Regulation of B1 cell migration by signals through Toll-like receptors

Abstract

Peritoneal B1 cells are known to generate large amounts of antibodies outside their residential site. These antibodies play an important role in the early defense against bacteria and viruses, before the establishment of adaptive immune responses. Although many stimuli, including antigen, lipopolysaccharide, or cytokines, have been shown to activate B1 cells and induce their differentiation into plasma cells, the molecular signals required for their egress from the peritoneal cavity are not understood. We demonstrate here that direct signals through Toll-like receptors (TLRs) induce specific, rapid, and transient down-regulation of integrins and CD9 on B1 cells, which is required for detachment from local matrix and a high velocity movement of cells in response to chemokines. Thus, we revealed an unexpected role for TLRs in governing the interplay between integrins, tetraspanins, and chemokine receptors required for B1 cell egress and, as such, in facilitating appropriate transition from innate to adaptive immune responses.

Figure 1.

Bacteria and bacterial components induce peritoneal B1 cell egress. (A) Bacteria identified by sequence analyses of the 16S rRNA genes in omentum (OM) and mesenterium (MES) after s.c. injection of indomethacin. Each color represents a specific sequence, and the numbers indicate percentages in sequenced clones. (B) Representative flow cytometric profiles of peritoneal cavity cells in control (Indo−) or mice treated 24 h previously with indomethacin (Indo+). Numbers indicate the percentages of B1 cells (IgM^hiMac-1⁺) and monocytes-MΦ (IgM⁻Mac-1^hi) in total cells. (C) Absolute numbers of B1 cells and monocytes-MΦ in the peritoneal cavity before and 24 h after indomethacin treatment. (D) Representative flow cytometric profiles and (E) total numbers of B220⁺DsRed⁺(B cells) and B220⁻DsRed⁺(MΦ) of cells from the peritoneal cavity 3 and 6 h after i.p. injection of 10⁷ E. coli (DH5α) expressing Ds-Red. (F) Total numbers of B220^lowIgM^hi Mac-1⁺ B1 cells from the peritoneal cavity and omentum at the indicated time points after i.p. injection of 10 μg lipid A. Mean ± standard error, n = 4, Student's t test. (G) Total numbers of B1 cells in the peritoneal cavity (PEC), spleen (SPL), and small intestine (SI) 14 h after i.p. injection of 10⁷ peritoneal cells from GFP Tg mice without (white bars) or with 10 μg lipid A (black bars). Mean ± standard error, n = 5, Mann-Whitney test. Competitive homing experiments between peritoneal B1 cells from GFP Tg mice (WT) and TLR-4^−/− mice. (H) Percentages of B220^lowGFP⁺ (white bars) and B220^lowGFP⁻ (black bars) B1 cells in the IgM⁺ gate, and (I) the homing index calculated by total B1 numbers in the indicated tissues 14 h after i.p. injection of peritoneal cells (1:1 ratio) into RAG2^−/− mice together with 10 μg lipid A. Error bars represent 95% confidence intervals. Data are representative of two independent experiments.

Figure 2.

TLR stimulation induces integrin down-regulation on B1 cells. (A) Representative flow cytometric profiles of cells isolated from the peritoneal cavity (PEC) and spleen (SPL) stained for IgM and the indicated integrins. Gates were set for PEC B1 (red) and SPL B2 (black). (B) Kinetics of surface integrin modulation on peritoneal B220^lowIgM^hi Mac-1⁺ B1 cells (red squares) and spleen (SPL) B220⁺IgM⁺ cells (black squares) after i.p. stimulation with 20 μg LPS. Mean ± standard error, n = 3 mice. (C) Representative flow cytometric profiles of peritoneal cavity cells stained for IgM and α4 and β1 integrins isolated from WT and TLR-4^−/− mice 5 h after i.p injection of PBS or 10 μg lipid A. Histograms show mean fluorescence intensities of surface integrins on IgM^hi Mac-1⁺ B1 cells (red gate) without (black line) and with (red line) stimulation. (D) Mean fluorescence intensities of α4, α6, and β1 integrins and CD9 of peritoneal B1 cells from MyD88^−/− mice and WT mice 5 h after i.p. injection of PBS (white bars), 1 μg PAM2CSK4 (black bars), or 10 μg lipid A (red bars). Mean ± standard error, n = 3 mice, Student's t test.

Figure 3.

Egress of B1 cells requires coordinated down-regulation of integrins and CD9. (A) Total number of B220^lowIgM^hi B1 cells recovered from the peritoneal cavity (PEC), omentum (OM), mesenterium (MES), and spleen (SPL) 3 h after i.p. injection (without any TLR stimulation) into RAG-2^−/− mice of peritoneal cells preincubated for 20 min with the indicated blocking antibodies. Mean ± standard error, n = 4–7, Student's t test. (B) CD9 and integrin α4, α6, and β1 flow cytometric profiles of IgM⁺ gated peritoneal cavity cells. (C) Representative IgM and CD9 profiles of B220⁺ gated peritoneal cavity cells 5 h after i.p. injection of PBS or 20 μg LPS. Numbers indicate the mean fluorescence intensity of CD9 in gated cells. (D) Kinetics of surface expression of CD9 (red line), integrin α4 (blue line), α6 (orange line), and β1 (black line) in WT mice (squares) and TLR-2,4^−/− mice (circles) after i.p. injection of 20 μg LPS. (E) Frequency distribution of peritoneal cell velocities toward CXCL13 after incubation with isotype control or 200 μg/ml anti-CD9 antibodies at the 15-min time point. p-value was measured by Student's t test. (F) Velocity of peritoneal cells from WT mice (black) or CD9^−/− mice (red) determined by measuring cell displacements with successive video frames (1 min) during 50-min chemotaxis. Data represent the mean ± standard error of a minimum of 40 cells tracked for each time point and cell phenotype. (G) Competitive migration between peritoneal B1 cells from GFP Tg mice (WT) and CD9^−/− mice. B1 cells (1:1 ratio) were preincubated for 30 min with anti-α4 or anti-α4 and 200 μg/ml anti-CD9 antibodies and injected i.p. into RAG2^−/− mice. Percentages of GFP⁺ (black bars) and GFP⁻ (white bars) cells in the B220^lowIgM^hi gate from the indicated tissues 3 h after injection. Error bars represent 95% confidence intervals, n = 2 mice/group.

Figure 4.

Involvement of chemokine and chemokine receptors for B1 cell egress. (A) Total number of IgM^hiMac1⁺B1 cells recovered from the peritoneal cavity (PEC), omentum (OM), mesenterium (MES), and spleen (SPL) of RAG2^−/− mice 8 h after i.p. injection (with 10 μg lipid A) of peritoneal cells treated for 2 h with 100 ng/ml oligomer B (white bars) or PTX (black bars). Mean ± standard error, n = 2. (B) Quantitative PCR analyses of the indicated chemokines in omentum (OM), mesothelial cells (Meso), and peritoneal MΦ (PEC MΦ) expressed as relative amounts of mRNA normalized to 36B4. Bars represent the mean ± standard error of three experiments. (C) Whole mount pictures of omentum stained for CXCL13 and lymphatic endothelium marker (Lyve-1). Bar, 50 μm. (D) Total numbers of B1 cells in the peritoneal cavity (PEC) and omentum (OM) 14 h after i.p. injection of 10⁷ peritoneal cells from GFP Tg mice without or with 10 μg lipid A into WT mice (white bars) or CXCL13^−/− mice (black bars). Mean ± standard error, n = 3, Student's t test.