This is a FIRST award application for support to study the interactions in vitro between leukocytes and glomerular mesangial and endothelial cells. In particular, the influence of leukotrienes and lipoxins on leukoctye adhesion to glomerular cells will be examined and the role of cell:cell adhesion molecules in this process will be determined. In addition, the ability of glomerular mesangial and endothelial cells to synthesize leukotrienes and lipoxins from leukocyte-derived lipoxygenase-intermediaries will be studied. There are three specific aims.
Specific Aim #1 seeks to determine the role of leukotrienes (LT) as stimuli for PMN and monocyte adhesion to glomerular mesangial cells (MC) and glomerular endothelial cells (GEC) using an in vitro leukocyte adhesion assay, and to identify the leukocyte adhesion molecules involved in this process using monoclonal antibodies. The role of lipoxins (LX) as inhibitors of LT-induced adhesion and cell injury will be assessed. The studies proposed under Specific Aim #2 seek to determine whether PMN interact with MC and GEC to generate LX and LT. It will be determined if MC generate LX from PMN derived arachidonate intermediates. The biosynthetic pathways will be defined by alcohol trapping of intermediates and product identification using RP-HPLC and gas chromatography-mass spectroscopy. The mechanisms by which LX are generated will be probed by (a) using inhibitors of LO activity, (b) by incubating MC with synthetic LTA4 or 5, (6)-epoxytetraene, the likely PMN-derived intermediates, and comparing product profiles with those generated during PMN-MC coincubation, and (c) by identifying the sources of oxygen for this biosynthesis process using 1802 and H218O. In addition, it will be determined whether peptidolipoxins (LXC4, LXD4, LXE4) and peptidoleukotrienes (LTC4, LTD4, LTE4) are generated by PMN-GEC interaction. The biosynthetic mechanism(s) will be defined by incubating GEC with exogenous LTA4 and 5, (6)-epoxytetraene, the likely intermediate for LTC4 and LXC4 production, respectively, and comparing product profiles to those generated during PMN-GEC coincubation. The source of glutathione for these reactions will be identified by 35S-cysteine labeling of PMN or GEC. The studies under Specific Aim #3 seek to determine whether cell-cell adhesion promotes LX and LT formation by transcellular routes. To this end, LX and LT production will be studied under conditions of reduced (adhesion-blocking monoclonal antibodies) or increased (cytokine-induction of adhesion molecules) PMN-glomerular cell adhesion.

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Brigham and Women's Hospital
United States
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Hebert, M J; Takano, T; Papayianni, A et al. (1998) Acute nephrotoxic serum nephritis in complement knockout mice: relative roles of the classical and alternate pathways in neutrophil recruitment and proteinuria. Nephrol Dial Transplant 13:2799-803
Hebert, M J; Gullans, S R; Mackenzie, H S et al. (1998) Apoptosis of endothelial cells is associated with paracrine induction of adhesion molecules: evidence for an interleukin-1beta-dependent paracrine loop. Am J Pathol 152:523-32
Brady, H R; Papayianni, A; Serhan, C N (1997) Transcellular pathways and cell adhesion as potential contributors to leukotriene and lipoxin biosynthesis in acute glomerulonephritis. Adv Exp Med Biol 400B:631-40
Papayianni, A; Serhan, C N; Brady, H R (1996) Lipoxin A4 and B4 inhibit leukotriene-stimulated interactions of human neutrophils and endothelial cells. J Immunol 156:2264-72
Hebert, M J; Takano, T; Holthofer, H et al. (1996) Sequential morphologic events during apoptosis of human neutrophils. Modulation by lipoxygenase-derived eicosanoids. J Immunol 157:3105-15
Gilligan, H M; Bredy, B; Brady, H R et al. (1996) Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apoptotic neutrophils in the absence of neutrophil priming. J Exp Med 184:2231-41
Mayadas, T N; Mendrick, D L; Brady, H R et al. (1996) Acute passive anti-glomerular basement membrane nephritis in P-selectin-deficient mice. Kidney Int 49:1342-9
Madore, F; Lazarus, J M; Brady, H R (1996) Therapeutic plasma exchange in renal diseases. J Am Soc Nephrol 7:367-86
Brady, H R; Serhan, C N (1996) Lipoxins: putative braking signals in host defense, inflammation and hypersensitivity. Curr Opin Nephrol Hypertens 5:20-7
Brady, H R; Lamas, S; Papayianni, A et al. (1995) Lipoxygenase product formation and cell adhesion during neutrophil-glomerular endothelial cell interaction. Am J Physiol 268:F1-12

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