Session Title: Cytokines, Mediators, and Gene Regulation
Session Type: Abstract Submissions (ACR)
Background/Purpose: SLE immune complexes (IC) induce IFN-α production by stimulation of TLR7 and 9 in plasmacytoid dendritic cells (pDC). Serum from normal human donors inhibits IC-induced IFN-α in vitro and both C1q and IgG have been identified as inhibitory serum components. Since pooled IgG (IVIG) is used for therapy of autoimmune diseases and the sialylated subfraction implicated in its mode of action, we addressed how IVIG regulates IFN-α production by pDC.
Methods: Normal human peripheral blood mononuclear cells (PBMC) or negatively selected pDC were stimulated with SLE IC or the TLR 7 and 9 agonists, Loxoribine (Lox) or CpG-A respectively. IFN-α was quantified in the supernatants by ELISA. Unsorted IVIG or IVIG that had been enriched (SNA+) or depleted (SNA-) of the sialylated subset by a lectin affinity column or digested into Fc or F(ab’)2 fragments were added to stimulated cultures. SLE IC containing Alexa Fluor 647 labeled SmRNP antigen was allowed to bind to PBMC with or without pretreatment with FcγRII blocking antibody or IVIG Fc, and the binding to pDC quantified by flow cytometry.
Results: IVIG dose-dependently inhibited SLE IC-induced IFN-α (~20%, 50%, and 95% inhibition by treatment with 50, 500, and 5000 μg/mL IVIG, respectively). Inhibition was Fc-dependent (500 μg/mL IVIG inhibited 50% of IFN-α compared to molar equivalents of F(ab’2), <10%, and Fc, 60%, p<0.01), but was sialylation-independent. IgG Fc inhibited SmRNP IC binding to pDC (50% of pDC bind IC, which was reduced to 20% with 167 μg/mL Fc, p<0.001). In contrast, TLR agonist-induced IFN-α was only modestly (10-20%) inhibited by high doses (5000 μg/mL) of IVIG and inhibition was significantly higher by the SNA+ versus SNA- fraction of IVIG (Lox: 90% and 40%, p<0.001; CpG 75% and 0%, p<0.001, for SNA+ versus SNA- respectively). Furthermore, the inhibitory activity was contained in the F(ab’)2 fragment. The inhibitory activity of sialylated IVIG was not direct on pDC, but required the presence of monocytes (depletion of CD14+ monocytes reduced inhibition by 500 μg/mL SNA+ IVIG after Lox stimulation of IFN-α from 70% to 0%, p<0.05 while depletion of CD19+ B cells or CD56+ NK/NKT cells had no effect). Monocytes produced prostaglandin E2 (PGE2) specifically in response to the sialylated IVIG subset (10-20 ng/mL with SNA+ IVIG compared to undetectable quantities with SNA- IVIG, p<0.05). Furthermore, blockade of PGE2 from the monocyte supernatants reduced inhibitory activity to <10%, and addition of PGE2 blocked IFN-α production.
Conclusion: IVIG Fc directly inhibits production of IFN-α in response to SLE IC by blocking IC binding to FcγRIIa on pDC. In contrast, the SNA+ subset of IVIG inhibits TLR agonist stimulation of IFN-α by inducing the production of PGE2 by monocytes. Understanding these disparate mechanisms of IVIG inhibition of IFN-α will provide novel methods for immunomodulation and may allow use of smaller amounts of subcomponents of IVIG for therapy.
K. B. Elkon,
Hoffman La Roche ,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/different-mechanisms-responsible-for-ivig-inhibition-of-immune-complex-versus-tlr-stimulated-interferon-alpha/