Methods: Four SLE patient serum samples, with varying levels of IFN-α activity, as measured by a specific IFN-α reporter bioassay, were used to stimulate normal PBMC in the presence or absence of anti-IFN-α mAb. Controls contained PBMC with normal donor serum. Following incubation for 4 or 18 h, PBMC were harvested for RNA isolation and Affymetrix whole genome arrays were used to measure the changes in gene expression. The degree of transcript upregulation was established relative to the control wells and the percent neutralization was determined by comparing the results of SLE patient sera-treated wells in the presence and absence of anti-IFN-α mAb.
Results: Treatment of normal PBMC with four individual SLE serum samples induced a significant increase in transcript levels in PBMC. The total number of transcripts induced after serum treatment for 4 h correlated with the IFN-α bioactivity in the serum. At the same time point, the majority of the most highly upregulated genes were type I IFN inducible. These transcripts were largely neutralized by an anti-IFN-α mAb in a dose dependent manner, but were not affected by an anti-IFN-γ mAb. After 18 h of stimulation, we observed a significant increase in the total number of SLE serum-induced transcripts compared to 4 h. Of interest were a large number of transcripts that were neutralized by the anti-IFN-α mAb pre-treatment. These late response genes may provide insight into pathways that might be downstream of the type I IFN response. Analysis of the genes uniquely activated at the 18 h time point revealed upregulation of genes involved in the innate immune response (TLR, NFκB), adaptive immune response (NFAT, IL-1/IL-6), complement activation as well as leukocyte chemotaxis and adhesion.
Conclusions: These results suggest that neutralization of the IFN-α pathway not only alters the upstream regulation of type I IFN inducible genes but also has the potential to modify downstream pathways that may significantly impact the pathogenesis of SLE.
W. Trigona, Yes, MedImmune, 1; Yes, MedImmune, 1; Yes, MedImmune, 3; Y. Yao, 2250 shares, 1; MedImmune, Inc., 3; C. Morehouse, Yes, 1; Full time, 3; M. de los Reyes, YES, 1; FULL, 3; B. Chen, Full, 2; J. Zmuda, Yes. MedImmune, 1; Yes. MedImmune, 1; Yes- MedImmune, 3; J. Hirsch, YES, 1; If 401K= yes, if not 401K then No, 1; YES, 3; A.J. Coyle, Yes, 1; Yes, 1; Full Time, 3; Yes, 6; P. Kiener, Yes (MedImmune), 1; YES, 3; Yes- Synovex, 6; W. White, Yes, 1; FULL, 3; B. Jallal, YES, 1; YES, 1; FULL, 3.
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