Role of B cell receptor signaling in IL-10 production by normal and malignant B-1 cells

Abstract

B-1 cells are considered innate immune cells, which produce the majority of natural antibodies. B-1 cell responses to B cell receptor (BCR) and Toll-like receptor ligation are tightly regulated owing to the cross-reactivity to self-antigens. CD5 has been shown to play a major role in downregulation of BCR responses in B-1 cells. Here, we provide evidence for another mechanism by which BCR response is regulated in B-1 cells. B-1 cells, as well as their malignant counterpart, B cell chronic lymphocytic leukemia (B-CLL) cells, produce interleukin-10 (IL-10) constitutively. IL-10 secretion by normal B-1 cells downregulates their proliferation responses to BCR ligation. However, we found that CLL cells appear to be unique in not responding to IL-10-mediated feedback-suppressive effects in comparison to normal B-1 cells. In addition, we describe a novel role of the BCR signaling pathway in constitutive IL-10 secretion by normal and malignant B-1 cells. We found that inhibition of Src family kinases, spleen tyrosine kinase, Syk, or Bruton's tyrosine kinase reduces constitutive IL-10 production by both normal and malignant B-1 cells.

Keywords: B cell receptor; B-1 cell; IL-10; Toll-like receptor; chronic lymphocytic leukemia.

Figure 1

Hyporesponsiveness of peritoneal B-1 cells to BCR ligation is due to high IL-10 production. (A) Culture supernatants of B-2S and B-1P cells were collected after stimulation with (Fab)₂ goat anti-μ for 24 h and assayed by ELISA for IL-10. At this time point, BCR stimulation did not increase the number of either B-1P or B-2S cells recovered, indicating that the increase in IL-10 levels by B-1P cells is not due to an increase in cell number. The asterisk denotes the statistical significance (P ≤ 0.05) of differences between the responses of B-1P and B-2S cells. (B) B-1P (left panel) or B-2S (right panel) cells were cultured with anti-μ antibody in the presence or absence of anti–IL-10R antibody, and proliferation was measured by ³[H]- thymidine incorporation. The P-values (* = P ≤ 0.05) indicate the statistical significance of the differences between proliferation responses with and without anti–IL-10R antibody. (C) B-2S cells were cultured with exogenous IL-10 and proliferation was measured by thymidine incorporation. The asterisk (P < 0.05) indicates that the difference between proliferation responses with and without exogenous IL-10 is statistically significant. ND, not detectable.

Figure 2

IL-10 inhibits B-1P cell response by arresting the cells in the G₁/S phase of cell cycle. (A) B-1P cells from C57BL/6 and Il10^–/– mice were cultured with anti-IgM or anti-CD40, and proliferation response was measured by ³[H]-thymidine incorporation. *** indicates statistical significance (P ≤ 0.0005) of the differences in response between wild-type (C57BL/6) B-1P and Il10^–/– _B-1P cells. (B) IL-10 mediated inhibition of wild-type and Il10^–/– _B-1P cell proliferation responses to multiple doses of anti-IgM. The P-values (* = P ≤ 0.05) denote the statistical significance of differences between the responses of B-1P from wild-type and Il10^–/– mice. (C) CFSE-labeled B-1P cells from C57BL/6 and Il10^–/– mice were cultured with anti-IgM and analyzed for CFSE dilution. The percentage of cells in the M1 regional gate represent CFSE dilution from the second and subsequent divisions of cells based on the ModFit analysis of CFSE fluorescence. (D) B-1P cells from C57BL/6 and Il10^–/– mice were cultured with anti-IgM or anti-CD40 and cell cycle analysis was performed by propidium iodide staining. The fraction of cells in S/G₂/M was determined and plotted. The statistical significance of difference in response between wild-type and Il10^–/– B-1P cells in response to anti-IgM is shown by * = P ≤ 0.005.

Figure 3

BCR-induced IL-10 production by B-1P cells requires p38 MAPK. (A) B-1P cells were stimulated with anti-μ and treated with SB-203580, a p38 MAPK inhibitor. Culture supernatants were assayed by ELISA for IL-10. The P-values (* = P ≤ 0.05) indicate the significance of differences between IL-10 levels with or without P38 MAPK inhibitor. (B) Dose dependence of cell proliferation responses to anti-μ in the presence or absence of SB-203580 (2 μM) was measured by ³[H]-thymidine incorporation. (C) Proliferation responses of B-1P cells were measured after treatment with p38 MAPK inhibitor or anti–IL-10R antibody alone or together. (D) B-1P and B-2S cell lysates were probed with the indicated antibodies. Phospho-protein levels were normalized to total protein levels and total protein levels were normalized to β-actin.

Figure 4

IL-10 production by both peritoneal and splenic Eμ-Tcl1 cells. (A) Normal B-1 cells and peritoneal and splenic Eμ-Tcl1 cells (10⁵ cells) were cultured in 200 μl of medium for 24 h. IL-10 was measured in the supernatant of the cells by ELISA. Values represent mean ± SE of triplicate cultures. (B) Eμ-Tcl1 cells (10⁵ cells) were cultured in the presence or absence of anti– IL-10R antibody or anti–IL-10 antibody and the survival was measured by MTT assay.

Figure 5

IL-10 may be transcriptionally regulated in the Eμ-Tcl1 CLL cells. Primary Eμ-Tcl1 CLL cells were cultured with 5μM BAY 61-3606, a Syk specific inhibitor, for 6 h. IL-10 mRNA expression levels were measured by qRT-PCR and normalized to 18S reference gene to control the variability in expression levels and were analyzed using the 2^-ΔΔCT method described by Livak and Schmittgen. The P-values (* = P ≤ 0.05) indicate the significance of differences between IL-10 mRNA levels with or without Syk inhibitor.