Background/Purpose: Previous experiments suggest that the B cells of lupus patients are hyper-responsive to B cell receptor engagement resulting in increased tyrosine phosphorylation and Ca²⁺mobilization.  However the precise B cell populations that are affected and the mechanisms leading to this hyper-responsiveness have yet to be determined.  In this study we have used Phosflow to address these questions.

Methods: PBMC were isolated from 27 healthy controls and 39 SLE patients with ≥ 4 ACR criteria.  Phosflow was used to assess the levels of p-SYK, p-PLCγ2, or p-ERK following Ig receptor engagement with goat anti-human IgM F(ab’)₂in distinct B cell subsets defined by anti-CD19,-CD27, -IgD, -IgM and -CD38.  B cell proliferation and apoptosis following anti-IgM stimulation were assessed by flow cytometry, using CFSE and annexin V staining, respectively.  For some experiments, healthy control B cells were incubated with IFN-α, or 50% plasma ± anti-IFN or irrelevant Ab.  Lupus associated SNPs were determined by TaqMan genotyping.

Results: There were increased basal levels of p-SYK and p-ERK in naïve B cells (CD19⁺CD27^–IgD⁺) from lupus patients as compared to controls.  The levels of basal p-SYK correlated with CD86 expression suggesting that these cells had already been activated in-vivo.  Following crosslinking with anti-IgM, there was a significant increase in the proportion of p-SYK⁺ cells above basal levels in the naïve B cell population of lupus patients as compared to controls. Similar trends were seen for the proportion of p-PLCγ2⁺ and p-ERK⁺ cells.  The increases seen in p-SYK⁺ cells were most marked for the transitional B cell subset (CD19⁺CD27^–IgD⁺CD38^hiIgM^hi), where the levels of p-SYK correlated with enhanced proliferation and survival.   There was no correlation between lupus associated SNPs in BLK, LYN, PTPN22, and CSK, and the proportion of p-SYK⁺ cells following IgM crosslinking.  The proportion of p-SYK⁺cells in the transitional B cell subset fluctuated between visits, suggesting a possible role for pro-inflammatory factors.  Consistent with this, incubation of lupus plasma with control B cells enhanced SYK phosphorylation following IgM crosslinking, which was blocked by pre-incubation of plasma with anti-IFN but not irrelevant Ab.  Incubation of healthy control cells with recombinant IFN-α enhanced SYK phosphorylation, proliferation, and survival following IgM crosslinking, particularly of the transitional B cell subset.

Conclusion: IFN-α alters transitional B cell function leading to enhanced survival and proliferation.  As purging of transitional B cells plays an important role in preventing autoreactive B cells from entering the mature B cell pool, it is likely that elevated levels of IFN-α exacerbate the breach of B cell tolerance in lupus.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/ifn-%ce%b1-induces-altered-transitional-b-cell-signaling-and-function-in-systemic-lupus-erythematosus/