Clin. exp. Immunol. (1990) 81, 496-500
Adhesion of polymorphonuclear cells to human endothelial cells. Adhesion-molecule-dependent, and Fc receptor-mediated adhesion-molecule-independent mechanisms J. F. M. LEEUWENBERG, G. M. A. A. JEUNHOMME & W. A. BUURMAN From the Department of Surgery, Biomedical Centre, University of Limburg, Maastricht, The Netherlands
(Acceptedfor publication 16 March 1990)
SUMMARY Activation of human umbilical vein endothelial (HUVE) cells with the inflammatory mediators tumour necrosis factor-alpha (TNF), interleukin-l (IL-1), lipopolysaccharide (LPS) and phorbol esters enhanced their adhesiveness for leucocytes. The appearance of an activation antigen ELAM- 1, recognized by a monoclonal antibody (MoAb) ENA1, parallels the kinetics of the enhanced adherence of leucocytes to endothelial cells. Adhesion of polymorphonuclear cells (PMN) to activated HUVE cells could be blocked by F(ab')2 fragments of MoAb ENA I up to 60%. An additive inhibition ofthe adhesion was established by pre-incubation ofthe PMN with anti-CD 18 MoAb and/ or leucocyte adhesion inhibitor (LAI), produced by endothelial cells. An opposite reaction, however, was observed when HUVE cells were pre-incubated with intact MoAb ENAl, resulting in an enhancement of the adhesion up to 200%. Apparently, the blocking effect of MoAb ENA1 could be bypassed by the strong interaction of the Fc part of the MoAb with the Fc receptor (FcR) on the PMN. Similarly, anti-CD18 MoAb and/or LAI reduced the adhesion observed if intact ENA I were used, and Fc-FcR interaction took place. The results presented in this study indicate that adhesion via ELAM-1, the CD1 8 antigen and via the receptor for LAI are different mechanisms. These mechanisms may act in concert to strengthen the binding of PMN to HUVE cells. Moreover, a strong adhesion could be established via the Fc part of MoAbs directed against HUVE cells with the FcR on the PMN. The phenomenon described may play a role in graft rejection and in diseases where antibodies directed against endothelium are involved. Keywords activation antigen adhesion endothelium neutrophils Fc receptor
(Hynes, 1987; Vedder & Harlan, 1988). The expression of CD18 antigens on PMN can be enhanced by inflammatory mediators (Tonnesen et al., 1989). Known adhesion molecules expressed on endothelial cells are ICAM-1, ICAM-2 and ELAM-1 (Rothlein et al., 1986; Bevilacqua et al., 1987; Staunton, Dustin & Springer, 1989). ICAM-1 is not endothelial specific, and its expression, which is normally at a low basal level, can be enhanced within 24 h by the lymphokines tumour necrosis factor (TNF), interleukin- I (IL- l) and interferon-gamma (IFNy) (Rothlein et al., 1988). Endothelial cells do not normally express ELAM-l, but ELAM-l expression can be rapidly induced by TNF, IL-I and LPS with an optimum after 4-6 h of activation, after which the expression declines (Pober et al., 1986). Blocking ICAM-1 and ELAM-l with antigen-specific antibodies results in an inhibition of the adhesion (Dustin & Springer, 1988; Bevilacqua et al., 1987). Furthermore, an endothelial-derived leucocyte adhesion inhibitor (LAI) has been described, which is generated by cytokine-stimulated endothelial cells (Wheeler et al., 1988), and which is identical to the
INTRODUCTION Acute inflammatory responses are characterized by an infiltration of leucocytes. This reaction is thought to play a role in pathophysiological processes such as graft rejection, septic shock, thermal injury, ischaemic organ injury and adult respiratory distress syndrome (Malech & Gallin, 1987). Several agents are known to mediate chemotaxis, adherence and migration of polymorphonuclear cells (PMN) into the vessel wall. The molecular basis for the mechanism of PMN adherence to endothelial cells in vitro has not been elucidated. PMN as well as endothelial cells may express surface molecules that mediate the adhesion. Known adhesion molecules on PMN belong to the integrin family: LFA-l, Mac-I (receptor for C3bi) and p150/95 (also called CD1 la, Cdl lb and CDI lc, respectively), consisting of a constant beta chain (CD18) and a variable alpha chain Correspondence: J. F. M. Leeuwenberg, University of Limburg, Department of General Surgery, Biomedical Centre, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
496
J. F. M. Leeuwenberg, G. M. A. A. Jeunhomme & W. A. Buurman neutrophil-activating factor, interleukin-8 (IL-8) (Gimbrone et al., 1989). We described recently a monoclonal antibody (MoAb) ENAl reactive with an inducible endothelial surface antigen with characteristics similar to those of ELAM-1 (Leeuwenberg, Jeunhomme & Buurman, 1989). Here we demonstrate that this MoAb, which reacts with ELAM-1, effectively blocks PMN adhesion to endothelial cells. We also studied the interaction of the CD18 molecule and LAI with ELAM-1 in adhesion. Using intact, not pepsin-digested IgG MoAb, we demonstrated an adhesion, independent ofthe so-called adhesion molecules. This process is shown to be Fc-dependent. MATERIALS AND METHODS Cells Human umbilical vein endothelial (HUVE) cells were obtained by collagenase treatment of the umbilical vein. The cells were cultured in fibronectin-coated (fibronectin kindly provided by Dr J. van Mourik, Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) tissue culture flasks (Costar, Cambridge, MA) in RPMI (GIBCO, Paisley, UK) supplemented with 20% heat-inactivated human serum, 50 Mg/ml heparin, 30 ug/ml endothelial growth supplement (Collaborative Research Incorporated, Bedford, MA) and antibiotics. The endothelial cells were characterized by positive staining with Factor VIII and with MoAbs directed against angiotensinconverting enzyme. For the experiments, cells of passage 3-4 were used. Other cells used were the monocytic cell line HL60, cultured in RPMI 1640 supplemented with 15% heat inactivated fetal calf serum (FCS) and antibiotics. Buffy coats, obtained after cytophoresis of healthy donors were kindly provided by the local blood bank (Blood Bank Zuid Limburg, head Dr D. Rhenen) and used to prepare peripheral blood lymphocytes (PBL) and PMN. PBL and PMN were isolated by buoyant density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). The PMN in the Lymphoprep fraction were separated from the erythrocytes by sedimentation in 33% Plasmasteril (Fresenius, Bad Homburg, FRG), followed by density gradient centrifugation on 75% Percoll. Monoclonal antibodies MoAb ENAl (IgG1) was obtained by immunizing mice with HUVE cells, pretreated with IL-I and TNF for 5 h as described elsewhere (Leeuwenberg et al., 1989). MoAb ENAl is specific for endothelial cells activated with TNF, IL-1, LPS or phorbol esters and is not reactive with other cell types. Cross-blocking studies with the labelled MoAbs ENA1 and 1.2B6 (kindly provided by Dr D. Haskard, Division of Medicine, UMDS, Guy's Hospital, London, UK), which is known to react with ELAM-1 (Wellicome et al., 1990), showed that MoAbs ENAl and 1.2B6 reacted with the same epitope on activated HUVE cells, being the ELAM-1 structure (data not shown). MoAb 1B4 (IgG2a, a generous gift from Prof. M. Daha, Department of Nephrology, University Hospital Leiden, The Netherlands) was used directed against the common beta chain (CD18) of LeuCAM (Wright et al., 1983). MoAb 7-24-1-2 (IgGI, kindly provided by Dr W. Tax, Department of Nephrology, St Radboud Hospital, Nijmegen, The Netherlands) reacted with MHC class I antigens. F(ab')2 fragments were prepared by
497
pepsin digestion (Immobilized Pepsin, Pierce, Rockford, IL) as described (Leeuwenberg et al., 1989). Reagents Recombinant (r) TNF was kindly provided by BASF/Knoll (Ludwigshafen, FRG). rIL-la was a kind gift from Dr S. Gillis, Immunex, Seattle, WA. LPS (Escherichia coli 055:B5) was purchased from Sigma, St Louis, MO.
Induction and detection of cell surface antigens The antigen expression was determined in ELISA. For the ELISA, I04 HUVE cells/well were grown for 48 h to confluency on 96-well, flat-bottomed tissue culture plates (Costar, Cambridge, MA) coated with fibronectin prior to the incubation with appropriate reagents for the experiments. The experiments were performed in RPMI 1640 supplemented with 8% FCS and antibiotics. After fixation (10' 0-025% glutaraldehyde) antigen expression was determined using goat anti-mouse peroxidase (Jackson, West Grove, PA) and o-phenylenediamine dihydrochloride (Sigma) as substrate. The plates were read on a Microtiter ELISA reader at 492 nm. The data were reported as the mean absorbance units from quadruplicate determinations after substraction of the background. The s.d. never exceeded 10% of the mean value. Adhesion assay HUVE cells were grown to confluency for 48 h in fibronectincoated 96-well plates. After washing, the cells were incubated with mediators as indicated in the experiments. The adhesion assay and the washings were carried out with RPMI/1 % FCS. The wells were rinsed twice before addition of appropriate MoAbs, and then 51Cr labelled PMN (2-5 x 105/well) were added. In some experiments the labelled cells were preincubated with MoAbs as described in the experiments. After a 3-min centrifugation and an incubation for 15 min at 37°C, the wells were washed three times to remove non-adherent cells. The remaining adherent cells were then solubilized (10% acetic acid) and the radioactivity was measured in a gamma-counter. Percentage adherence was calculated according to the formula: %= (ct/min harvested)/(ct/min added) x 100. The results were presented as % relative adhesion= % adherence (experiment)/ % adhesion of untreated PMN to activated HUVE cells (=control) x 100%. The absolute percentage of adherent PMN to activated HUVE cells varied in the different experiments (range 15-35%). All tests were carried out in quadruplicate and the data were expressed as the mean percentage cell binding to HUVE cells+ s.d. Preparation of LAI LAI was prepared as described by Wheeler et al. (1988). Briefly, HUVE cells were incubated with rIL-la (10 U/ml) in RPMI 1640 supplemented with 1% FCS and antibiotics. After 4 h the cells were washed with HBSS and further incubated for S h in the medium without cytokine. The supernatant containing LAI was centrifuged and frozen before use.
RESULTS Inhibition of adhesion by MoAb ENAI HUVE cells were activated by rIL-la for various periods of time. The adhesion of 51Cr-labelled PMN to these HUVE cells
J. F. M. Leeuwenberg, G. M. A. A. Jeunhomme & W. A. Buurman
498
I-
5
Table 1. Effects of pretreatment of activated HUVE cells by F(ab')2 fragments or intact MoAb on adhesion of PMN
2000
T/
Adhesion of polymorphonuclear cells to human endothelial cells. Adhesion-molecule-dependent, and Fc receptor-mediated adhesion-molecule-independent mechanisms J. F. M. LEEUWENBERG, G. M. A. A. JEUNHOMME & W. A. BUURMAN From the Department of Surgery, Biomedical Centre, University of Limburg, Maastricht, The Netherlands
(Acceptedfor publication 16 March 1990)
SUMMARY Activation of human umbilical vein endothelial (HUVE) cells with the inflammatory mediators tumour necrosis factor-alpha (TNF), interleukin-l (IL-1), lipopolysaccharide (LPS) and phorbol esters enhanced their adhesiveness for leucocytes. The appearance of an activation antigen ELAM- 1, recognized by a monoclonal antibody (MoAb) ENA1, parallels the kinetics of the enhanced adherence of leucocytes to endothelial cells. Adhesion of polymorphonuclear cells (PMN) to activated HUVE cells could be blocked by F(ab')2 fragments of MoAb ENA I up to 60%. An additive inhibition ofthe adhesion was established by pre-incubation ofthe PMN with anti-CD 18 MoAb and/ or leucocyte adhesion inhibitor (LAI), produced by endothelial cells. An opposite reaction, however, was observed when HUVE cells were pre-incubated with intact MoAb ENAl, resulting in an enhancement of the adhesion up to 200%. Apparently, the blocking effect of MoAb ENA1 could be bypassed by the strong interaction of the Fc part of the MoAb with the Fc receptor (FcR) on the PMN. Similarly, anti-CD18 MoAb and/or LAI reduced the adhesion observed if intact ENA I were used, and Fc-FcR interaction took place. The results presented in this study indicate that adhesion via ELAM-1, the CD1 8 antigen and via the receptor for LAI are different mechanisms. These mechanisms may act in concert to strengthen the binding of PMN to HUVE cells. Moreover, a strong adhesion could be established via the Fc part of MoAbs directed against HUVE cells with the FcR on the PMN. The phenomenon described may play a role in graft rejection and in diseases where antibodies directed against endothelium are involved. Keywords activation antigen adhesion endothelium neutrophils Fc receptor
(Hynes, 1987; Vedder & Harlan, 1988). The expression of CD18 antigens on PMN can be enhanced by inflammatory mediators (Tonnesen et al., 1989). Known adhesion molecules expressed on endothelial cells are ICAM-1, ICAM-2 and ELAM-1 (Rothlein et al., 1986; Bevilacqua et al., 1987; Staunton, Dustin & Springer, 1989). ICAM-1 is not endothelial specific, and its expression, which is normally at a low basal level, can be enhanced within 24 h by the lymphokines tumour necrosis factor (TNF), interleukin- I (IL- l) and interferon-gamma (IFNy) (Rothlein et al., 1988). Endothelial cells do not normally express ELAM-l, but ELAM-l expression can be rapidly induced by TNF, IL-I and LPS with an optimum after 4-6 h of activation, after which the expression declines (Pober et al., 1986). Blocking ICAM-1 and ELAM-l with antigen-specific antibodies results in an inhibition of the adhesion (Dustin & Springer, 1988; Bevilacqua et al., 1987). Furthermore, an endothelial-derived leucocyte adhesion inhibitor (LAI) has been described, which is generated by cytokine-stimulated endothelial cells (Wheeler et al., 1988), and which is identical to the
INTRODUCTION Acute inflammatory responses are characterized by an infiltration of leucocytes. This reaction is thought to play a role in pathophysiological processes such as graft rejection, septic shock, thermal injury, ischaemic organ injury and adult respiratory distress syndrome (Malech & Gallin, 1987). Several agents are known to mediate chemotaxis, adherence and migration of polymorphonuclear cells (PMN) into the vessel wall. The molecular basis for the mechanism of PMN adherence to endothelial cells in vitro has not been elucidated. PMN as well as endothelial cells may express surface molecules that mediate the adhesion. Known adhesion molecules on PMN belong to the integrin family: LFA-l, Mac-I (receptor for C3bi) and p150/95 (also called CD1 la, Cdl lb and CDI lc, respectively), consisting of a constant beta chain (CD18) and a variable alpha chain Correspondence: J. F. M. Leeuwenberg, University of Limburg, Department of General Surgery, Biomedical Centre, P.O. Box 616, 6200 MD Maastricht, The Netherlands.
496
J. F. M. Leeuwenberg, G. M. A. A. Jeunhomme & W. A. Buurman neutrophil-activating factor, interleukin-8 (IL-8) (Gimbrone et al., 1989). We described recently a monoclonal antibody (MoAb) ENAl reactive with an inducible endothelial surface antigen with characteristics similar to those of ELAM-1 (Leeuwenberg, Jeunhomme & Buurman, 1989). Here we demonstrate that this MoAb, which reacts with ELAM-1, effectively blocks PMN adhesion to endothelial cells. We also studied the interaction of the CD18 molecule and LAI with ELAM-1 in adhesion. Using intact, not pepsin-digested IgG MoAb, we demonstrated an adhesion, independent ofthe so-called adhesion molecules. This process is shown to be Fc-dependent. MATERIALS AND METHODS Cells Human umbilical vein endothelial (HUVE) cells were obtained by collagenase treatment of the umbilical vein. The cells were cultured in fibronectin-coated (fibronectin kindly provided by Dr J. van Mourik, Red Cross Blood Transfusion Service, Amsterdam, The Netherlands) tissue culture flasks (Costar, Cambridge, MA) in RPMI (GIBCO, Paisley, UK) supplemented with 20% heat-inactivated human serum, 50 Mg/ml heparin, 30 ug/ml endothelial growth supplement (Collaborative Research Incorporated, Bedford, MA) and antibiotics. The endothelial cells were characterized by positive staining with Factor VIII and with MoAbs directed against angiotensinconverting enzyme. For the experiments, cells of passage 3-4 were used. Other cells used were the monocytic cell line HL60, cultured in RPMI 1640 supplemented with 15% heat inactivated fetal calf serum (FCS) and antibiotics. Buffy coats, obtained after cytophoresis of healthy donors were kindly provided by the local blood bank (Blood Bank Zuid Limburg, head Dr D. Rhenen) and used to prepare peripheral blood lymphocytes (PBL) and PMN. PBL and PMN were isolated by buoyant density gradient centrifugation on Lymphoprep (Nycomed, Oslo, Norway). The PMN in the Lymphoprep fraction were separated from the erythrocytes by sedimentation in 33% Plasmasteril (Fresenius, Bad Homburg, FRG), followed by density gradient centrifugation on 75% Percoll. Monoclonal antibodies MoAb ENAl (IgG1) was obtained by immunizing mice with HUVE cells, pretreated with IL-I and TNF for 5 h as described elsewhere (Leeuwenberg et al., 1989). MoAb ENAl is specific for endothelial cells activated with TNF, IL-1, LPS or phorbol esters and is not reactive with other cell types. Cross-blocking studies with the labelled MoAbs ENA1 and 1.2B6 (kindly provided by Dr D. Haskard, Division of Medicine, UMDS, Guy's Hospital, London, UK), which is known to react with ELAM-1 (Wellicome et al., 1990), showed that MoAbs ENAl and 1.2B6 reacted with the same epitope on activated HUVE cells, being the ELAM-1 structure (data not shown). MoAb 1B4 (IgG2a, a generous gift from Prof. M. Daha, Department of Nephrology, University Hospital Leiden, The Netherlands) was used directed against the common beta chain (CD18) of LeuCAM (Wright et al., 1983). MoAb 7-24-1-2 (IgGI, kindly provided by Dr W. Tax, Department of Nephrology, St Radboud Hospital, Nijmegen, The Netherlands) reacted with MHC class I antigens. F(ab')2 fragments were prepared by
497
pepsin digestion (Immobilized Pepsin, Pierce, Rockford, IL) as described (Leeuwenberg et al., 1989). Reagents Recombinant (r) TNF was kindly provided by BASF/Knoll (Ludwigshafen, FRG). rIL-la was a kind gift from Dr S. Gillis, Immunex, Seattle, WA. LPS (Escherichia coli 055:B5) was purchased from Sigma, St Louis, MO.
Induction and detection of cell surface antigens The antigen expression was determined in ELISA. For the ELISA, I04 HUVE cells/well were grown for 48 h to confluency on 96-well, flat-bottomed tissue culture plates (Costar, Cambridge, MA) coated with fibronectin prior to the incubation with appropriate reagents for the experiments. The experiments were performed in RPMI 1640 supplemented with 8% FCS and antibiotics. After fixation (10' 0-025% glutaraldehyde) antigen expression was determined using goat anti-mouse peroxidase (Jackson, West Grove, PA) and o-phenylenediamine dihydrochloride (Sigma) as substrate. The plates were read on a Microtiter ELISA reader at 492 nm. The data were reported as the mean absorbance units from quadruplicate determinations after substraction of the background. The s.d. never exceeded 10% of the mean value. Adhesion assay HUVE cells were grown to confluency for 48 h in fibronectincoated 96-well plates. After washing, the cells were incubated with mediators as indicated in the experiments. The adhesion assay and the washings were carried out with RPMI/1 % FCS. The wells were rinsed twice before addition of appropriate MoAbs, and then 51Cr labelled PMN (2-5 x 105/well) were added. In some experiments the labelled cells were preincubated with MoAbs as described in the experiments. After a 3-min centrifugation and an incubation for 15 min at 37°C, the wells were washed three times to remove non-adherent cells. The remaining adherent cells were then solubilized (10% acetic acid) and the radioactivity was measured in a gamma-counter. Percentage adherence was calculated according to the formula: %= (ct/min harvested)/(ct/min added) x 100. The results were presented as % relative adhesion= % adherence (experiment)/ % adhesion of untreated PMN to activated HUVE cells (=control) x 100%. The absolute percentage of adherent PMN to activated HUVE cells varied in the different experiments (range 15-35%). All tests were carried out in quadruplicate and the data were expressed as the mean percentage cell binding to HUVE cells+ s.d. Preparation of LAI LAI was prepared as described by Wheeler et al. (1988). Briefly, HUVE cells were incubated with rIL-la (10 U/ml) in RPMI 1640 supplemented with 1% FCS and antibiotics. After 4 h the cells were washed with HBSS and further incubated for S h in the medium without cytokine. The supernatant containing LAI was centrifuged and frozen before use.
RESULTS Inhibition of adhesion by MoAb ENAI HUVE cells were activated by rIL-la for various periods of time. The adhesion of 51Cr-labelled PMN to these HUVE cells
J. F. M. Leeuwenberg, G. M. A. A. Jeunhomme & W. A. Buurman
498
I-
5
Table 1. Effects of pretreatment of activated HUVE cells by F(ab')2 fragments or intact MoAb on adhesion of PMN
2000
T/
