Reactivity of endothelial cell intermediate filaments with human and mouse monoclonal IgM antiphospholipid antibodies

Abstract

s A.63 REACTIVITY OF ENDOTHELIAL CELL INTERMEDIATE FILAMENTS WITH HUMAN AND MOUSE MONOCLONAL IgM ANTIPHOSPHOLIPID ANTIBODIES. N.S. Rote, L. Lin, L. Shroyer, T.W. Lyden, and A.K. Ng, Departments of Microbiology & Immunology, and Obstetrics & Gynecology, Wright State University School of Medicine, Dayton, OH, USA. Antibodies against phospholipids (aPLs) have been associated with recurrent thrombosis, possibly through modulation of endothelial cell function. In the current study we investigate the antl-endothelial cell reactivity of IgM monoclonal aPLs and IgM aPL-positive sera from patients with the antiphospholipid antibody syndrome. Using immunofluorescence and acetone fixed human endothelial cells we investigated the reactivity of three monoclonal antibodies that differentiate between cardiolipin (CL) and phosphatidylserine (PS) (BA3B5C4 [CL+/PS+]; 3SB9b [CL-/PS+]; DIIA4 [CL+/PS-]). 3SB9b reacted strongly with cytoskeletal-like cytoplasmic components, whereas DIIA4 and BA3B5C4 were unreactive. The patients' sera also contained IgM that reacted with cytoskeletal-like structures in the same pattern. Blocking experiments were performed using antibodies against actin (microfilaments), tubulin (microtubules), and vimentin (intermediate filaments), proteins that are representative of the three primary endothelial cell cytoskeletal structures. Only anti-vimentin blocked the binding of aPLs, either monoclonal or human, western blots of NP40 extracts of endothelial cells were probed with aPLs. 3SBgb reacted strongly with bands at 112 and 51 kDa, BA3B5C4 reacted with a 50 kDa band, and DIIA4 was unreactive. Sera from four patients also reacted against the 112 kDa, and two also reacted against the 51 kDa band. These results suggest that PS-like epitopes, identified by both monoclonal and patient's antibodies, are associated with endothelial intermediate filaments, aPL-mediated pathophysiology may, therefore, require endothelial cell damage or activation as a necessary cofactor to expose PS to circulating aPLs. EXPRESSION OF IL-IB AND IL-6 PROTEIN AND mRNA IN AMNIOCHORIONIC MEMBRANE. N.S. Rote, R. Menon, K.F. Swan, T.W. Lyden, and S.J. Fortunato, Departments of Microbiology & Immunology, and obstetrics & Gynecology, Wright State University School of Medicine, Dayton, OH, USA. A proposed mechanism for preterm labor is that intraamniotic infections induce cytokines that increase prostaglandin production. The role of fetal membranes in this mechanism has not been determined. We investigated whether cells of the amniochorionic membranes produce cytokines and whether that production is increased with exposure to endotoxin. Membranes collected by elective caesarian section from women without infection or pregnancy-related complications were washed in sterile buffer, cut into 5 mm diameter pieces, and maintained for 72 hr in media (DMEM:Nutrient mixture FI2 Ham) with antibiotics. IL-IB and IL-6 gene expression was measured with or without exposure to endotoxin (i00 pg/ml to i000 ng/ml) by PCR and in situ hybridization using short (20-35 mer) primers/probes for each cytokine and the sense strand as a negative control and by immunocytochemistry using immunoperoxidase labelling. Probes/primers for GAPDH were used as positive controls. Both chorion and amnion cells normally produced IL1B message or protein, or both, but not IL-6. Endotoxin (optimum at i0 ng/ml) induced IL-6 message and protein in both chorion and amnion. This study suggests that fetal membranes may be direct sources of IL-I and IL-6 in situ and contribute to infection-induced preterm labor.