Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1307-1314
August 14, 1992
ADHESION PROMOTES TRANSCELLULAR LEUKOTRIENE BIOSYNTHESIS DURING NEUTROPHIL-GLOMERULAR ENDOTHELIAL CELL INTERACTIONS: INHIBITION BY ANTIBODIES AGAINST CD18 AND L-SELECTIN Hugh R. Brady*.# and Charles N. Serhan** *Renat and **Hematology/Oncology Divisions, Department of Medicine Brigham & Women's Hospital, Harvard Medical School, *75 Francis St and *'221 Longwood Avenue., Boston, MA 02115 Received June 19, 1992
Summary: Eicosanoid formation by transcellular routes can amplify the levels and types of lipid mediators within a local milieu. To evaluate the role of adhesion in this process, we assessed the influence of rnAb against adhesion molecules on LTC4 generation by PMN-endothelial cell interaction. Transcellular LTC4 generation was initiated by addition of fMLP to coincubations of GM-CSFprimed PMN and TNF-activated endothelial cells cultured from kidney glomeruli. Both PMN-endothelial cell adhesion and transcellular LTC4 generation were inhibited by mAb against leukocyte L-selectin and CD18. These results indicate that cytokine-treated PMN and endothelial cells generate LTC4 via transcellular routes by receptor-triggered mechanisms. They suggest that adhesion promotes transcellular eicosanoid biosynthesis and that adhesion molecules may also be targets for blockade of transcellular biosynthesis of lipid mediators. ~ ~2 Aoademio Press, inc.
Eicosanoids, generated by the initial actions of lipoxygenases with unesterified arachidonic acid, are considered important inflammatory mediators and modulators of smooth muscle tone in many disease states (1). Eicosanoid generation by resident tissue cells and platelets, from PMN-derived intermediates, appears to be an important mechanism for lipoxygenase product biosynthesis (teukotrienes and lipoxins) under these circumstances (2-6). For example both endothelial cells and platelets, which themselves lack 5#To whom correspondence should be addressed. Abbreviations: Polymorphonuclear leukocytes, PMN; leukotriene A4, LTA4; leukotriene C4, LTC4; recombinant human granulocytemacrophage colony-stimulating factor, GM-CSF; recombinant human tumor necrosis factor alpha, TNF; formyl-met-leu-phe, fMLP; monoclonal antibodies, mAb; prostaglandin B2, PGB2.
1307
0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
lipoxygenase activity, can generate peptidoleukotrienes, such as LTC4, from PMN-derived LTA4 in PMN-endothelial cell (2-4) or PMN-platelet (5,6) coincubations. Adhesion between PMN and endothelial cells via interaction of cell surface adhesion molecules is a central event in PMN trafficking to sites of inflammation (7-9). Adhesion might also be expected to enhance transcellular eicosanoid biosynthesis by approximating PMN and endothelial cell membranes and promoting transfer of substrates, such as the intermediate LTA4, from PMN to acceptor cells. To test this hypothesis, we monitored LTC4 formation during interaction of PMN and microvascular endothelial cells cultured from kidney glomeruli as a marker of transcellular eicosanoid biosynthesis and assessed the impact of mAb against PMN adhesion molecules on this process. I ~ rih o d s PMN isolation. PMN were isolated at 4oc from heparinized venous blood drawn from normal volunteers by the standard procedure of density gradient centrifugation on Lymphocyte Separation Medium (Organon Tecknika, Durham, NC) and dextran sedimentation, as described previously (10,11). Contaminating red blood cells were removed by hypotonic lysis. Following isolation, PMN were suspended in Du!becco's phosphate buffered saline (PBS, pH 7.4) containing 1% bovine serum albumin (BSA). These suspensions contained 97 _+ 2% PMN, as determined by light microscopy. The integrity of PMN in suspension was assessed by determining the exclusion of trypan blue. Suspensions in which greater than 97% of PMN excluded Trypan Blue were used for studies of PMN-endotheliat cell interaction. Glomerular Endothefial Cell Culture. Glomerular endothelial cells were isolated from bovine calf kidney gtomeruli and cultured as reported previously (10,11) by slight modification of the technique described by Ballermann (12). Individual clones of endothelial cells were characterized by their uniform uptake of fluorescein-labeled acetylated low density lipoproteins, by expression of Factor VIII antigen, and by expression of angiotensin-1 converting enzyme activity. Cells were grown in RPMI 1640 medium supplemented with L-glutamine, 15% iron-supplemented bovine calf serum (BCS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 bg/ml endothelial cell growth factor (Biomedical Tech Inc, Stoughton, MA). Endothelial cells were used at passage levels 8 - 12. PMN-Endothelial Cell Adhesion Assays. PMN adhesion to glomerular endothelial cells was assayed at 37oC with nonstatic conditions in a quantitative monolayer adhesion assay using 111 indium-labelled PMN (11). In experiments designed to define PMN adhesion molecules involved in this process, PMN were incubated with saturating concentrations of mAb directed against PMN ligands (vide infra) for 10 minutes, prior to PMN-endothelial cell coincubations. Analysis of eicosanoids. PMN-endothelial cell coincubations were terminated by addition of ethanol (2 vol) with PGB2 as internal standard, and LTC4 levels were analysed by RP-HPLC with UV detection, or RIA (New England Nuclear, Boston, MA) following extraction and chromatography with Sep Pack C18 cartridges (Waters Associates, Millipore Co., Milford, MA). For HPLC analysis of 1308
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
LTC4, the methanol fractions were injected into a HPLC system consisting Beckman pump (model 11A), an Altex Ultrasphere -ODS (4.6 mm x 25 column, injector, and a Perkin Elmer LC-75 spectrophotometric detector. column was eluted with MeQH:H20: acetic acid (65:35:0.01, pH 5.7) at a rate of 1 ml/min, and the UV detector was set to monitor 280 nm.
of a cm) The flow
Reagents and Monoclonal Antibodies. Purified mAb against the common CD18 of leukocyte CD11/CD18 beta-2 integrins (anti-CD18, R15.7, IgG1)was a gift from Dr. R. Rothlein (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT) and was used at a concentration of 20 ug/ml. MAb against leukocyte L-selectin (anti-LAM1-3 mAb, IgG1 ) was a gift from Dr. T.F. Tedder (Dana Farber Cancer Institute, Boston, MA) (13). These mAb have been shown previously to inhibit CD18-mediated and L-selectin-mediated PMN adhesion to glomerular endothelial cells (10,11 ). MAb OKM1 (IgG1) and anti-LAMl-10 (IgG1) recognize a functionally epitopes on CD11/CD18 and L-selectin which do not mediate adhesion (gifts from Drs. F.W. Luscinskas, Brigham and Womens Hospital, and T.F. Tedder, Dana Farber Cancer Institute, respectively), and were used as control mAb. Statistical analysis. Data were analyzed by Student's t-test, and p values of
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1307-1314
August 14, 1992
ADHESION PROMOTES TRANSCELLULAR LEUKOTRIENE BIOSYNTHESIS DURING NEUTROPHIL-GLOMERULAR ENDOTHELIAL CELL INTERACTIONS: INHIBITION BY ANTIBODIES AGAINST CD18 AND L-SELECTIN Hugh R. Brady*.# and Charles N. Serhan** *Renat and **Hematology/Oncology Divisions, Department of Medicine Brigham & Women's Hospital, Harvard Medical School, *75 Francis St and *'221 Longwood Avenue., Boston, MA 02115 Received June 19, 1992
Summary: Eicosanoid formation by transcellular routes can amplify the levels and types of lipid mediators within a local milieu. To evaluate the role of adhesion in this process, we assessed the influence of rnAb against adhesion molecules on LTC4 generation by PMN-endothelial cell interaction. Transcellular LTC4 generation was initiated by addition of fMLP to coincubations of GM-CSFprimed PMN and TNF-activated endothelial cells cultured from kidney glomeruli. Both PMN-endothelial cell adhesion and transcellular LTC4 generation were inhibited by mAb against leukocyte L-selectin and CD18. These results indicate that cytokine-treated PMN and endothelial cells generate LTC4 via transcellular routes by receptor-triggered mechanisms. They suggest that adhesion promotes transcellular eicosanoid biosynthesis and that adhesion molecules may also be targets for blockade of transcellular biosynthesis of lipid mediators. ~ ~2 Aoademio Press, inc.
Eicosanoids, generated by the initial actions of lipoxygenases with unesterified arachidonic acid, are considered important inflammatory mediators and modulators of smooth muscle tone in many disease states (1). Eicosanoid generation by resident tissue cells and platelets, from PMN-derived intermediates, appears to be an important mechanism for lipoxygenase product biosynthesis (teukotrienes and lipoxins) under these circumstances (2-6). For example both endothelial cells and platelets, which themselves lack 5#To whom correspondence should be addressed. Abbreviations: Polymorphonuclear leukocytes, PMN; leukotriene A4, LTA4; leukotriene C4, LTC4; recombinant human granulocytemacrophage colony-stimulating factor, GM-CSF; recombinant human tumor necrosis factor alpha, TNF; formyl-met-leu-phe, fMLP; monoclonal antibodies, mAb; prostaglandin B2, PGB2.
1307
0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS
lipoxygenase activity, can generate peptidoleukotrienes, such as LTC4, from PMN-derived LTA4 in PMN-endothelial cell (2-4) or PMN-platelet (5,6) coincubations. Adhesion between PMN and endothelial cells via interaction of cell surface adhesion molecules is a central event in PMN trafficking to sites of inflammation (7-9). Adhesion might also be expected to enhance transcellular eicosanoid biosynthesis by approximating PMN and endothelial cell membranes and promoting transfer of substrates, such as the intermediate LTA4, from PMN to acceptor cells. To test this hypothesis, we monitored LTC4 formation during interaction of PMN and microvascular endothelial cells cultured from kidney glomeruli as a marker of transcellular eicosanoid biosynthesis and assessed the impact of mAb against PMN adhesion molecules on this process. I ~ rih o d s PMN isolation. PMN were isolated at 4oc from heparinized venous blood drawn from normal volunteers by the standard procedure of density gradient centrifugation on Lymphocyte Separation Medium (Organon Tecknika, Durham, NC) and dextran sedimentation, as described previously (10,11). Contaminating red blood cells were removed by hypotonic lysis. Following isolation, PMN were suspended in Du!becco's phosphate buffered saline (PBS, pH 7.4) containing 1% bovine serum albumin (BSA). These suspensions contained 97 _+ 2% PMN, as determined by light microscopy. The integrity of PMN in suspension was assessed by determining the exclusion of trypan blue. Suspensions in which greater than 97% of PMN excluded Trypan Blue were used for studies of PMN-endotheliat cell interaction. Glomerular Endothefial Cell Culture. Glomerular endothelial cells were isolated from bovine calf kidney gtomeruli and cultured as reported previously (10,11) by slight modification of the technique described by Ballermann (12). Individual clones of endothelial cells were characterized by their uniform uptake of fluorescein-labeled acetylated low density lipoproteins, by expression of Factor VIII antigen, and by expression of angiotensin-1 converting enzyme activity. Cells were grown in RPMI 1640 medium supplemented with L-glutamine, 15% iron-supplemented bovine calf serum (BCS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 bg/ml endothelial cell growth factor (Biomedical Tech Inc, Stoughton, MA). Endothelial cells were used at passage levels 8 - 12. PMN-Endothelial Cell Adhesion Assays. PMN adhesion to glomerular endothelial cells was assayed at 37oC with nonstatic conditions in a quantitative monolayer adhesion assay using 111 indium-labelled PMN (11). In experiments designed to define PMN adhesion molecules involved in this process, PMN were incubated with saturating concentrations of mAb directed against PMN ligands (vide infra) for 10 minutes, prior to PMN-endothelial cell coincubations. Analysis of eicosanoids. PMN-endothelial cell coincubations were terminated by addition of ethanol (2 vol) with PGB2 as internal standard, and LTC4 levels were analysed by RP-HPLC with UV detection, or RIA (New England Nuclear, Boston, MA) following extraction and chromatography with Sep Pack C18 cartridges (Waters Associates, Millipore Co., Milford, MA). For HPLC analysis of 1308
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICALRESEARCHCOMMUNICATIONS
LTC4, the methanol fractions were injected into a HPLC system consisting Beckman pump (model 11A), an Altex Ultrasphere -ODS (4.6 mm x 25 column, injector, and a Perkin Elmer LC-75 spectrophotometric detector. column was eluted with MeQH:H20: acetic acid (65:35:0.01, pH 5.7) at a rate of 1 ml/min, and the UV detector was set to monitor 280 nm.
of a cm) The flow
Reagents and Monoclonal Antibodies. Purified mAb against the common CD18 of leukocyte CD11/CD18 beta-2 integrins (anti-CD18, R15.7, IgG1)was a gift from Dr. R. Rothlein (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT) and was used at a concentration of 20 ug/ml. MAb against leukocyte L-selectin (anti-LAM1-3 mAb, IgG1 ) was a gift from Dr. T.F. Tedder (Dana Farber Cancer Institute, Boston, MA) (13). These mAb have been shown previously to inhibit CD18-mediated and L-selectin-mediated PMN adhesion to glomerular endothelial cells (10,11 ). MAb OKM1 (IgG1) and anti-LAMl-10 (IgG1) recognize a functionally epitopes on CD11/CD18 and L-selectin which do not mediate adhesion (gifts from Drs. F.W. Luscinskas, Brigham and Womens Hospital, and T.F. Tedder, Dana Farber Cancer Institute, respectively), and were used as control mAb. Statistical analysis. Data were analyzed by Student's t-test, and p values of
