Immune deficiencies, infection, and systemic immune disorders
Amplification of Toll-like receptor–mediated signaling through spleen tyrosine kinase in human B-cell activation

https://doi.org/10.1016/j.jaci.2012.03.014Get rights and content

Background

B cells are activated by combined signals through the B-cell receptor (BCR) and CD40. However, the underlying mechanisms by which BCR signals synergize with Toll-like receptor (TLR) signaling in human B cells remain unclear.

Objective

We sought to elucidate a role of spleen tyrosine kinase (Syk), a key molecule of BCR signaling, in TLR-mediated activation of human B cells.

Methods

Human naive and memory B cells were stimulated with combinations of anti-BCR, soluble CD40 ligand, and CpG. Effects of the Syk inhibitors on several B-cell functions and expression of TLR9, TNF receptor–associated factors (TRAFs), and phospho–nuclear factor κB in B cells were assessed.

Results

Activation of BCR synergized with CD40- and TLR9-mediated signals in driving robust proliferation, cell-cycle progression, expression of costimulatory molecules, cytokine production, and immunoglobulin production of human B-cell subsets, especially memory B cells. However, the Syk inhibitors remarkably abrogated these B-cell functions. Notably, after stimulation through all 3 receptors, B-cell subsets induced marked expression of TLR9, TRAF6, and phospho–nuclear factor κB, which was again significantly abrogated by the Syk inhibitors.

Conclusion

Syk-mediated BCR signaling is a prerequisite for optimal induction of TLR9 and TRAF6, allowing efficient propagation of TLR9-mediated signaling in memory B cells. These results also underscore the role of Syk in aberrant B-cell activation in patients with autoimmune diseases.

Section snippets

Reagents

Syk inhibitor I, Syk inhibitor II, Syk inhibitor IV, BAY61-3606, PP1, and PP2 were purchased from Merck (Darmstadt, Germany). Lyn peptide inhibitor was purchased from Tocris Bioscience (Ellisville, Mo). PF-956980 (JAK3 kinase inhibitor) was provided from Pfizer, Inc (New York, NY). Anti-BCR mAbs (anti-Igλ and anti-Igk), recombinant human IL-2, recombinant human CD40 ligand, and phosphorothioate-protected CpG-oligonucleotide 2006 (CpG-ODN 2006; 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′) were from BD

Syk is critical for proliferation and cell-cycle progression in memory B cells

We investigated the effect of BCR, CD40, and TLR9 stimulation on the proliferation of B-cell subsets. BCR stimulation alone remarkably induced Syk phosphorylation; however, it had only marginal effects on DNA synthesis in B cells (Fig 1, A and B). Combined stimulation of BCR, CD40, and TLR9 strongly induced DNA synthesis in both naive and memory B cells, although significantly more so in the latter. This robust proliferation was inhibited by Syk inhibitor IV (BAY61-3606) in a dose-dependent

Discussion

In this study we demonstrate that engagement of BCR in conjunction with ligation of CD40 and TLR9 induces remarkable proliferation, expression of costimulatory molecules, cytokine production, and immunoglobulin production in human B cells, especially the memory subset. Moreover, the Syk inhibitor suppresses all of these functions to background levels, at least in part through inhibition of expression of TLR9 and TRAF6, resulting in decreased phosphorylation of NF-κB.

We show that combined

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    Supported in part by a Research Grant-in-Aid for Scientific Research from the Ministry of Health, Labor and Welfare of Japan; the Ministry of Education, Culture, Sports, Science and Technology of Japan; and the University of Occupational and Environmental Health, Japan.

    Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

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