Abstract 2112 Poster Session III, Monday, 5/3 (poster 168)

We have previously demonstrated that immune complexes isolated from synovial fluids of children with juvenile rheumatoid arthritis (JRA) vary in their size, composition, and pro-inflammatory activity. Furthermore, we have demonstrated that size and composition are interdependent variables regulating the pro-inflammatory activities of immune complexes and that at least some of the effects are mediated through the leukocyte complement receptor, CR1 (CD35). The current studies were undertaken to examine in further detail how complement binding to immune complexes regulates the activation of leukocytes.

Undifferentiated monomyelocytic HL-60 cells were incubated with either unopsonized BSA-anti-BSA immune complexes or immune complexes to which C4b had been bound through the fluid-phase cleavage of C4 by C1s ("C4b complexes"). After specific time periods, cell were lysed in the presence of protease and phosphatase inhibitors. The complement C4b/C3b receptor, CR1, was then immunoprecipitated from cell lysates, subjected to SDS-PAGE, transfered to nitrocellulose membranes, and analysed for the presence of phosphorylated serine-threonine residues by western blotting. Similarity, membranes containing immunoprecipatated CR1 were analyzed for the presence of syk, a protein tyrosine kinase critical in the signaling events mediated through Fc gamma receptors on leukocytes.

Immunoprecipitation studies demonstrated that CR1 is not constitutively phosphorylated on HL-60 cells Incubation of HL-60 cells with immune complexes was associated with phosphorylation of CR1 on serine or threonine residues within 5-10 minutes. Phosphorylation of CR1 occurred whether or not CR1 was specifically ligated. That is, both unopsonized and C4b-bearing immune complexes were associated with CR1 phosphorylation. Furthermore, although CR1 contains no intracellular tyrosine residues, its phosphorylation could be inhibited by genestein, a tyrosine kinase inhibitor. This suggests the prior phosphorylation of another, tyrosine-containing, protein is required for CR1 phosphorylation.

Immunoprecipitated CR1 was associated with syk after immune complex stimulation of HL-60 cells. Syk association with CR1 occured with both unopsonized or opsonized immune complexes. These findings are consistent with previous observations of capping of CR1 on neutrophils incubated with heat-aggregated IgG. We conclude that the complement receptor, CR1, may act as a regulator of immune complex-mediated activation of leukocytes. This regulation may involve the activation of specific protein tyrosine kinases, such as syk, used in the signaling of Fc gamma receptor on leukocytes. Thus, these data provide new insight into the inflammatory mechanisms activated by high molecular weight, complement fixing immune complexes.

Supported by the National Institutes of Health and the Children's Medical Research Foundation of Oklahoma City