R. Deans

Elevated spontaneous IgG production is characteristic of SLE. To identify the factors that support it, IL-6, a cytokine with an important role in the differentiation of IgG-secreting cells, was studied in SLE patients. Higher than normal levels of IL-6 were found, by a B9 assay, in sera of 63 of 70 patients (p less than 0.05). IL-6 was detected in 36 of 37 active SLE sera in higher titers (p = 0.009) than those for inactive SLE (n = 33), which were higher (p less than 0.05) than healthy controls (n = 15). IL-6 mRNA was detected in freshly isolated PBMC of 11 of 11 patients but not in normal PBMC, whereas IL-1 mRNA was detected only in patients with active disease. IL-6 activity was recovered from PBMC of four SLE patients, but not from four normal donors. By immunoperoxidase, IL-6 was detected in the cytoplasm of SLE monocytes and lymphocytes. When SLE PBMC were grown in short term cultures with no deliberate stimulation, expression of the IL-6 gene declined rapidly. Accordingly, the spontaneous production of IgG by SLE PBMC could be enhanced by exogenous IL-6. Spontaneous IgG production was diminished by 20 to 65% in the presence of neutralizing antibodies to IL-6, TNF-alpha, or IL-1. In contrast, neutralization of endogenous IL-4 increased production by approximately 40%. Anti-TNF-alpha treatment decreased IL-6 content of PBMC cultures, whereas anti-IL-4 augmented it, and exogenous IL-6 reversed anti-TNF-alpha effects on IgG production. Therefore, it is possible that the neutralization of TNF-alpha and IL-4 affected IgG production by modulating the synthesis/activity of IL-6. These results support the concept that SLE B cell hyperactivity is promoted by dysregulation of endogenous cytokines and suggest that IL-6, in particular, has an important pathogenic role.

Northwestern blot analysis in the presence of competitor RNA was used to examine the interaction between the mouse hepatitis virus (MHV) nucleocapsid protein (N) and virus-specific RNAs. Our accompanying article demonstrates that anti-N monoclonal antibodies immunoprecipitated all seven MHV-specific RNAs as well as the small leader-containing RNAs from infected cells. In this article we report that a Northwestern blotting protocol using radiolabeled viral RNAs in the presence of host cell competitor RNA can be used to demonstrate a high-affinity interaction between the MHV N protein and the virus-specific RNAs. Further, RNA probes prepared by in vitro transcription were used to define the sequences that participate in such high-affinity binding. A specific interaction occurs between the N protein and sequences contained with the leader RNA which is conserved at the 5' end of all MHV RNAs. We have further defined the binding sites to the area of nucleotides 56 to 65 at the 3' end of the leader RNA and suggest that this interaction may play an important role in the discontinuous nonprocessive RNA transcriptional process unique to coronaviruses.

The interaction of the mouse hepatitis virus (MHV) nucleocapsid protein (N) and viral RNA was examined. Monoclonal antibody specific for N protein coimmunoprecipitated MHV genomic RNA as well as all six MHV subgenomic mRNAs found in MHV-infected cells. In contrast, monoclonal antibodies to the MHV E2 or E1 envelope glycoproteins, an anti-I-A monoclonal antibody, and serum samples from lupus patients did not immunoprecipitate the MHV mRNAs. Moreover, the anti-N monoclonal antibody did not coimmunoprecipitate vesicular stomatitis virus RNA or host cell RNA under conditions which immunoprecipitated all MHV RNAs. These data suggest a specific interaction between the N protein and the virus-specific mRNAs. Both the membrane-bound and cytosolic small MHV leader-specific RNAs of greater than 65 nucleotides long were immunoprecipitated only by anti-N monoclonal antibody. These data suggest that an N binding site is present within the leader RNA sequences at a site at least 65 nucleotides from the 5' end of genomic RNA and all six subgenomic mRNAs. The larger leader-containing RNAs originating from mRNA 1 and mRNA 6, as well as the MHV negative-stranded RNA, were also immunoprecipitated by the anti-N monoclonal antibody. These data indicate that the MHV N protein is associated with MHV-specific RNAs and RNA intermediates and may play an important functional role during MHV transcription and replication.