BACTERIAL CONTRIBUTIONS TO SYSTEMIC LUPUS PATHOGENESIS

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Systemic Lupus Erythematosus (SLE) is an autoimmune arthritic disease of unknown etiology. Increased incidence of SLE in defined geographic regions suggests that this autoimmune disease may have an environmental basis. As the immune system functions largely as a microbial adversary, it has been suggested that autoimmunity may arise as a disease state prompted by microbial molecular mimicry of immune-tolerable, human components. The antigenic components associated with SLE, namely the Sm antigen associated with small nuclear ribonucleoprotein particles (snRNPs), are highly conserved, intracellular molecules within eukaryotes, but are not thought to exist in prokaryotic organisms. Evidence for coincident molecular sequences suggesting mimicry between Sm and prokaryotic components is absent. However recent evidence suggests that ribonucleoprotein particles displaying high homology to human snRNPs do exist among two groups of eubacteria, although neither of these bacteria are known to be infectious in humans. The existence of snRNPs among infectious prokaryotes could provide an antigenic stimulus which might lead to autoimmunity in susceptible individuals. To test this hypothesis, this proposal intends to use the polymerase chain reaction (PCR) to determine whether commonly infectious eubacteriaI organisms also possess genes associated with snRNPs.. Both U1 and U6snRNA-specific primers will be reacted with genomic DNA from a variety of prokaryotic microorganisms in an effort to discover the extent of the snRNA genes among the eubacteria. Southern hybridization with PcR probes from the snRNP-positive eubacterial organisms will also be used to screen for snRNA genes in genomic DNA. To address the question of whether the prokaryotics snRNAs represent some form of spontaneous molecular mimicry or whether they have a functional role in prokaryotes, RNA splicing assays will be developed, both in vitro and in vivo, using a plasmid with an SV4O intron and both eukaryotic and prokaryotic promoter sites. The plasmid will be transcribed in vitro and incubated in either human or bacterial extracts presumed to contain the splicing components; the RNA products of the splicing reaction will be amplified by reverse transcriptase-PCR (RT-PCR) and visualized on electrophoretic separations. Southern hybridizations with sequence-defined probes for the intron (unspliced) or spliced exons (spliced) should verify any splicing activity. Human extracts depleted of U1snRNA by antisense hybridization will be incubated in varying quantities of extracts derived from U1snRNA- positive prokaryotic cells to determine whether "splicing complementation" can occur between prokaryotic components and the eukaryotic splicing system. In addition, transformation of the prokaryotic cells with the intron-containing plasmid will be used as the in vivo assay system. Extraction of the RNA from these transformed bacteria and amplification with RT-PCR should allow assessment of in vivo splicing performance. Finally, the ability of snRNP-containing prokayotic cell extracts to stimulate SLE will be assessed by immunization of SLE- model mice, MRL/lpr. The MRL/lpr mice, which spontaneously develop SLE around five months of age, will be inoculated with varying amounts of snRNP-containing prokaryotic extract, and the time course of development of anti-Sm antibodies will be determined in comparison to genetically similar mice which are not prone to develop SLE or anti-Sm antibodies, i.e. MRL/+. Mice. inoculated with extracts from bacteria not containing snRNPs, i.e. E. coli, may also be evaluated. Production of anti-Sm will be monitored by ELISA and immunofluorescence assays. The outcome of these experiments should provide a clearer indication of: the extent of snRNPs in prokaryotic cells; whether the prokaryotic snRNPs are "fortuitous" molecular mimics or functional splicing components; and whether inoculation with prokaryotic snRNPs contributes to the pathogenesis of SLE.