TLR4 signals in B lymphocytes are transduced via the B cell antigen receptor and SYK

Abstract

Toll-like receptors (TLRs) play an important role in immune responses to pathogens by transducing signals in innate immune cells in response to microbial products. TLRs are also expressed on B cells, and TLR signaling in B cells contributes to antibody-mediated immunity and autoimmunity. The SYK tyrosine kinase is essential for signaling from the B cell antigen receptor (BCR), and thus for antibody responses. Surprisingly, we find that it is also required for B cell survival, proliferation, and cytokine secretion in response to signaling through several TLRs. We show that treatment of B cells with lipopolysaccharide, the ligand for TLR4, results in SYK activation and that this is dependent on the BCR. Furthermore, we show that B cells lacking the BCR are also defective in TLR-induced B cell activation. Our results demonstrate that TLR4 signals through two distinct pathways, one via the BCR leading to activation of SYK, ERK, and AKT and the other through MYD88 leading to activation of NF-κB.

Figure 1.

SYK is required for TLR-induced B cell activation. (A and B) Number of live control (Syk^fl/+MCM) and mutant (Syk^fl/−MCM) B cells after 48-h (A) or 72-h (B) culture with the indicated stimuli, normalized to the number in control B cells stimulated with anti-IgM, which was set to 100. Medium only (med), lipid A from S. minnesota (LAS) or E. coli (LAE), synthetic lipid A (SLA), and CD40L and IL4 (C+I). A, n = 6 (Syk^fl/+MCM) and 8 (Syk^fl/−MCM); B, n = 10 (Syk^fl/+MCM) and 14 (Syk^fl/−MCM). (C) Cell surface expression of CD86 (as measured by mean fluorescence intensity [MFI]) on control or SYK-deficient B cells cultured for 24 h with the indicated stimuli; one of three independent experiments. n = 8 (Syk^fl/+MCM) and 6 (Syk^fl/−MCM). (D) Fraction of B cells that had divided after 72 h of culture with the indicated stimuli. n = 10 (Syk^fl/+MCM) and 14 (Syk^fl/−MCM). (E) Histogram of expression of intracellular SYK measured by flow cytometry in control B cells or mutant B cells that had divided or remained undivided in cultures with LPS; representative of experiments with 11 Syk^fl/+MCM and 12 Syk^fl/−MCM mice. (F) B cells were isolated from radiation chimeras reconstituted with BM cells of the indicated genotypes infected with a retroviral vector expressing GFP and Bcl-x_L. Histograms on the left show overlay of GFP expression in B cells taken straight out of mice (ex vivo) or after culture for 72 h with LPS or in medium only (med), indicating an increase in GFP expression in cultured Syk^fl/−MCM B cells. 2D dot plots on the right show dilution of the CPD plotted against GFP expression, showing LPS-induced proliferation in Syk^fl/+MCM but not Syk^fl/−MCM B cells; numbers indicate percentage of cells in each quadrant; representative of three experiments (total n = 10 of each genotype). (G) Control or SYK-deficient B cells labeled with CFSE or CPD, respectively, were injected into TLR4-deficient mice that were treated with LPS. Flow cytometric analysis of CFSE⁺ or CPD⁺Syk⁻ B cells isolated from the spleen of recipient mice 3 d later shows that some B cells had undergone division as seen by dilution of dye (boxes). Percentages of cells that had divided are indicated; representative of two experiments. (H) Number of live B cells remaining after 48-h culture of wild-type B cells with a range of concentrations of three SYK inhibitors, BAY 61-3606 (BAY), PRT062607 (PRT), or R406, or with concentrations of vehicle (DMSO) equivalent to those used for the inhibitors. Cells were cultured in medium only or with LPS or CD40L + IL4, and cell numbers were normalized to cells treated with no inhibitor, which was set to 100. Representative of three independent experiments. Graph shows mean ± SEM of triplicates. For comparison, the numbers of control (Syk^fl/+MCM, n = 6) and mutant (Syk^fl/−MCM, n = 8) B cells remaining after 48 h of culture are also shown, normalized to the number of control cells, which was set to 100% (data taken from experiment in A). Graphs show mean ± SEM. Statistical analysis was performed using a Mann–Whitney test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 2.

SYK is required for TLR4-induced cytokine secretion. (A) Levels of mRNA for IL10, IL6, and TNF in control and mutant B cells cultured for the indicated times with LPS or of cytokines secreted by B cells cultured for 16 h in the absence (−) or presence (+) of LPS. Cytokine mRNA levels were normalized to levels of Hprt mRNA. AU, arbitrary units; nd, not detectable. (B) Fraction of B cells positive for intracellular IL10 after culture for 5 h in the presence (+) or absence (−) of LPS. (C) Fraction of splenic B cells that were CD5⁺CD1d⁺. (D) Fraction of CD5⁺CD1d⁺ B cells positive for intracellular IL10 after culture for 5 h in the presence (+) or absence (−) of LPS. Graphs show mean ± SEM. Sample numbers: A, 3 (mRNA), 4 (cytokine protein, Syk^fl/+MCM), and 6 (Syk^fl/−MCM); B, 5; C, 4; and D, 5. Statistical analysis was performed using two-way ANOVA (p-values for effect of genotype; A, mRNA) and a Mann–Whitney test (A, cytokine protein; C): *, P < 0.05; **, P < 0.01.

Figure 3.

SYK transduces TLR4 signals leading to activation of ERK and AKT but not NF-κB. (A–C) Immunoblots of lysates from control and mutant B cells stimulated with LPS (A), CD40L (B), or LAS (C) for the indicated times. Blots were probed with antibodies to phospho-Ser473 AKT (pAKT), phospho-Thr202/Tyr204 ERK1/ERK2 (pERK), SYK, IκBα, and ERK2. Graphs show mean ± SEM levels of pAKT, pERK, and IκBα normalized to ERK2 and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: A, 3 (pAKT), 9 (pERK), and 3 (IκBα); B, 3 (pAKT), 7 (pERK), and 6 (IκBα); C, 4 (pERK) and 4 (IκBα). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using two-way ANOVA (p-values for effect of genotype): *, P < 0.05; ****, P < 0.0001.

Figure 4.

BCR is required for TLR4-induced activation of SYK. (A) Immunoblot of lysates from wild-type B cells stimulated for various times with LPS (left) or LAS (right). Blots were probed with antibodies to pSYK and SYK. Graphs show mean ± SEM levels of pSYK normalized to SYK or tubulin and then to unstimulated cells (time 0), quantitated from multiple immunoblots. n = 4 (LPS) and 3 (LAS). (B–E) Immunoblots of lysates from IgM⁺ or IgM⁻ B cells (B–D) or Myd88^+/+ or Myd88^−/− B cells (E) stimulated with LPS (B and E), CD40L (C), or LAS (D) for the indicated times. Blots were probed with antibodies to pSYK, pAKT, pERK, SYK, ERK2, and IκBα. Graphs show mean ± SEM levels of pSYK, pAKT, pERK, and IκBα normalized to SYK or ERK2, and then to the levels in unstimulated control B cells (time 0), quantitated from multiple immunoblots. Minimum number of repeats per time point: B, 9 (pSYK except 3 for 15 min), 8 (pAKT), 7 (pERK), and 5 (IκBα); C, 3 (pERK), 3 (IκBα), and 2 (pAKT); D, 3 (pSYK), 4 (pERK), and 4 (IκBα); E, 9 (pSYK) and 7 (pAKT); 2 (pERK and IκBα in WT), 4 (pERK and IκBα in Myd88^−/−). Numbers on righthand edge of all blots indicate molecular mass makers (kilodaltons). Statistical analysis was performed using a Mann–Whitney test (A and B, pAKT) or two-way ANOVA (all other graphs; p-values for effect of genotype): *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Figure 5.

BCR is required for TLR-induced B cell activation. (A) Fraction of control (IgM⁺) and mutant (IgM⁻) B cells that had divided after 72 h of culture with the indicated stimuli; abbreviations of stimuli as in Fig. 1 A. Plots show combined results from three experiments with at least nine replicates for each sample, except for IgM⁻ cells stimulated with CD40L alone, for which there were six replicates. (B) Cell surface expression of CD86 (mean fluorescence intensity [MFI]) on IgM⁺ or IgM⁻ B cells cultured for 24 h with the indicated stimuli; one of two independent experiments with n = 5 and 6 replicates for IgM⁺ and IgM⁻ cells, respectively. Graphs show mean ± SEM. Statistical analysis was performed using a Mann–Whitney test; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.