BCCHNER, B. S., F. W. LUSKINCAS, M. A. GIMBRONE, W. NEWMAN, S. A. STERBINSKY, C. P.DERSE-ANTHONY, D. KLUNK & R P. SCHLEIMER. 1991. Adhesion of human basophils, eosinophilsand neutrophils to IG1 activated human vascular endothelial cell adhesion molecules. J. Exp. Med. 173: 1553-1556. 13. PICKER, L. J., T. K. KISHIMOTO,C. W. SMITH,R A. WARNOCK & E. C. BUTCHER. 1991. ELAM-1 is an adhesion molecule for skin-homing T cells. Nature 349: 796-799. 14. SHIMIZU, Y., S. SHAW,N. GRABBR, T. V. GOPAL,K. J. HORGAN,G. A. VAN SEVENTER & W. NEWMAN. 1991. Activation-independentbinding of human memory T cells to adhesion molecule ELAM-1. Nature 349 799-802. 15. BEVILACQUA,M. P., S. STENGELIN, M. A. GIMBRONE & B. SEED.1989. ELAM-1: An inducible receptor fbr neutrophils related to complement regulatory proteins and lectins. Science 243: 1160-1165. 16. POLLEY, M. J., M. L. PHILLIPS,E. WAYNER, E. NUDBLMAN, A. K. SINGHAL,S. HAKOMORI & J. C. PAUWN. 1991. CD62 and ELAM-1 recognise the same carbohydrate ligand, sialyl-lewis x. Proc. Natl. Acad. Sci. USA.88: 6224-6228. 17. BRANDLEY,B. K., S. J. SWIEDLER & P. W. ROBBINS.1990. Carbohydrate ligands of the LEC cell adhesion molecules. Cell 63: 861-863. 18. JOHNSTON, G. I., R G. CQOK& R P. MCEVER.1989. Cloning of GMP140, a granule membrane protein of platelets and endothelium: Sequencesimilarity to proteins involved in cell adhesion and inflammation. Cell 56: 1033-1044. 19. ADAMS, D. H., S. G. HUBSCHEB, J. SHAW,R ROTHLEIN & J. M. NEUBERGER. 1989. ICAM-1 on liver allografts during rejection. Lancet 2: 1122-1125. 20. FAULL,R J. & G. R Rvss. 1989. lhbular expression of ICAM-1 during renal allograft rejection. Transplantation 48: 226-230. 21. SOBEL,R A., M. E. MITCHELL& G. FONDRBN. 1990. ICAM-1 in central immune reactions in the human CNS. Am. J. Pathol. 136: 1309-1316. 22. NORRIS,P., R N. POSTON,D. S. THOMAS,M. THORNHILL, J. HAWK& D. 0. HASKARD. 1991. The expression of ELAM-1, ICAM-1 and VCAM-1 in experimental cutaneous inflammation: A comparison of UVb erythema and delayed hypersensitivity. J. Invest. Dermatol. 9 6 763-770. 1991. Vascular and non23. RICE, G. E., J. M. MUNRO,C. C o w & M. P. BEVXLACQUA. vascular expression of INCAM-110. Am. J. Pathol. 138: 385-393.
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24. LEUNG,D. Y., J. S. POBER& R S. COTRAN.1991. Expression of ELAM-1 in elicited late phase allergic reactions. J. Clin. Invest. 87: 1805-1809. 25. MWNRO, J. M., J. S. POBER&R S. COTRAN. 1989. Tumour necrosis hctor and interferon y induce distinct patterns of endothelid activation and associated leucocyte accumulation in skin of PapM a n A . Am. J. Pathol. 135: 121-133. R P I m , A. STOPPACCW, F. MONARDO, S. UCCINI,D. 26. Rvco, L. P., D. POMPONI, BORASCHI,A. TAGLIABUE, A. SANTONI,E. DEJANA & C. D. BARONI. 1990. Cytohe production ( I L l a , I L l s and TNFa) and endothelid cell activation (ELAM-1 and HLADr) in reactive lymphadenitis, Hodgkins disease, and in non-Hodgkms lymphoma: An immunochemical study. Am. J. Pathol. 137: 1163-1171. 27. RICE,E. G. & M. P.BEVILACQUA. 1989. An inducible endothelial cell sumce glycoprotein mediates melanoma adhesion. Science 246: 1303-1306. P. REILLY,L. G. HAYNES & R RDTHLEIN. 1990. ICAM-1 28. WEGNER, C. D., R H. GUNDEL, in the pathogenesis of asthma. Science 247: 456-459. 29. MULLIGAN, M. S.,J. VARANI, M. K. DAME,C. L. LANE,C. W. SMITH,D. C. ANDERSON & P. A. WARD.1991. Role of ELAM-1 in neutrophil mediated lung injury in rats. J. Clin. Invest. 88: 1396-1406. 30. MARLIN,S. D., D. E. STAUNTON, T. A. SPRINGER,C. S T R A ~ WW. A , SOMMERGRUBER & V. J. MERLUZZI. 1990. A soluble form of ICAM-1 inhibits rhinovirus inkction. Nature 344: 70-72. 31. ~ B B R, R, G. CHI-ROW, D. R LEONE,M. D. ROSA, S. BxmR, B. M. NEWMAN, S. LUHOWSKY, C. D. BENJAMIN,I. G. DOUGLAS, S. E. Go=, C. HESSION & E. P. CHOW.1991. Expression and functional characterisation of a soluble form of ELAM-1. J. Immunol. 147: 124-129. 32. WATSON, S. R, C. FENNIE& L. A. LASKY.1991. Neutrophil influx into an inflammatory site inhibited by a soluble homing receptor chimaera. Nature 349: 164-167. 33. Lo, S. K., S. LEE,R A. RAMos, R LOBB,M. ROSA, G. CHI-ROSSO& S. D. WRIGHT. 1991. ELAM-1 stimulates the adhesive activity of leucocyte integrin CR3 (CDllbKD18, Mac-1, am82) on human neutrophils. J. Exp. Mcd. 173: 1493-1500. 34. SETH, R , F. D. RAYMOND & M. W. MAKGOBA. 1991. Cirrulating ICAM-1 isoforms: Diagnostic prospects for inflammatory and immune disorders. Lancet 338: 83-84. 35. HAWING,R , E. MAINOLPI,J-C. BYSTRYN, M. HENN,V. J. MERLUZZI & R ROTHLEIN. 1991. Serum levels of circulating ICAM-1 in human m a l p a n t melanoma. Cancer Rcs. 51: 5003-5005. 36. RDSENSTEIN,Y., J. K. PARK,W. C. HAHN, F. S. ROSEN, B. E. BIERW&S.J. BURAKOFF. 1991. CD43, a molecule defective in Wiskott-Aldrich syndrome, binds ICAM-1. Nature 354: 233-235.
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TABLE1. Properties of the Vascular Adhesion Molecules, ICAM-1,VCAM-I, and E-Selectin ICAM-1
property
Structure
immunoglobulin superfamily 5 Ig domains
VCAM-1
E-Selectin
immunoglobulin superfamily 6 and 7 Ig domains
selectinlLECCAM family 6 CRP domains
Expression
Broad
endothelid cells dendritic cells
endothelid cells
Induction
IL-1, TNF, IFN-y, LPS
IL1, TNF, IL4, LPS
IL1, TNF, LPS
Ligand
@2intcgrins, CD43
a401
? s k x , sLea
of an alternatively spliced soluble form of P-Selectin (GMP140),'* which is related to E-Selectin, prompted us to construct ELISAs capable of detecting soluble forms of either ICAM-1, VCAM-1, or E-Selectin. These ELISAs have been used to demonstrate the existence of soluble adhesins in culture supernatants of cytokine-activated endothelial cells, and in normal and pathological human sera. Assays for soluble forms of the adhesins may be useful diagnostic or prognostic tools.
EXPRESSION OF ESELECTIN, ICAM AND VCAM IN V N O A number of studies have investigated the distribution of E-Selectin, ICAM-1, and VCAM-1 on healthy and diseased tissues. ICAM-1 shows a very broad distribution. It is basally expressed on vascular endothelium and shows marked upregulation on most tissues in acute and chronic inflammatory diseases.4J-22 VCAM-1 has a more restricted distribution than ICAM-1, being expressed on vascular endothelium and on dendritic cells.22.23 E-Selectin is only expressed on vascular endothelium, predominantly on post-capillary venules.22,24,25 The relative expression of ICAM-1, VCAM-1 and E-Selectin on sections of inflamed human tonsil is illustrated in FIGURES1, 2, and 3. Expression of adhesion molecules on vascular endothelium can be observed in the proximity of cells expressing inflammatory cytokines such as interleukin (IL-1) and tumor necrosis factor (TNF),stimuli which, in vim, are known to cause upregulation of adhesins.26 Other studies have shown correlation between the time of expression of particular adhesins and the type of leukocyte infiltration. These results support the hypothesis that inflammatory stimuli, such as cytokines, induce the expression of adhesion molecules on the vascular endothelium, which control the emigration of leukocytes to the inflammatory site. Recent evidence has also implicated adhesion molecules in the metastasis of some tumors. Melanoma cells have been shown to adhere to endothelium via VCAM-1, and colonic carcinoma cells via E - S e l e ~ t i n . ~ ~ A critical role fbr these adhesins in acute and chronic inflammation is indicated by the results obtained in a number of in PiPo models using adhesion blockers. Most studies reported to date have used monoclonal antibodies which bind either to ICAM-1 or its p2 integrin lignds. These have shown therapeutic effects in models of graft rejection, graft versus host disease, asthma, and local or systemic reperfhion injury.28 Clinical trials with anti-LFA-1 (anti-lymphocyte function-associated antigen 1)have given
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FIGURE 1. Indirect immunopemxidase stained section of human tonsil with anti-ICAM-1 monoclonal antibody. Staining of cells within the germinal center (GC)and a small vessel (V). encouraging results in bone marrow transplantation. Anti-E-Selectin antibodies have proved effective in models of immune complex-mediated lung injury and asthma.29 In most if not all of these cases the protective effects are thought to be mediated by prevention of leukocyte infiltration into the tissue.
RECOMBINANT SOLUBLE ADHESINS
A number of groups have reported the construction of recombinant soluble brms of ICAM-1, VCAM-1 and E S e l e ~ t i n .Most ~ ~ . ~of~ these have been constructed tiom the full length cDNA by insertion of a stop codon immediately external to the transmembrane region. Secretion of these soluble adhesins has only been reported from mammalian expression systems. The soluble adhesins are all capable, when bound to a plastic surface, of binding appropriate target cells in a specific fashion. However, they are relatively ineffective as adhesion blockers in solution when compared to monoclonal antibodies. This probably reflects the multiple array display of adhesins on planar surfaces in cell-cell binding, a process which would be &cult to disrupt with a monomeric soluble adhesin. One approach to make effective adhesion-blockers has been used by Genentech who have constructed fusion proteins of Selectins and immunoglobulin. Their standard construct consists of the terminal lectin, epidermal
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FIGURE 2. Indirect immunopemxidase stained section of human tonsil with anti-VCAM-1 monoclonal antibody. The whole germinal center is visible. VCAM-1 is present on the dendritic cells of the germinal center (GC)and in the mantle mne (MZ).
gmwth fictor and two complement binding protein repeat domains of the selectin fused to the CH2 and CH3 domains of I@. The fusion protein forms a dimeric structure which has been demonstrated to block adhesion in vitro and in pipo.32 Some p r o p erties of the adhesins are displayed by soluble monomers. These include blockade of rhinovirus binding to ICAM-1 by soluble ICAM-1, and even by truncated forms consisting of the two N-terminal domains of ICAM-1. Soluble ESelectin has been shown to stimulate the activation of Mac-1 on neutrophils.33
NATURAL SOLUBLE ADHESINS There is no published evidence for naturally occurring soluble splice-variants of VCAM-1 or E-Selectin, nor of soluble material being found in culture supernatants or bodily fluids. It has recently been shown that a soluble material immunologically related to ICAM-1 is present in normal human serum, and is elevated in serum from patients with melanoma.34.35 We have developed two-site ELISAs which can detect recombinant soluble forms of ICAM-1, VCAM-1 and ESelectin. Standard preparations of the adhesins have been aliquoted and stored at -7OOC to establish a unitage. The assays were also found to
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ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 3. Indirect immunoperoxidase stained section of human tonsil with anti-E-Selectin monoclonal antibody. Only the endothelial cells of small venules are stained 0.
detect adhesins solubdized with detergent from endothelial cell membranes. We were also surprised to find that soluble forms of ICAM-1 and VCAM-1 were released by cytokine-stimulated endothelial cells. E-Selectin was also detected but at very low levels. Samples of human serum from healthy volunteers were also found to contain immunoreactive ICAM-1, VCAM-1, and E-Selectin. In a sample of 62 individuals, mean levels of ICAM-1 were 56.5 U/ml,VCAM-1 54.7 U/mland of ErSelectin 10.8 U/ml. We also examined serum samples from groups of patients with Diabetes mellitus, impaired renal function (IRF), on chronic ambulatory peritoneal dialysis (CAPD), hemodialysis, and hypertension. The results in TABLE 2 clearly show that the mean levels of soluble ICAM-1, V W - 1 , and E-Selectin are elevated above control levels in each of the patient groups, except those with hypertension. Patients with impaired renal function or on chronic ambulatory peritoneal dialysis consistently gave elevations of all three adhesins, hernodialysis patients had high levels of E-Selectin and VCAM-1, and diabetics had elevated E-Selectin and ICAM-1.
BIOLOGICAL SIGNIFICANCE OF SOLUBLE ADHESINS The appearance of soluble fbrms of adhesins in serum begs the question ‘Why are they there?” Expression of adhesins on the sudace of the endothelium or circulating
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TABLE 2. Levels of Soluble Adhesins in Human Serum Sample Normal Diabetes IRF CAPD Haemodialysis Hypertension
ICAM-1
VCAM-1
E-Selectin
56.5 94.5 (0.0002)9/31 106.4 (0.0003)8/26 141.2 (0.0001) 11/19 88.7 (0.0002) 3/20 69.5 (0.5304)2/10
54.7 69.6 (0.0393)4/31 134.6 (0.OOOl) 17/26 134.2 (0.OOOl) 14/19 124.5 (0.0001) 16/20 73.9 (0.1363) 3/10
10.8 23.4 (0.0001) 14/31 17.9 (0.0144) 9/26 19.7 (0.0091)6/19 20.1 (0.0048) 6/20 15.2 (0.0829) 2/10
Values quoted are: mean Unitslml (p value compared to control group), number of individuals with levels greater than 2 standard deviations above the mean value for controldtotal number of individuals in the group.
cells is tightly regulated, both temporally and spatially, in order to avoid uncontrolled cell emigration.22-25Release of these adhesins from cultured endothelium requires cytokine treatment to upregulate expression, however the mechanism of release is unclear (Pigott, unpublished results). There is no evidence for alternativelyspliced soluble variants of these adhesins, which would suggest proteolytic cleavage fiom the cell s u d c e as a likely mechanism. The finding of detectable ICAM-1, VCAM-1, and E-Selectin in normal serum is surprising given the relative lack of E-Selectin, VCAM-1 and even ICAM-1 on normal tissue sections, but may reflect minor inflammatory insults which the body suffers on a daily basis. Release of cell s u d c e adhesins may simply be a mechanism for breaking adhesive interactions between cells, or may provide a means for clearing the cell s u d c e of adhesins to control adhesivity. The soluble forms may themselves have b i o l o g d functions. Recombinant soluble adhesins can partially block adhesion of leukocytes to surface bound adhesins; soluble forms at locally high concentrations may therefore also act to damp down adhesive interactions. Binding of soluble adhesins to their taxget cells may also deliver an activating stimulus, recombinant soluble ESeleain has been shown to be a neutrophil chemoattractant, and to activate the integrin CR3.j3 A further intriguing possibility is that as soluble ICAM-1 has been shown to inhibit rhinovirus infection of cells, release of ICAM-1 from the upper respiratory tract could have an antiviral action. The elevated levels of soluble adhesins in serum from patients with inflammatory diseases may provide some useful diagnostic or prognostic information. The broad tissue distribution of ICAM-1 would only be expected to give general information on the overall level of inflammation; VCAM-1 is more resmcted to endothelium and dendritic cell populations, however, and may have more particular uses. E-Selectin has only been described on endothelial cells; levels of &selectin may therebre provide a circulating sumgate b r measurement of endothelial damage or activation. Our results from diabetic patients, who frequently develop vascular pathology, would suggest that measurement of ESelectin levels could have prognostic utility. Prospective studies are needed to establish this point. REFERENCES 1. SPRINGER,T. A. 1990. Adhesion receptors of the immune system. Nature 346:425-434. 2. PI^, R , L. A. NEEDHAM,R M. EDWARDS, C. A. WALKER& C. POWER. 1991. Stmctural and hnctional studies of the activation antigen ELAM-1 using a panel of monoclonal antibodies. J. Immunol. 147: 130-135.
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