Inflammation controls B lymphopoiesis by regulating chemokine CXCL12 expression

Abstract

Inflammation removes developing and mature lymphocytes from the bone marrow (BM) and induces the appearance of developing B cells in the spleen. BM granulocyte numbers increase after lymphocyte reductions to support a reactive granulocytosis. Here, we demonstrate that inflammation, acting primarily through tumor necrosis factor alpha (TNFalpha), mobilizes BM lymphocytes. Mobilization reflects a reduced CXCL12 message and protein in BM and changes to the BM environment that prevents homing by cells from naive donors. The effects of TNFalpha are potentiated by interleukin 1 beta (IL-1beta), which acts primarily to expand the BM granulocyte compartment. Our observations indicate that inflammation induces lymphocyte mobilization by suppressing CXCL12 retention signals in BM, which, in turn, increases the ability of IL-1beta to expand the BM granulocyte compartment. Consistent with this idea, lymphocyte mobilization and a modest expansion of BM granulocyte numbers follow injections of pertussis toxin. We propose that TNFalpha and IL-1beta transiently specialize the BM to support acute granulocytic responses and consequently promote extramedullary lymphopoiesis.

Figure 1.

Immunization depletes BM B cells but increases BM granulocyte numbers. BL/6 mice were immunized with NP-CGG/IFA. Lymphocytes and granulocytes from femur and tibia were analyzed by flow cytometry; representative FACS^® profiles of BM cells (A, B220 and CD93; C, B220 and Gr-1) 3 or 4 d after immunization. Percentages of gated CD93⁺B220^lo, CD93⁻B220^hi, and Gr-1⁺B220⁻ cells are given. 3 d after immunization, CD93⁺B220^lo and CD93⁻B220^hi cell numbers fall in the BM (A), whereas Gr-1⁺B220⁻ cell numbers change little (C). Dynamics of BM B cell (B) or granulocyte (D) populations indicate that B lymphopenia persists for ≥12 d (B), whereas granulocyte numbers increase (D). Points represent mean ± SEM of CD93⁺B220^lo (•), CD93⁻B220^hi (▪), and Gr-1⁺B220⁻ cells (♦). Asterisks indicate significant differences from controls: *, P < 0.05; **, P < 0.01.

Figure 2.

Appearance of CD93⁺B220^lo cells in the periphery after immunization. CD93⁺B220^lo B cells in the blood (BL) and spleen (SPL) of immunized BL/6 mice at days 3–16 were characterized and enumerated by flow cytometry. (A) Representative FACS^® profiles of CD93⁺B220^lo and CD93⁻B220^hi cells in BL and SPL. Percentages of gated CD93⁺B220^lo cells are indicated. (B) Kinetics of CD93⁺B220^lo cell numbers in BL and SPL after immunization. Asterisks indicate significant differences from controls: *, P < 0.05; **, P < 0.01. (C) Surface IgM expression by CD93⁺B220^lo cells in BL and SPL. Shaded areas, solid lines, and dashed lines represent surface IgM expression by CD93⁺B220^lo, CD93⁻ B220^hi, and B220⁻ cells, respectively. Percentages of IgM⁺ cells in CD93⁺B220^lo cell gates are shown.

Figure 3.

Immunization increases splenic CFU-B numbers. BL/6 mice were immunized with 5 × 10⁸ SRBCs in 200 μl IFA. 4–16 d later, spleen and BM cells were harvested, and CFU-B were enumerated after culture for 7 d. (A) Femoral BM CFU-B. (B) Splenic CFU-B numbers after immunization. Asterisks indicate significant differences from controls: *, P < 0.05; **, P < 0.01.

Figure 4.

TNFα mobilizes CD93⁺B220^loIgM⁻ cells. (A) Representative FACS^® profiles of blood (BL) and spleen (SPL) cells from PBS- and TNFα-injected mice. Percentages indicate CD93⁺B220^lo cells in the lymphocyte gate. (B) IgM expression by CD93⁺B220^lo B cells from BL and SPL. Shaded areas, solid lines, and dashed lines represent surface IgM expression by CD93⁺B220^lo, CD93⁻B220^hi, and B220⁻ cells, respectively. Numbers indicate percentages of IgM⁺ cells in CD93⁺B220^lo cell gates.

Figure 5.

Adjuvant-induced BM lymphopenia and B cell mobilization in TNFα^−/− mice. TNFα^−/− mice and congenic controls were immunized with NP-CGG/IFA. 3 d after immunization, B cells in BM and spleen (SPL) were analyzed by flow cytometry. (A) Adjuvant-induced BM lymphopenia is mitigated in TNFα^−/− mice. Bars represent mean ± SD of B220^lo and B220^hi cell numbers from two femurs of naive (unshaded) or immunized (shaded) mice. Asterisks indicate significant differences from controls: *, P < 0.05; **, P < 0.01. (B) Mobilization of CD93⁺B220^lo cells is absent in TNF^−/− mice. Representative FACS^® profiles for CD93⁺B220^lo and CD93⁻B220^hi cells in the blood (BL) and SPL of naive and immunized mice are shown. Percentages of BL and SPL CD93⁺B220^lo lymphocytes (CD11c-, Gr-1–, Mac-1–, CD8-, and TER-119–negative) are shown.

Figure 6.

Adjuvants and TNFα suppress BM CXCL12. (A) Adjuvants and TNFα reduce BM CXCL12 protein, but do not alter total protein levels. Bars represent mean ± SD protein concentrations. (B) CXCL12 protein levels in BM plasma of BL/6 mice after immunization. Points represent mean ± SD CXCL12 concentrations. Asterisks indicate significant differences from controls: *, P < 0.05; **, P < 0.01. (C) Adjuvant-induced suppression of BM CXCL12 is mitigated in TNFα^−/− mice. 3 d after immunization, CXCL12 protein concentrations in BM plasma of TNFα^−/− and control mice were determined. Experimental groups are as follows: naive TNF^+/+, untreated B6.129SF2; NP-CGG/IFA TNF^+/+, immunized B6.129SF2; naive TNF^−/−, untreated TNFα^2/−; and NP-CGG/IFA TNF^−/−, immunized TNF^−/−.

Figure 7.

Transferred BM B cells do not home efficiently in immunized recipients. (A) BM cells from naive B6.SJL (CD45.1) animals were transferred into naive or immunized, congenic (CD45.2) recipients. 1 d after transfer, donor B220^lo and B220^hi cells in the BM and SPL of recipients were enumerated by flow cytometry. (B) Homing of TNFR^−/− BM B cells in immunized recipients is impaired. BM cells from naive, TNFR^−/− (CD45.2) mice were transferred into naive and immunized (CD45.1) recipients. 1 d after transfer, donor B cells in the BM and SPL of recipients were enumerated. Bars indicate mean ± SD numbers of CD45.2 B cells recovered from naive (unshaded) and immunized (shaded) recipients. Asterisks indicate significant differences from controls: *, P < 0.05; **, P < 0.01.

Figure 8.

PTX elicits BM lymphopenia and mobilizes CD93⁺B220^loIgM⁻ cells. BM, blood (BL), and spleen (SPL) cells were harvested from BL/6 mice 0–24 d after injection of 300–500 ng PTX and analyzed by flow cytometry. (A) Dynamics of BM B cell subsets (left) and granulocytes (right). Points represent mean ± SEM numbers of B220^lo (•), B220^hi (▪), and Gr-1⁺ (⋄) cells from two femurs. (B) Appearance of CD93⁺B220^loIgM⁻ cells in the periphery after PTX treatment. FACS^® profiles of CD93⁺B220^lo and CD93⁻B220^lo cells from BL and SPL in PBS- and PTX-treated mice are shown. Percentages of cells in the CD93⁺B220^lo gate are indicated. (C) PTX elicits populations of CD93⁺B220^loIgM⁻ cells in BL and SPL. Representative FACS^® profiles of surface IgM expression by CD93⁺B220^lo cells. Shaded areas, solid lines, and dashed lines represent surface IgM expression by CD93⁺B220^lo, CD93⁻B220^hi, and B220⁻ cells, respectively. Percentages of IgM⁺ cells in CD93⁺B220^lo cell gates are shown.