Faculty Bibliography

It has been reported that antiphospholipid autoantibodies do not recognize phospholipid alone, but rather the plasma protein beta 2-glycoprotein I (beta 2GPI), or a beta 2GPI-phospholipid complex. In vitro beta 2GPI binds to anionic phospholipids and inhibits the prothrombinase activity of procoagulant membranes. In light of the fact that lupus anticoagulants, a type of antiphospholipid antibody, have similar anticoagulant properties, the relationship of beta 2GPI to lupus anticoagulant activity was investigated. IgG from patients with autoimmune diseases or syphilis were tested for anticardiolipin reactivity and lupus anticoagulant activity in the presence and absence of beta 2GPI. As expected, anti-cardiolipin reactivity associated with autoimmune disease was beta 2GPI dependent. In contrast, IgG from a patient with syphilis recognized cardiolipin alone and binding was inhibited by beta 2GPI. Autoimmune antiphospholipid antibodies prolonged the dilute Russell viper venom time of normal plasma, but had no effect on beta 2GPI-depleted plasma. Antiphospholipid antibodies associated with syphilis had no anticoagulant effect. RP-1, an anti-beta 2GPI mAb, had anticoagulant effects similar to those of autoimmune antiphospholipid antibodies. These data demonstrate that antiphospholipid autoantibodies exert lupus anticoagulant activity via an interaction with beta 2GPI. These antibodies and RP-1 appear to amplify the anticoagulant effect of beta 2GPI itself

OBJECTIVE. To determine whether anti-Ro/SS-A antibodies selectively bind to neonatal cardiac cells and alter membrane repolarization. METHODS. An in vitro electrophysiologic and immunocytochemical experimental model contrasting neonatal and rabbit cardiac tissue was employed. RESULTS. Sera and IgG-enriched fractions from anti-Ro/SS-A antibody-positive mothers of infants with neonatal lupus erythematosus and congenital heart block bind to neonatal, rather than adult, rabbit cardiac tissue and alter the transmembrane action potential (i.e., inhibit repolarization). The additional presence of anti-La/SS-B antibodies was not additive or synergistic for these immunocytochemical and electrophysiologic features. Sera containing other antibody specificities (i.e., anti-native DNA, cardiolipin, Sm, and nuclear RNP) failed to stain the neonatal cardiac tissue or produced alterations in membrane repolarization. CONCLUSION. Anti-Ro/SS-A antibodies may play a pathophysiologic role in the development of congenital heart block in neonatal lupus

OBJECTIVE. To assess the activity of systemic lupus erythematosus (SLE) during pregnancy and to distinguish it from preeclampsia. METHODS. We prospectively measured the complement activation products Ba, Bb, SC5b-9, and C4d, as well as the conventional complement determinants C3, C4, and CH50, during pregnancy in 14 patients with SLE and 10 women with preeclampsia. RESULTS. Four of the 14 SLE patients were considered to have disease flares, 3 occurring in the second trimester and 1 postpartum. In these patients, significant abnormalities of Ba, Bb, SC5b-9, and CH50 were noted. In contrast, measures of C4d did not distinguish between pregnant patients who had flares and those whose SLE remained stable. Although decreased values of C3 were rarely seen in the patients with stable disease, normal values of C3 during lupus pregnancy were not reliably associated with stable disease. Three of 10 non-SLE patients with preeclampsia had elevated levels of Ba; however, in each case, the CH50 level was close to or within the normal range. This was in sharp contrast to the findings observed in the 4 patients with active SLE, in whom high levels of plasma Ba were always associated with low CH50 values. Moreover, the ratio of CH50 to Ba was significantly lower in the patients with lupus flares than in the non-SLE patients with preeclampsia. CONCLUSION. While a decline in the CH50 level alone could otherwise be attributed to decreased synthesis of complement components, these data demonstrate that ongoing activation of the alternative complement pathway can accompany disease flares in pregnant women with SLE

C receptor CR3 (iC3b-receptor, CD11b/CD18) plays an essential role in several phagocytic and adhesive neutrophil functions. Recent evidence suggests that stimulus-induced phosphorylation of the CR3 beta-chain, CD18, may mediate certain neutrophil functions by transiently converting the molecule to an activated state. Staurosporine, a protein kinase C inhibitor that blocks PMA-induced CD18 phosphorylation, was used to study the functional relevance of this event. Neutrophils adhered to glass were assayed for binding and phagocytosis of iC3b-opsonized sheep E (EC3bi) in the presence or absence of PMA and/or staurosporine. Binding of EC3bi was markedly increased, not only by PMA, but also by staurosporine and by a combination of both agents (three- to sevenfold). The enhancement of rosetting by staurosporine was likely caused by increased surface expression of CR3 via exocytosis of specific granular contents. In contrast, staurosporine alone did not stimulate phagocytosis of EC3bi and markedly inhibited PMA-induced phagocytosis. Staurosporine also inhibited phagocytosis of yeast beta glucan particles, a CR3 ligand that, in contrast to EC3bi, is bound and ingested without additional prior treatment with PMA. beta glucan phagocytosis was associated with a low level of CD18 phosphorylation. Staurosporine did not block phagocytosis in general, because this agent had relatively little effect on FcR-mediated phagocytosis. These data demonstrate that phagocytosis mediated by CR3 requires activation of CR3 via a staurosporine-sensitive pathway. Increased binding of EC3bi, a function of increased surface expression of CR3, does not require activation of CR3 by such a pathway, confirming previous evidence for the independence of these two phenomena. A direct role for CD18 phosphorylation in the activation of CR3 for phagocytosis is consistent with these data

Serum SS-A/Ro autoantibodies are commonly found in patients with Sjogren's syndrome, systemic lupus erythematosus, neonatal lupus, and subacute cutaneous lupus. Two proteins of 60 and 52 kD have been described as targets for these autoantibodies. To define the 52-kD component unambiguously, cDNA clones were isolated from human HepG2 and MOLT-4 cell cDNA libraries. The identity of cDNA was established by (a) the specificity of the antibody affinity purified from the recombinant protein, (b) the reactivity of the purified recombinant protein with prototype SS-A/Ro sera in immunoblot and ELISA, and (c) two-dimensional gel comigration of MOLT-4 cell 52-kD protein and the recombinant protein. A 1.9-kb cDNA encoded the complete 52-kD protein containing 475 amino acids (Mr 54,082). Putative zinc-finger domains and a leucine zipper motif were identified in the amino-terminal half of the 52-kD protein, implicating its possible association with DNA/RNA. Sequence homology detected between the 52-kD protein and human ret transforming protein, and mouse T cell gene expression down-regulatory protein rpt-1, may provide leads to the functional role of the 52-kD protein in addition to the possibility that these proteins might constitute members of a subfamily of finger proteins

The characteristics of homotypic neutrophil aggregation, mediated by the adhesion molecule CD11b/CD18, differ according to whether activation takes place via intracellular protein kinase C(PKC) inducers or chemoattractants. In response to diacylglycerol (DAG) analogues such as PMA and 1,2-dioctanoyl-sn-glycerol, a prolonged cellular aggregation occurs that is associated with intense phosphorylation of the CD18 beta-chain. In response to the chemoattractant FMLP, a more transient aggregation event results that is associated with minimal beta-chain phosphorylation. By using the PKC inhibitor staurosporine, we now show that these differences are likely to reflect two different pathways of activation. Both aggregation and phosphorylation induced by DAG analogues are completely abolished by staurosporine in a parallel dose-dependent manner. Conversely, FMLP-induced aggregation is enhanced and prolonged by staurosporine whereas the associated minimal phosphorylation event is further diminished by staurosporine. Accordingly, activation of neutrophil aggregation by DAG analogues is associated with and presumably due to phosphorylation of the CD18 beta-chain. This intense phosphorylation occurs via a staurosporine-sensitive kinase such as PKC. FMLP, on the other hand, appears to activate CD11b/CD18 by a distinct mechanism. This latter mechanism does not seem to be dependent on what may be a minor PKC-induced phosphorylation of the beta-chain, and indeed is enhanced by inhibition of PKC. Of note, staurosporine was also found to cause selective release of specific granules with concomitant increase in surface display of CD11b/CD18. These data further support previous observations that up-regulation of this adhesive molecule is not the primary event in the induction of cellular adhesiveness