Vascular Cell Adhesion Molecule-l and Eosinophil Adhesion to Cultured Human Umbilical Vein Endothelial Cells In Vitro u. Kyan-Aung, Dorian O. Haskard, and Tak H. Lee Departments of Allergy and Allied Respiratory Disorders and Rheumatology, United Medical and Dental Schools, Guy's Hospital, London, United Kingdom
Cultured human umbilical vein endothelial cell (EC) monolayers stimulated with 10 ng/ml tumor necrosis factor demonstrate a time-dependent increase in the expression of the vascular cell adhesion molecule-l (VCAM-l) with maintained maximal expression at 24 h following EC activation. A monoclonal antibody (mAb) directed against VCAM-l (1Gl1) significantly inhibited the adhesion of eosinophils, but not neutrophils, to EC, which had been activated by tumor necrosis factor-a for 24 h, but only when eosinophils had been pretreated with an mAb directed against the common (3 chain of the CD11/CDI8 complex. In the absence of pretreatment with anti-CDI8, mAb IG11 had no significant effect on eosinophil adhesion. These results suggest that eosinophils bind to VCAM-l. However, the functional capacity in this model of the eosinophil receptor for VCAM-l is likely to be minor compared with the activity of the CD11/CDI8 leukocyte adhesion molecules.
Preferential accumulation of eosinophils is a characteristic feature of allergic airway inflammation. Because eosinophil adherence to vascular endothelium is the first step in their emigration into tissues, changes in endothelial adhesiveness for eosinophils could be critical in the mechanism for selective recruitment. In vitro studies with cultured human endothelial cells (EC) have shown that certain cytokines and inflammatory mediators increase the adhesiveness of cultured EC for leukocytes. Interleukin (IL)-I, tumor necrosis factor-a (TNF) , and bacterial lipopolysaccharide (LPS) form a group of agents with similar effects in enhancing EC adhesiveness for eosinophils (1), neutrophils (2-4), monocytes (2), lymphocytes (5-8), and basophils (9). IL-4 and interferon-y (IFN-')') are more selective in that they increase EC adhesiveness for T cells (10-12) but not for neutrophils (11, 12) or eosinophils (13). All these molecules enhance EC adhesiveness by a mechanism that requires protein synthesis and is dependent on the expression of EC adhesion molecules: endothelial leukocyte adhesion molecules-l (ELAM1) (14, 15), intercellular adhesion molecules-l (lCAM-l) (Received in original form January 9, 1991 and in revised form April 9, 1991) Address correspondence to: Prof. Tak H. Lee, Department of Allergy and Allied Respiratory Disorders, UMDS Guy's Hospital, London SEI 9RT, United Kingdom. Abbreviations: endothelial cell(s), EC; endothelial leukocyte adhesion molecule-l, ECAM-I; enzyme-linked immunosorbent assay, ELISA; fluorescence-activated cell sorter, FACS; fetal calf serum, FCS; Hanks' buffered salt solution, HBSS; HBSS without calcium or magnesium, HBSS=; intercellular adhesion molecule-l, ICAM-I; interferon-v, IFN-'Y; interleukin, IL; lipopolysaccharide, LPS; monoclonal antibody, mAb; tumor necrosis factor-a, TFN-a; vascular cell adhesion molecule-l, VCAM-l. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 445-450, 1991
(16), and vascular cell adhesion molecule-l (VCAM-l) (17-19). ELAM-l expression is induced by IL-l, TNF, and LPS. Its expression is maximal within 4 to 6 h and returns to the basal level by 24 h (14). ELAM-1 mediates the adhesion of eosinophils (13) and neutrophils (14, 15) via the interaction with a receptor on granulocytes, which has recently been shown to contain a sialyl-fucosyl pentasaccharide sequence (20-22). ELAM-l has been found to be involved in neutrophil, eosinophil, and monocyte adhesion to EC. It probably plays little role in lymphocyte-EC interactions, although there is one recent report suggesting that lymphocytes may also adhere to this molecule (23). ICAM-l is constitutively expressed on EC and is upregulated to a plateau level within 24 h when stimulated with IL-l, TNF, LPS, or IFN-')' (24). ICAM-l expression is partially suppressed by IL-4 (25). ICAM-l has been shown to be involved in eosinophil (13, 26), lymphocyte (27), and neutrophil (13, 28) adhesion to EC via interaction with CDI1/CD18 heterodimers on leukocytes. VCAM-l expression is induced by IL-l, TNF, LPS, and IL-4 but not by IFN-')' (18, 25, 29) and, in contrast to ELAM-l, expression on the EC following TNF stimulation is maintained as long as this cytokine is in the medium (29). The VCAM-l receptor on lymphocytes has been identified as VLA-4 (30), which is not found on the surface of neutrophils (31). As part of a study investigating whether differences exist between neutrophils and eosinophils in mechanisms of adhesion to EC, we have recently shown that ICAM-l and ELAM-1 are used in a similar manner by the two cell types during adhesion to EC stimulated for 6 h (13). We have now extended this analysis to look at EC stimulated with TNF for 24 h, at which time point the contribution ofELAM-l would
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be expected to be considerably less. We have found that at this time point it is possible to demonstrate a selective involvement of VCAM-1 in eosinophil but not in neutrophil adhesion.
Materials and Methods Culture of Human Umbilical Vein EC EC were obtained from human umbilical veins by collagenase (Type II; Sigma Chemical Co., Poole, Dorset, UK) treatment, using a modification of a method of Jaffe and associates (5,32). The EC were cultured at 37° C in 5% CO 2 in RPMI 1640 with L-glutamine (GIBCO, Paisley, Renfrewshire, Scotland) supplemented with 15 % heatinactivated fetal calf serum (FCS) (Sera-Lab, Crawley, Down, UK), 10% heat-inactivated (56° C, 30 min) human serum from normal donors (basic medium), 10 U/ml preservative-free heparin (33) (Leo, Princess Risborough, Bucks, UK), 15 J1.g/ml EC growth factor (Sigma), and antibiotics (100 U/ml penicillin, 100 J1.g/ml streptomycin; GIBCO) (complete medium) in tissue culture flasks pretreated with 1% gelatin (Sigma). Confluent EC were detached by using 0.125 % trypsinEDTA (GIBCO) in Puck's saline A (GIBCO), and subcultured up to the fourth passage in complete medium by 1:3 split ratio. The cells were confirmed to be EC by immunofluorescent staining with the EC-specific monoclonal antibody (mAb) EN4 (Sanbio, Uden, the Netherlands) (34) and fluorescence-activated cell sorter (FACS) analysis. As determined by FACS, less than 1% of the cells at this passage were stained by the monocyte- and macrophage-specific mAb M02 (Coulter, Hialeah, FL) (35). Plating and Stimulation of EC Confluent fourth passage EC were removed from culture flasks with 0.125 % trypsin-EDTA in Puck's saline A and resuspended in basic medium at 0.2 x 1{)6/ml. Aliquots (0.2 ml) were then cultured overnight or longer in 1% gelatin-coated, flat-bottomed, 96-well microtiter plates (Costar, Cambridge, MA) to produce confluent monolayers. TNF with a specific activity of 3.2 x 107 U/mg (gift from the Chiron Corp., Emeryville, CA) was diluted in Hanks' balanced salt solution (HBSS) (GIBCO) containing 30 mM Hepes and 4 mg/ml ovalbumin (Sigma). Stimulation of EC was performed by addition to the cultures of22 J1.1 TNF solution at 10 times the final concentration. In some experiments, EC were fixed with 2 % paraformaldehyde (BDH Ltd., Poole, UK)/L-Iysine monohydrochloride (Sigma)/sodium meta-periodate (Sigma) (PLP fixative) as described (29) and kept at 4° C for later use. This procedure had been shown to preserve the antigenicity of many protein and carbohydrate determinants (36) and to retain the increased adhesiveness ofEC stimulated by IL-l (5), TNF (6), and LPS (7) for leukocytes. Preparation of Neutrophils Neutrophils were isolated from heparinized (25 U/ml) peripheral blood by venipuncture. Erythrocytes were sedimented for 45 min by mixing with 0.2 vol of 6% dextran (Dextran 110; Fisons plc, Loughborough, Leicestershire, UK). The white cell-rich supernatant was then layered over Ficoll/Hy-
paque (Lymphoprep; Nycomed, Oslo, Norway), and the neutrophils separated by density gradient centrifugation at 350 x g for 30 min as described by Boyum (37). Erythrocytes contaminating the neutrophil pellet were removed by incubating for 10 min on ice with lysing solution (10 mM potassium bicarbonate containing 0.155 M ammonium chloride and 0.1 mM EDTA) and washing with HBSS without calcium and magnesium (HBSS=) (GIBCO). The cells were then washed again before radiolabeling. The purity of cells was more than 95% as determined by Kimura stain (38), and viability was greater than 95 % by try pan blue exclusion. Preparation of Eosinophils Eosinophils were separated from the blood of mildly hypereosinophilic (eosinophils > 5 % of total white cell count) patients by the method of Vadas and associates (39). A total of 100 ml of blood was anticoagulated with 5 ml 0.2 M EDTA, and erythrocytes were sedimented with dextran. The white cell-rich plasma was washed twice by centrifugation at 200 x g for 10 min, at 20° C, in Eagle's minimal essential medium with Earle's salts, supplemented with 25 mM Hepes, 2% heat-inactivated FCS (GIBCO), 2 mM L-glutamine (GIBCO) , and 3 mg/ml deoxyribonuclease-l (Sigma) (washing buffer). Metrizamide (Sigma) solution was prepared in Tyrode's solution containing 1 mg/ml gelatin (BDH). From 30% stock solution, 18%,20%,22%,23%, and 24 % solutions were made. Aliquots (2 ml) were taken and carefully overlayed one after another in 15-ml conical Falcon tubes, with the 24 % lowest and the 18% highest. A 2-ml leukocyte suspension was then layered on top of the metrizamide. Eosinophils were then separated by centrifugation for 45 min at 1,200 x g (2,200 rpm) at 20° C. The bands 18 and 20 were discarded, and bands 22, 23, and 24 were pooled and washed with washing buffer. The purity of cells from each band was determined by Kimura stain (38) and eosinophil preparations of> 85 % purity were used in the study. Radiolabeling of Leukocytes and Leukocyte-EC Adhesion Assay Leukocytes were suspended in 150 to 300 J1.1 of the buffer solution in which they had been prepared and 75 to 150 J1.1 51Cr (1 mCi/ml) as sodium chromate (CJS l.P; Amersham International, Amersham, UK) added to make up a 2:1 suspension. They were then incubated at 37°C and 5% CO 2 for 1 h with intermittent shaking. The cells were then washed 4 times in HBSS= and resuspended in HBSS at 2 x 106/ml. Leukocyte-EC adhesion was assessed as previously described (13). After washing EC with warm HBSS, 100 J1.1 of mAb IG11 or mAb 1.4C3 (1:100 dilution) or RPMI 1640 containing 10% FCS were added to respective wells and incubated for 30 min at 37° C. Leukocytes were pretreated with assay buffer or anti-CDl8 mAb TSI/18 (10 J1.g/ml) for 15 min and transferred to each well in the continuous presence of mAb and coincubated for 30 min at 37° C and 5 % CO 2 to allow cells to settle. After the incubation period, the medium was aspirated and the monolayers were washed twice. The cells were then lysed by addition of 200 J1.1 1% Nonidet-P40 (BDH) for 10 min. One hundred-microliter aliquots were taken from each well and the radioactivity was counted in a gamma counter (LKB 1260 Multigamma). The
Kyan-Aung, Haskard, and Lee: VCAM-l and Eosinophil Adhesion
percentage of adhesion of leukocytes was calculated as follows : % adhesion =
cpm in 100 Itl of lysate x 100. cpm in 100 Itl of original leukocyte suspension
Enzyme-linked Immunosorbent Assay (ELISA) for Adhesion Molecules on EC Monolayers After stimulation with TNF, EC were fixed with 2 % paraformaldehydellysine/sodium periodate and kept at 4 0 C. A total of 50 ILl of the anti-ELAM-l, anti-ICAM-l, or anti-VCAM-l mAb solution was added to each well and allowed to react for 30 min at room temperature. The excess mAb was then aspirated and EC washed once with PBS with 0.1% bovine serum albumin (A7906; Sigma) . A total of 50 ILl F(ab')2 biotinylated anti-mouse IgG (Seratek Laboratories, Kidlington, Oxford, UK) or undigested biotinylated anti-mouse Fe fragment (Binding Site, Birmingham, UK) was then added into each well and allowed to bind with primary antibody for 30 min before washing with PBS/OJ % bovine serum albumin. High-molecular-weight complex of streptavidin-biotin horseradish peroxidase (Dako or Sera-Lab) that had been preincubated for 30 min on ice was added into each well (50 Ill/well) and left for 20 min. After three washes, the integrity of the EC monolayer was checked under the phase-contrast microscope. The ELISA was then developed by addition of substrate solution (200 Ill) containing 20 mg ortho-phenylenediamine (Sigma) in 20 ml 0.1 M citric acid (pH 3.5),20 ml 0.1 M disodium phosphate buffer (pH 9.6) (final pH of buffer = 5), and 1 ILl/ml 30% hydrogen peroxide; 2 M sulfuric acid was used to stop the reaction. The color change (as reflected in a change in optical density) was read on an ELISA plate reader (Multiskan MO; Titertek) at 492 nm using 690 nm as a reference. Monoclonal Antibodies mAb 1.4C3 and IGll are both ofIgOl isotype as shown by double immunogel diffusion against isotype-specific polyclonal antisera. mAb 1.4C3 was originally characterized as binding to a novel activation epitope on EC (29) and together with mAb IGll, has now been shown to bind to COS cells transfected with VCAM-l eDNA (40). mAb TSI/18 is a mouse IgGI mAb directed against the {3 chain (CDI8) of LFA-l (41, 42) and was obtained as a cell line (American Type Culture Collection, Rockville, MD). mAb TSlI18 (IgGl) was isolated from ascites fluid by passage over protein A-Sepharose (43) and concentrated to 1 mg/ml in a dialysis sac by extraction of water using polyethylene glycol (molecular weight, 35,000) (Fluka Chemie AG, Buchs, Switzerland) followed by removal of excess salt by dialysis against phosphate-buffered saline. Before use in the adhesion assay, mAb 1.4C3 and IGll were titrated by ELISA on TNFstimulated EC, and mAb TSl/18 was titrated on neutrophils by flow cytometry. mAb were then used in excess of the saturating concentration. Statistics The difference in leukocyte-EC adhesion in the absence of mAb and in the presence of mAb was compared by using the two-tailed Student's t test.
447
Results Stimulation of human EC monolayers with 10 ng/ml TNF produced a time-dependent change in the expression of ELAM-l, ICAM-I, and VCAM-l. Three experiments demonstrated similar results and one representative experiment is shown in Figure 1. ELAM -1 was maximally expressed on EC 6 h after stimulation with TNF and had returned to near basal levels by 24 h. In contrast, ICAM-I and VCAM-I were maintained at maximal levels at both time points (Figure I). Because VCAM-l expression was still maximal on EC at 24 h after stimulation with TNF, all experiments tested the inhibitory effect of mAb IGll on EC that had been stimulated with TNF for 24 h. The results are presented in Table 1. In the absence of pretreatment of leukocytes with antiCDI8, the adhesion of eosinophils to TNF activated EC at 24 h was 46.1%, 52.1 %, and 41.1% in the presence of medium alone, mAb 1.4C3, and mAb IGll, respectively. These values were not significantly different. Following pretreatment with anti-CDI8, the adhesion of eosinophils to TNFstimulated EC at 24 h was 24.8% in the presence of mAb IGll. This was significantly less (P < 0.05) than the 40.6% adhesion of eosinophils to EC in the presence of control mAb 1.4C3. Under these conditions therefore, mAb IGII inhibited eosinophil adhesion by approximately 30% compared with mAb 1.4C3 of medium alone. mAb 1011 did not inhibit eosinophil adhesion to 6-h TNF-stimulated EC (data not shown). The level of neutrophil adhesion to 24-h TNFstimulated EC was generally lower than that of eosinophils. There was no significant inhibition of neutrophil adhesion to 24-h TNF-stimulated EC by mAb IGII in the absence of or in the presence of anti-CDl8 pretreatment.
Discussion In our previous report comparing the adhesion of neutrophils and eosinophils to cytokine-stimulated EC, we demonstrated that the mechanisms of adhesion to 6-h TNF-stimulated EC are very similar (13). Thus, both cell types used ELAM-l and ligand(s) for CDIl/CDIS integrins on the EC surface, and blocking both pathways of adhesion with mAb resulted
•
ELAM - 1
II
VCAM -l
o
EC ANT IGEN
ICAM- I
N (7\ ~
o o
o
6
24
TNF sl1mulal1on (hour s)
Figure 1. Expression of adhesion molecules on tumor necrosis factor-a (TNF)-stimulated endothelial cells (EC). EC monolayers were stimulated with TNF 10 ng/ml for 0, 6, or 24 h and then fixed with PLP fixative. EC antigen expression was measured by enzymelinked immunosorbent assay. Data points are mean OD ± SEM of triplicates in one representative experiment.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
TABLE 1
The inhibitory effect of anti-VCAM-l mAb on EOS and PMN adhesion to 24-h TNF-stimulated human EC monolayerss" Coincubation Experiment No.
EOS Without anti-C018 pretreatment 1 2 3 4
Mean With anti-C018 pretreatment 1 2 3 4 Mean PMN Without anti-CO18 pretreatment 1 2 3 4 5 Mean With anti-C018 pretreatment 1 2 3 4 5 Mean
Medium Control
± ± ± ± 46.1 ±
0.8 0.6 1.7 5.1
± ± ± ± 35.7 ±
0.8 3.3 0.5 4.4
8.0 2.0 1.9 0.7 1.8
47.7 36.3 47.5 30.8
± ± ± ±
7.4
40.6 ± 8.4
24.8 ± 7.4*
± 0.5 ± 1.0 ± 1.3 ± 1.6 ± 1.0
29.0±2.1 42.5 ± 2.4 32.4±4.1 16.3 ± 0.9 45.6 ± 1.7
21.8 41.6 33.3 14.6 47.4
33.2 ± 11.7
31.7 ± 13.6
39.4 30.3 44.3 28.7
24.2 ± 10.7 4.0 18.0 8.8 5.4 17.8
Anti-YCAM-1 mAb 1G11
± ± ± ± 52.1 ±
43.4 38.4 57.3 45.4
12.2 34.4 19.0 18.7 36.5
Anti-YCAM-1 rnAb 1.4C3 (control)
± ± ± ± ±
0.9 0.8 1.3 0.2 0.6
10.8 ± 6.7
58.4 47.0 59.5 43.3
11.7 26.0 7.2 7.8 24.1
8.2
± ± ± ± 41.1 ±
9.6
2.4 3.1 2.9 0.9
18.5 25.0 35.0 20.8
± ± ± ±
2.3 1.6 0.5 2.0
± ± ± ±
1.8 1.0 1.2 0.2 ± 0.5
15.4 ± 9.0
50.8 33.8 47.8 31.8
6.2 23.6 8.4 7.6 22.5
1.5 2.8 2.2 9.3
± 1.6 ± ± ± ±
2.1 3.2 1.3 0.2
± 1.4 ± 0.4 ± 1.3 ± 0.5 ± 0.9
13.7 ± 8.6
Definition ofabbreviations: YCAM-1 = vascular cell adhesion molecule-l ; mAb = monoclonal antibody;TNF = tumor necrosis factor-a; EOS = eosinophils; PMN = neutrophils; EC = endothelial cells. * Values are mean percent adhesion ± SD. t EC were treated with TNF 10 ng/rnl for 24 h and washed before incubating with mAb (1: 100 dilution). "Cr-labeled eosinophils or neutrophils that had been pretreated with buffer or anti-CD18 mAb TSl/18 were added to each well and co-cultured for 30 min in the continuous presence of mAb. Results are expressed as mean percent adhesion ± SD of triplicates. Inhibition of eosinophil adhesion to 24-h TNF-stimulated EC by anti-YCAM-1 rnAb 1Gl1 was significant when EOS were pretreated with anti-CD 18 rnAb. Mean percent adhesion ± SD of EOS to unstimulated EC monolayers was 15.2 ± 9.6 and 10.5 ± 1.9 in the absence and presence of anti-CD18 (data not shown). Mean percent adhesion ± SD adhesion of PMN to unstimulated EC monolayers was 8.4 ± 4.9 and 4.1 ± 1.5 in the absence and presence of anti-CD18 (data not shown). P < 0.05.
*
in almost complete inhibition of neutrophil and eosinophil binding. These studies were unable, however, to test a role for VCAM-l because mAb 1.4C3, the antibody used in the previous experiments, is not thought to be directed against a functional epitope on the molecule (40). In contrast to mAb 1.4C3, mAb IGll is fully effective in inhibiting VCAM-l-dependent adhesion of T cells (40). In this report, we have shown that mAb IGII is also able to inhibit eosinophil adhesion to 24-h TNF-stimulated EC monolayers, at which time point the ELAM-l expression on the EC surface has returned toward baseline low levels. Inhibition was only demonstrable, however, in the presence of antiCD18 mAb, suggesting that interactions between CDll/ CD18moiecuies and their ligands on EC can compensate for the absence of VCAM-1.
The ligand for VCAM-l on leukocytes is VLA-4 (CD49d/ CD29), which is a {31 integrin (30). It is expressed on thymocytes, monocytes, and activated peripheral blood lymphocytes (31). The recent finding that the eosinophil but not the neutrophil expresses VLA-4 (44, 45) suggests that the selective effect ofVCAM-l-i~duced adhesion for the eosinophil is mediated via VLA-4. The degree of inhibition by mAb IGll and anti-CDl8 of eosinophil adhesion to 24-h TNF-stimulated EC was only 30%, suggesting that molecules other than ICAM-l, ICAM-2, and VCAM-l might be involved. We cannot however eliminate the possibility that greater inhibition might have been obtained if Ftab'), fragments of mAb IGII had been used, thereby excluding a possible effect of the Fc part of the mAb in promoting nonspecific eosinophil-EC adhesion.
Kyan-Aung, Haskard, and Lee: VCAM-land Eosinophil Adhesion
In view of the evidence that increased adhesion of lymphocytes to IL-4-stimulated EC is largely attributable to VCAM-l induction by this cytokine (40, 46), it is perhaps surprising that we have not detected increased eosinophil adhesion to IL-4-stimulated Ee. Our experiments with eosinophils and lymphocytes were performed under similar experimental conditions, and it is possible that the strength of binding of lymphocytes to EC that express VCAM-l is greater than that of eosinophils. We have not therefore excluded the possibility that eosinophils might show enhanced binding to IL-4-stimulated EC when less stringent washing conditions are used to remove nonadherent cells. Whereas anti-VCAM-1 mAb led to a modest inhibition of eosinophil adhesion, no inhibition of neutrophil adhesion to 24-h TNF-stimulated EC was observed under the same conditions. These experiments suggest that the binding of eosinophils to VCAM-1 may contribute to the selective accumulation of eosinophils during allergic inflammation, especially later on during the response. Although we have not shown VCAM-1 expression at 6 h after the initiation of the late-phase cutaneous reaction to allergen in vivo, it remains to be determined whether VCAM-1 is enhanced at later time points. It is also possible that binding of eosinophils to VCAM-1 may explain how eosinophils and not neutrophils are found in chronic inflammatory tissues in patients with leukocyte adhesion deficiency, in which cells lack surface expression of CDll/CD18 molecules (47). Previous work has shown that infusions of ICAM-1 antibodies into monkeys reduces airways eosinophilia and hyperresponsiveness (26). It remains to be established whether VCAM-1 antibodies produce the same effects. In summary, we have demonstrated that eosinophils but not neutrophils may use VCAM-1 in adhesion to TNFstimulated EC. This may be an important mechanism for determining the more prolonged presence of eosinophils compared with neutrophils in ongoing allergic reactions. Acknowledgments: This work was supported by the Medical Research Council and by Schering-Plough, United Kingdom. Dr. Kyan-Aung is a fellow of the World Health Organization. Dr. Haskard was a Wellcome Trust Senior Research Fellow.
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ture of human endothelial cells derived from umbilical veins: identification by morphologic and immunologic criteria. J. Clin. Invest. 52:2745- 2756. Thornton, S. C., S. N. Mueller, andE. M. Levine. 1983. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science 222:623-625. Cui, Y. C., P. C. Tai, K. C. Gatter, D. Y. Mason, and C. 1. E Spry. 1983. A vascular endothelial antigen with restricted distribution in human foetal, adult and malignant tissues. Immunology 49:183-189. Todd, R. E, III, and S. E Schlossman. 1982. Analysis of antigenic determinants on human monocytes and macrophages. Blood 59:775-786. McLean, I. w., and P. K. Nakane. 1974. Periodate-lysine-paraformaldehyde fixative. A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22:1077-1083. Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from blood. Scand. J. Clin. Lab. Invest. 97(Suppl.):77-89. Kimura, I., Y. Moritani, and Y. Tanizaki. 1973. Basophils in bronchial asthma with reference to reagin-type allergy. Clin. Allergy 3:195-202. Vadas, M. A., 1. R. David, A. Butterworth, N. T. Pisani, and T. A. Siongok. 1979. A new method for the purification of human eosinophils and neutrophils, and a comparison of the ability of these cells to damage schistosomula of Schistosoma masoni. J. Immunol. 122:1228-1236. Thornhill, M. H., S. M. Wellicome, D. Mahiouz, 1. S. S. Lanchbury, U. Kyan-Aung, and D. O. Haskard. 1991. TNF combined with IL-4 or IFN--y selectively enhance endothelial cell adhesion for T cells: the contribution ofVCAM-l dependent and independent binding mechanisms. J. Immunol. 146:592-598.
41. Sanchez-Madrid, E, A. M. Krensky, C. E Ware et al. 1982. Three distinct antigens associated with human T lymphocyte mediated cytolysis: LFA-1, LFA-2 and LFA-3. Proc. Natl. Acad. Sci. USA 79:7489-7493. 42. Krensky, A. M., E Sanchez-Madrid, E. Robbins, 1. A. Nagy, T. A. Springer, and S. 1. Burakoff. 1983. The functional significance, distribution and structure of LFA-l, LFA-2, LFA-3: cell surface antigens associated with CTL target interactions. J. Immunol. 131:611-616. 43. Ey, P. L., S. 1. Prowse, and C. R. Jenkin. 1978. Isolation of pure IgGl, IgG2a and IgG2b immunoglobulins from mouse serum using protein A sepharose. Immunochemistry 15:429-436. 44. Walsh, G. M., J.-J. Merrnod, A. Hartnell, A. R Kay, and A. 1. Wardlaw. 1991. Human eosinophil but not neutrophil adherence to IL-1-stimulated human umbilical vascular endothelial cells is 0I4{3\ (very late antigen-4) dependent. J. Immunol. 146:3419-3423. 45. Bochner, B. S., E R. Luscinskas, M. A. Gimbrone, Jr. et al. 1991. Adhesion of human basophils, eosinophils and neutrophils to IL-1-activated human vascular endothelial cells: contributions of endothelial cell adhesion molecules. J. Exp. Med. 173:1553-1556. 46. Masinovsky, R, D. Urdal, and W. M. Gallatin. 1990. IL-4 acts synergistically with IL-1 to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-l, J. Immunol. 145:2886-2895. 47. Anderson, D. C., E C. Schmalsteig, M. 1. Finegold et al. 1985. The severe and moderate phenotypes of heritable Mac-l, LFA-1 deficiency: their quantitative definition and relation to leukocyte dysfunction and clinical features. J. Infect. Dis. 152:668-689.
Cultured human umbilical vein endothelial cell (EC) monolayers stimulated with 10 ng/ml tumor necrosis factor demonstrate a time-dependent increase in the expression of the vascular cell adhesion molecule-l (VCAM-l) with maintained maximal expression at 24 h following EC activation. A monoclonal antibody (mAb) directed against VCAM-l (1Gl1) significantly inhibited the adhesion of eosinophils, but not neutrophils, to EC, which had been activated by tumor necrosis factor-a for 24 h, but only when eosinophils had been pretreated with an mAb directed against the common (3 chain of the CD11/CDI8 complex. In the absence of pretreatment with anti-CDI8, mAb IG11 had no significant effect on eosinophil adhesion. These results suggest that eosinophils bind to VCAM-l. However, the functional capacity in this model of the eosinophil receptor for VCAM-l is likely to be minor compared with the activity of the CD11/CDI8 leukocyte adhesion molecules.
Preferential accumulation of eosinophils is a characteristic feature of allergic airway inflammation. Because eosinophil adherence to vascular endothelium is the first step in their emigration into tissues, changes in endothelial adhesiveness for eosinophils could be critical in the mechanism for selective recruitment. In vitro studies with cultured human endothelial cells (EC) have shown that certain cytokines and inflammatory mediators increase the adhesiveness of cultured EC for leukocytes. Interleukin (IL)-I, tumor necrosis factor-a (TNF) , and bacterial lipopolysaccharide (LPS) form a group of agents with similar effects in enhancing EC adhesiveness for eosinophils (1), neutrophils (2-4), monocytes (2), lymphocytes (5-8), and basophils (9). IL-4 and interferon-y (IFN-')') are more selective in that they increase EC adhesiveness for T cells (10-12) but not for neutrophils (11, 12) or eosinophils (13). All these molecules enhance EC adhesiveness by a mechanism that requires protein synthesis and is dependent on the expression of EC adhesion molecules: endothelial leukocyte adhesion molecules-l (ELAM1) (14, 15), intercellular adhesion molecules-l (lCAM-l) (Received in original form January 9, 1991 and in revised form April 9, 1991) Address correspondence to: Prof. Tak H. Lee, Department of Allergy and Allied Respiratory Disorders, UMDS Guy's Hospital, London SEI 9RT, United Kingdom. Abbreviations: endothelial cell(s), EC; endothelial leukocyte adhesion molecule-l, ECAM-I; enzyme-linked immunosorbent assay, ELISA; fluorescence-activated cell sorter, FACS; fetal calf serum, FCS; Hanks' buffered salt solution, HBSS; HBSS without calcium or magnesium, HBSS=; intercellular adhesion molecule-l, ICAM-I; interferon-v, IFN-'Y; interleukin, IL; lipopolysaccharide, LPS; monoclonal antibody, mAb; tumor necrosis factor-a, TFN-a; vascular cell adhesion molecule-l, VCAM-l. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 445-450, 1991
(16), and vascular cell adhesion molecule-l (VCAM-l) (17-19). ELAM-l expression is induced by IL-l, TNF, and LPS. Its expression is maximal within 4 to 6 h and returns to the basal level by 24 h (14). ELAM-1 mediates the adhesion of eosinophils (13) and neutrophils (14, 15) via the interaction with a receptor on granulocytes, which has recently been shown to contain a sialyl-fucosyl pentasaccharide sequence (20-22). ELAM-l has been found to be involved in neutrophil, eosinophil, and monocyte adhesion to EC. It probably plays little role in lymphocyte-EC interactions, although there is one recent report suggesting that lymphocytes may also adhere to this molecule (23). ICAM-l is constitutively expressed on EC and is upregulated to a plateau level within 24 h when stimulated with IL-l, TNF, LPS, or IFN-')' (24). ICAM-l expression is partially suppressed by IL-4 (25). ICAM-l has been shown to be involved in eosinophil (13, 26), lymphocyte (27), and neutrophil (13, 28) adhesion to EC via interaction with CDI1/CD18 heterodimers on leukocytes. VCAM-l expression is induced by IL-l, TNF, LPS, and IL-4 but not by IFN-')' (18, 25, 29) and, in contrast to ELAM-l, expression on the EC following TNF stimulation is maintained as long as this cytokine is in the medium (29). The VCAM-l receptor on lymphocytes has been identified as VLA-4 (30), which is not found on the surface of neutrophils (31). As part of a study investigating whether differences exist between neutrophils and eosinophils in mechanisms of adhesion to EC, we have recently shown that ICAM-l and ELAM-1 are used in a similar manner by the two cell types during adhesion to EC stimulated for 6 h (13). We have now extended this analysis to look at EC stimulated with TNF for 24 h, at which time point the contribution ofELAM-l would
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be expected to be considerably less. We have found that at this time point it is possible to demonstrate a selective involvement of VCAM-1 in eosinophil but not in neutrophil adhesion.
Materials and Methods Culture of Human Umbilical Vein EC EC were obtained from human umbilical veins by collagenase (Type II; Sigma Chemical Co., Poole, Dorset, UK) treatment, using a modification of a method of Jaffe and associates (5,32). The EC were cultured at 37° C in 5% CO 2 in RPMI 1640 with L-glutamine (GIBCO, Paisley, Renfrewshire, Scotland) supplemented with 15 % heatinactivated fetal calf serum (FCS) (Sera-Lab, Crawley, Down, UK), 10% heat-inactivated (56° C, 30 min) human serum from normal donors (basic medium), 10 U/ml preservative-free heparin (33) (Leo, Princess Risborough, Bucks, UK), 15 J1.g/ml EC growth factor (Sigma), and antibiotics (100 U/ml penicillin, 100 J1.g/ml streptomycin; GIBCO) (complete medium) in tissue culture flasks pretreated with 1% gelatin (Sigma). Confluent EC were detached by using 0.125 % trypsinEDTA (GIBCO) in Puck's saline A (GIBCO), and subcultured up to the fourth passage in complete medium by 1:3 split ratio. The cells were confirmed to be EC by immunofluorescent staining with the EC-specific monoclonal antibody (mAb) EN4 (Sanbio, Uden, the Netherlands) (34) and fluorescence-activated cell sorter (FACS) analysis. As determined by FACS, less than 1% of the cells at this passage were stained by the monocyte- and macrophage-specific mAb M02 (Coulter, Hialeah, FL) (35). Plating and Stimulation of EC Confluent fourth passage EC were removed from culture flasks with 0.125 % trypsin-EDTA in Puck's saline A and resuspended in basic medium at 0.2 x 1{)6/ml. Aliquots (0.2 ml) were then cultured overnight or longer in 1% gelatin-coated, flat-bottomed, 96-well microtiter plates (Costar, Cambridge, MA) to produce confluent monolayers. TNF with a specific activity of 3.2 x 107 U/mg (gift from the Chiron Corp., Emeryville, CA) was diluted in Hanks' balanced salt solution (HBSS) (GIBCO) containing 30 mM Hepes and 4 mg/ml ovalbumin (Sigma). Stimulation of EC was performed by addition to the cultures of22 J1.1 TNF solution at 10 times the final concentration. In some experiments, EC were fixed with 2 % paraformaldehyde (BDH Ltd., Poole, UK)/L-Iysine monohydrochloride (Sigma)/sodium meta-periodate (Sigma) (PLP fixative) as described (29) and kept at 4° C for later use. This procedure had been shown to preserve the antigenicity of many protein and carbohydrate determinants (36) and to retain the increased adhesiveness ofEC stimulated by IL-l (5), TNF (6), and LPS (7) for leukocytes. Preparation of Neutrophils Neutrophils were isolated from heparinized (25 U/ml) peripheral blood by venipuncture. Erythrocytes were sedimented for 45 min by mixing with 0.2 vol of 6% dextran (Dextran 110; Fisons plc, Loughborough, Leicestershire, UK). The white cell-rich supernatant was then layered over Ficoll/Hy-
paque (Lymphoprep; Nycomed, Oslo, Norway), and the neutrophils separated by density gradient centrifugation at 350 x g for 30 min as described by Boyum (37). Erythrocytes contaminating the neutrophil pellet were removed by incubating for 10 min on ice with lysing solution (10 mM potassium bicarbonate containing 0.155 M ammonium chloride and 0.1 mM EDTA) and washing with HBSS without calcium and magnesium (HBSS=) (GIBCO). The cells were then washed again before radiolabeling. The purity of cells was more than 95% as determined by Kimura stain (38), and viability was greater than 95 % by try pan blue exclusion. Preparation of Eosinophils Eosinophils were separated from the blood of mildly hypereosinophilic (eosinophils > 5 % of total white cell count) patients by the method of Vadas and associates (39). A total of 100 ml of blood was anticoagulated with 5 ml 0.2 M EDTA, and erythrocytes were sedimented with dextran. The white cell-rich plasma was washed twice by centrifugation at 200 x g for 10 min, at 20° C, in Eagle's minimal essential medium with Earle's salts, supplemented with 25 mM Hepes, 2% heat-inactivated FCS (GIBCO), 2 mM L-glutamine (GIBCO) , and 3 mg/ml deoxyribonuclease-l (Sigma) (washing buffer). Metrizamide (Sigma) solution was prepared in Tyrode's solution containing 1 mg/ml gelatin (BDH). From 30% stock solution, 18%,20%,22%,23%, and 24 % solutions were made. Aliquots (2 ml) were taken and carefully overlayed one after another in 15-ml conical Falcon tubes, with the 24 % lowest and the 18% highest. A 2-ml leukocyte suspension was then layered on top of the metrizamide. Eosinophils were then separated by centrifugation for 45 min at 1,200 x g (2,200 rpm) at 20° C. The bands 18 and 20 were discarded, and bands 22, 23, and 24 were pooled and washed with washing buffer. The purity of cells from each band was determined by Kimura stain (38) and eosinophil preparations of> 85 % purity were used in the study. Radiolabeling of Leukocytes and Leukocyte-EC Adhesion Assay Leukocytes were suspended in 150 to 300 J1.1 of the buffer solution in which they had been prepared and 75 to 150 J1.1 51Cr (1 mCi/ml) as sodium chromate (CJS l.P; Amersham International, Amersham, UK) added to make up a 2:1 suspension. They were then incubated at 37°C and 5% CO 2 for 1 h with intermittent shaking. The cells were then washed 4 times in HBSS= and resuspended in HBSS at 2 x 106/ml. Leukocyte-EC adhesion was assessed as previously described (13). After washing EC with warm HBSS, 100 J1.1 of mAb IG11 or mAb 1.4C3 (1:100 dilution) or RPMI 1640 containing 10% FCS were added to respective wells and incubated for 30 min at 37° C. Leukocytes were pretreated with assay buffer or anti-CDl8 mAb TSI/18 (10 J1.g/ml) for 15 min and transferred to each well in the continuous presence of mAb and coincubated for 30 min at 37° C and 5 % CO 2 to allow cells to settle. After the incubation period, the medium was aspirated and the monolayers were washed twice. The cells were then lysed by addition of 200 J1.1 1% Nonidet-P40 (BDH) for 10 min. One hundred-microliter aliquots were taken from each well and the radioactivity was counted in a gamma counter (LKB 1260 Multigamma). The
Kyan-Aung, Haskard, and Lee: VCAM-l and Eosinophil Adhesion
percentage of adhesion of leukocytes was calculated as follows : % adhesion =
cpm in 100 Itl of lysate x 100. cpm in 100 Itl of original leukocyte suspension
Enzyme-linked Immunosorbent Assay (ELISA) for Adhesion Molecules on EC Monolayers After stimulation with TNF, EC were fixed with 2 % paraformaldehydellysine/sodium periodate and kept at 4 0 C. A total of 50 ILl of the anti-ELAM-l, anti-ICAM-l, or anti-VCAM-l mAb solution was added to each well and allowed to react for 30 min at room temperature. The excess mAb was then aspirated and EC washed once with PBS with 0.1% bovine serum albumin (A7906; Sigma) . A total of 50 ILl F(ab')2 biotinylated anti-mouse IgG (Seratek Laboratories, Kidlington, Oxford, UK) or undigested biotinylated anti-mouse Fe fragment (Binding Site, Birmingham, UK) was then added into each well and allowed to bind with primary antibody for 30 min before washing with PBS/OJ % bovine serum albumin. High-molecular-weight complex of streptavidin-biotin horseradish peroxidase (Dako or Sera-Lab) that had been preincubated for 30 min on ice was added into each well (50 Ill/well) and left for 20 min. After three washes, the integrity of the EC monolayer was checked under the phase-contrast microscope. The ELISA was then developed by addition of substrate solution (200 Ill) containing 20 mg ortho-phenylenediamine (Sigma) in 20 ml 0.1 M citric acid (pH 3.5),20 ml 0.1 M disodium phosphate buffer (pH 9.6) (final pH of buffer = 5), and 1 ILl/ml 30% hydrogen peroxide; 2 M sulfuric acid was used to stop the reaction. The color change (as reflected in a change in optical density) was read on an ELISA plate reader (Multiskan MO; Titertek) at 492 nm using 690 nm as a reference. Monoclonal Antibodies mAb 1.4C3 and IGll are both ofIgOl isotype as shown by double immunogel diffusion against isotype-specific polyclonal antisera. mAb 1.4C3 was originally characterized as binding to a novel activation epitope on EC (29) and together with mAb IGll, has now been shown to bind to COS cells transfected with VCAM-l eDNA (40). mAb TSI/18 is a mouse IgGI mAb directed against the {3 chain (CDI8) of LFA-l (41, 42) and was obtained as a cell line (American Type Culture Collection, Rockville, MD). mAb TSlI18 (IgGl) was isolated from ascites fluid by passage over protein A-Sepharose (43) and concentrated to 1 mg/ml in a dialysis sac by extraction of water using polyethylene glycol (molecular weight, 35,000) (Fluka Chemie AG, Buchs, Switzerland) followed by removal of excess salt by dialysis against phosphate-buffered saline. Before use in the adhesion assay, mAb 1.4C3 and IGll were titrated by ELISA on TNFstimulated EC, and mAb TSl/18 was titrated on neutrophils by flow cytometry. mAb were then used in excess of the saturating concentration. Statistics The difference in leukocyte-EC adhesion in the absence of mAb and in the presence of mAb was compared by using the two-tailed Student's t test.
447
Results Stimulation of human EC monolayers with 10 ng/ml TNF produced a time-dependent change in the expression of ELAM-l, ICAM-I, and VCAM-l. Three experiments demonstrated similar results and one representative experiment is shown in Figure 1. ELAM -1 was maximally expressed on EC 6 h after stimulation with TNF and had returned to near basal levels by 24 h. In contrast, ICAM-I and VCAM-I were maintained at maximal levels at both time points (Figure I). Because VCAM-l expression was still maximal on EC at 24 h after stimulation with TNF, all experiments tested the inhibitory effect of mAb IGll on EC that had been stimulated with TNF for 24 h. The results are presented in Table 1. In the absence of pretreatment of leukocytes with antiCDI8, the adhesion of eosinophils to TNF activated EC at 24 h was 46.1%, 52.1 %, and 41.1% in the presence of medium alone, mAb 1.4C3, and mAb IGll, respectively. These values were not significantly different. Following pretreatment with anti-CDI8, the adhesion of eosinophils to TNFstimulated EC at 24 h was 24.8% in the presence of mAb IGll. This was significantly less (P < 0.05) than the 40.6% adhesion of eosinophils to EC in the presence of control mAb 1.4C3. Under these conditions therefore, mAb IGII inhibited eosinophil adhesion by approximately 30% compared with mAb 1.4C3 of medium alone. mAb 1011 did not inhibit eosinophil adhesion to 6-h TNF-stimulated EC (data not shown). The level of neutrophil adhesion to 24-h TNFstimulated EC was generally lower than that of eosinophils. There was no significant inhibition of neutrophil adhesion to 24-h TNF-stimulated EC by mAb IGII in the absence of or in the presence of anti-CDl8 pretreatment.
Discussion In our previous report comparing the adhesion of neutrophils and eosinophils to cytokine-stimulated EC, we demonstrated that the mechanisms of adhesion to 6-h TNF-stimulated EC are very similar (13). Thus, both cell types used ELAM-l and ligand(s) for CDIl/CDIS integrins on the EC surface, and blocking both pathways of adhesion with mAb resulted
•
ELAM - 1
II
VCAM -l
o
EC ANT IGEN
ICAM- I
N (7\ ~
o o
o
6
24
TNF sl1mulal1on (hour s)
Figure 1. Expression of adhesion molecules on tumor necrosis factor-a (TNF)-stimulated endothelial cells (EC). EC monolayers were stimulated with TNF 10 ng/ml for 0, 6, or 24 h and then fixed with PLP fixative. EC antigen expression was measured by enzymelinked immunosorbent assay. Data points are mean OD ± SEM of triplicates in one representative experiment.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991
TABLE 1
The inhibitory effect of anti-VCAM-l mAb on EOS and PMN adhesion to 24-h TNF-stimulated human EC monolayerss" Coincubation Experiment No.
EOS Without anti-C018 pretreatment 1 2 3 4
Mean With anti-C018 pretreatment 1 2 3 4 Mean PMN Without anti-CO18 pretreatment 1 2 3 4 5 Mean With anti-C018 pretreatment 1 2 3 4 5 Mean
Medium Control
± ± ± ± 46.1 ±
0.8 0.6 1.7 5.1
± ± ± ± 35.7 ±
0.8 3.3 0.5 4.4
8.0 2.0 1.9 0.7 1.8
47.7 36.3 47.5 30.8
± ± ± ±
7.4
40.6 ± 8.4
24.8 ± 7.4*
± 0.5 ± 1.0 ± 1.3 ± 1.6 ± 1.0
29.0±2.1 42.5 ± 2.4 32.4±4.1 16.3 ± 0.9 45.6 ± 1.7
21.8 41.6 33.3 14.6 47.4
33.2 ± 11.7
31.7 ± 13.6
39.4 30.3 44.3 28.7
24.2 ± 10.7 4.0 18.0 8.8 5.4 17.8
Anti-YCAM-1 mAb 1G11
± ± ± ± 52.1 ±
43.4 38.4 57.3 45.4
12.2 34.4 19.0 18.7 36.5
Anti-YCAM-1 rnAb 1.4C3 (control)
± ± ± ± ±
0.9 0.8 1.3 0.2 0.6
10.8 ± 6.7
58.4 47.0 59.5 43.3
11.7 26.0 7.2 7.8 24.1
8.2
± ± ± ± 41.1 ±
9.6
2.4 3.1 2.9 0.9
18.5 25.0 35.0 20.8
± ± ± ±
2.3 1.6 0.5 2.0
± ± ± ±
1.8 1.0 1.2 0.2 ± 0.5
15.4 ± 9.0
50.8 33.8 47.8 31.8
6.2 23.6 8.4 7.6 22.5
1.5 2.8 2.2 9.3
± 1.6 ± ± ± ±
2.1 3.2 1.3 0.2
± 1.4 ± 0.4 ± 1.3 ± 0.5 ± 0.9
13.7 ± 8.6
Definition ofabbreviations: YCAM-1 = vascular cell adhesion molecule-l ; mAb = monoclonal antibody;TNF = tumor necrosis factor-a; EOS = eosinophils; PMN = neutrophils; EC = endothelial cells. * Values are mean percent adhesion ± SD. t EC were treated with TNF 10 ng/rnl for 24 h and washed before incubating with mAb (1: 100 dilution). "Cr-labeled eosinophils or neutrophils that had been pretreated with buffer or anti-CD18 mAb TSl/18 were added to each well and co-cultured for 30 min in the continuous presence of mAb. Results are expressed as mean percent adhesion ± SD of triplicates. Inhibition of eosinophil adhesion to 24-h TNF-stimulated EC by anti-YCAM-1 rnAb 1Gl1 was significant when EOS were pretreated with anti-CD 18 rnAb. Mean percent adhesion ± SD of EOS to unstimulated EC monolayers was 15.2 ± 9.6 and 10.5 ± 1.9 in the absence and presence of anti-CD18 (data not shown). Mean percent adhesion ± SD adhesion of PMN to unstimulated EC monolayers was 8.4 ± 4.9 and 4.1 ± 1.5 in the absence and presence of anti-CD18 (data not shown). P < 0.05.
*
in almost complete inhibition of neutrophil and eosinophil binding. These studies were unable, however, to test a role for VCAM-l because mAb 1.4C3, the antibody used in the previous experiments, is not thought to be directed against a functional epitope on the molecule (40). In contrast to mAb 1.4C3, mAb IGll is fully effective in inhibiting VCAM-l-dependent adhesion of T cells (40). In this report, we have shown that mAb IGII is also able to inhibit eosinophil adhesion to 24-h TNF-stimulated EC monolayers, at which time point the ELAM-l expression on the EC surface has returned toward baseline low levels. Inhibition was only demonstrable, however, in the presence of antiCD18 mAb, suggesting that interactions between CDll/ CD18moiecuies and their ligands on EC can compensate for the absence of VCAM-1.
The ligand for VCAM-l on leukocytes is VLA-4 (CD49d/ CD29), which is a {31 integrin (30). It is expressed on thymocytes, monocytes, and activated peripheral blood lymphocytes (31). The recent finding that the eosinophil but not the neutrophil expresses VLA-4 (44, 45) suggests that the selective effect ofVCAM-l-i~duced adhesion for the eosinophil is mediated via VLA-4. The degree of inhibition by mAb IGll and anti-CDl8 of eosinophil adhesion to 24-h TNF-stimulated EC was only 30%, suggesting that molecules other than ICAM-l, ICAM-2, and VCAM-l might be involved. We cannot however eliminate the possibility that greater inhibition might have been obtained if Ftab'), fragments of mAb IGII had been used, thereby excluding a possible effect of the Fc part of the mAb in promoting nonspecific eosinophil-EC adhesion.
Kyan-Aung, Haskard, and Lee: VCAM-land Eosinophil Adhesion
In view of the evidence that increased adhesion of lymphocytes to IL-4-stimulated EC is largely attributable to VCAM-l induction by this cytokine (40, 46), it is perhaps surprising that we have not detected increased eosinophil adhesion to IL-4-stimulated Ee. Our experiments with eosinophils and lymphocytes were performed under similar experimental conditions, and it is possible that the strength of binding of lymphocytes to EC that express VCAM-l is greater than that of eosinophils. We have not therefore excluded the possibility that eosinophils might show enhanced binding to IL-4-stimulated EC when less stringent washing conditions are used to remove nonadherent cells. Whereas anti-VCAM-1 mAb led to a modest inhibition of eosinophil adhesion, no inhibition of neutrophil adhesion to 24-h TNF-stimulated EC was observed under the same conditions. These experiments suggest that the binding of eosinophils to VCAM-1 may contribute to the selective accumulation of eosinophils during allergic inflammation, especially later on during the response. Although we have not shown VCAM-1 expression at 6 h after the initiation of the late-phase cutaneous reaction to allergen in vivo, it remains to be determined whether VCAM-1 is enhanced at later time points. It is also possible that binding of eosinophils to VCAM-1 may explain how eosinophils and not neutrophils are found in chronic inflammatory tissues in patients with leukocyte adhesion deficiency, in which cells lack surface expression of CDll/CD18 molecules (47). Previous work has shown that infusions of ICAM-1 antibodies into monkeys reduces airways eosinophilia and hyperresponsiveness (26). It remains to be established whether VCAM-1 antibodies produce the same effects. In summary, we have demonstrated that eosinophils but not neutrophils may use VCAM-1 in adhesion to TNFstimulated EC. This may be an important mechanism for determining the more prolonged presence of eosinophils compared with neutrophils in ongoing allergic reactions. Acknowledgments: This work was supported by the Medical Research Council and by Schering-Plough, United Kingdom. Dr. Kyan-Aung is a fellow of the World Health Organization. Dr. Haskard was a Wellcome Trust Senior Research Fellow.
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