Regulation of inflammatory monocyte/macrophage recruitment from the bone marrow during murine cytomegalovirus infection: role for type I interferons in localized induction of CCR2 ligands

Abstract

Monocytes/macrophages are critical early innate immune responders during murine CMV (MCMV) infection. It has been established that inflammatory monocyte/macrophages are released from the bone marrow and into the peripheral blood before entry into infected tissue sites. We previously reported a role for IFN-alpha/beta in promotion of CCR2-mediated recruitment of monocyte/macrophages into the liver in response to MCMV infection. However, the mechanisms that support the migration of monocyte/macrophages from the bone marrow and into the peripheral blood under conditions of MCMV infection have not been elucidated. Herein, we demonstrate an accumulation of monocyte/macrophages in the bone marrow of MCMV-infected CCR2-deficient mice, whereas circulating monocyte/macrophages are profoundly diminished. The CCR2 ligands MCP-1, MCP-3, and MCP-5 are detected in bone marrow and in serum from MCMV-infected mice. Furthermore, bone marrow leukocytes from naive mice produce high levels of MCP-1 and MCP-5, and moderate levels of MCP-3, when stimulated with recombinant IFN-alpha in culture. We identify bone marrow F4/80(+) cells as major producers of MCP-1, MCP-3, and MCP-5. Moreover, induction of CCR2 ligands is dependent on IFN-alpha/beta-mediated signals and MCMV infection. Taken together, the results reveal a critical role for inflammatory cytokines in stimulating production of CCR2-binding chemokines from F4/80(+) cells in the bone marrow, and they suggest that local production of chemokines supports monocyte/macrophage egress from the bone marrow into the blood during a virus infection.

FIGURE 1

CCR2-mediated effects on monocyte/macrophage accumulation in bone marrow and blood during MCMV infection. Samples were prepared from C57BL/6 (WT) or mice genetically deficient in CCR2 (CCR2^−/−) that were uninfected (day 0) or infected with MCMV for 48 h. To characterize the accumulation of monocyte/macrophages, bone marrow (A–C) and blood (D–F) leukocytes were labeled with F4/80 and CD11b and examined by flow cytometry. Representative dot plots for each group are shown after gating on live lymphocyte/monocyte populations (A and D). The percentages (B and E) and total numbers (C and F) of F4/80⁺CD11b⁺ cells are shown. Data shown represent the means ± SE of six to nine mice from at least two independent experiments. *, p ≤ 0.04 as compared with WT controls.

FIGURE 2

Characterization of CCR2-mediated effects on inflammatory monocyte/macrophage accumulation in the bone marrow and blood. Samples were prepared from C57BL/6 (WT) or mice genetically deficient in CCR2 (CCR2^−/−) that were uninfected (day 0) or infected with MCMV for 48 h. To characterize the accumulation of inflammatory monocyte/macrophages, bone marrow (A–C) and blood (D–F) leukocytes were labeled with F4/80, CD11b, and Ly6C and examined by flow cytometry. Inflammatory monocyte/macrophages were identified by analysis of Ly6C high expression after gating on the F4/80⁺CD11b⁺ cells. Representative dot plots and histograms for each group are shown (A and D). The percentages (B and E) and total numbers (C and F) of Ly6C^highF4/80⁺CD11b⁺ cells are shown. Data shown represent the means ± SE of six to nine mice from two independent experiments. *, p ≤ 0.04; **, p = 0.008 as compared with WT controls.

FIGURE 3

Kinetics of MCP-1, MCP-3, and MCP-5 in the bone marrow and serum during MCMV infection. Serum samples and bone marrow leukocytes were prepared from C57BL/6 mice that were uninfected or infected with MCMV after the indicated hours. For generation of leukocyte conditioned media, bone marrow cells were cultured for 24 h in medium without additional stimulation. Spontaneous release of MCP-1, MCP-3, or MCP-5 in cell-free supernatants (A) or in serum (B) was measured by standard sandwich ELISA. Data represent the means ± SE (n = 2–3 mice tested individually). Results are representative of three independent experiments.

FIGURE 4

IFN-α/β effects on CCR2 ligand production. Total bone marrow leukocytes from naive C57BL/6 (WT) or IFN-α/βR^−/− mice were pooled (n = 3–4 mice/group) and stimulated with rIFN-α at the doses indicated. Leukocytes were cultured overnight, and collected cell-free su-pernatants were evaluated for production of MCP-1, MCP-3, and MCP-5 by sandwich ELISA. Data represent the means ± SE of two independent experiments.

FIGURE 5

IFN-α/β effects on CCR2 ligand production during MCMV infection. Total bone marrow leukocytes were collected from uninfected or 40 h MCMV-infected WT or IFN-α/βR^−/− mice. Conditioned media was generated from leukocyte cultures as described in Materials and Methods and evaluated for (A) MCP-1, (B) MCP-5, and (C) MCP-3 production by ELISA. Shown are the means ± SE (n = 2–3 mice/group). Data are representative of three experiments. ‡, Below the level of detection.

FIGURE 6

Characterization of IFN-α/β-responding cells in bone marrow for CCR2 ligand production. Bone marrow leukocytes were prepared from uninfected C57BL/6 mice and enriched for F4/80 as described in Materials and Methods. Leukocyte-conditioned media were generated from pooled total, F4/80-enriched (F4/80⁺), and F4/80-depleted (F4/80⁻) cellular fractions after 24 h of incubation with or without rIFN-α at the doses indicated. Collected cell-free supernatants were evaluated in duplicate or triplicate for production of MCP-1 (A), MCP-3 (C), and MCP-5 (B) using ELISA. Data shown represent the means ± SE. Results are representative of two independent experiments. ‡, Below the level of detection.

FIGURE 7

Effects of IFN-α/β signaling on CCR2 ligand production by F4/80⁺ bone marrow cells. Bone marrow leukocytes were prepared from C57BL/6 (WT) or IFN-α/β receptor-deficient (IFN-α/βR^−/−) mice that were uninfected or infected for 40 h with MCMV. Bone marrow leukocytes were pooled from two to three mice per group and enriched or depleted of F4/80⁺ cells and plated to generate leukocyte conditioned media. Collected cell-free supernatants were evaluated in duplicate or triplicate for production of (A) MCP-1, (B) MCP-5, and (C) MCP-3 using ELISA. Data shown represent the means ± SE. Results are representative of at least two independent experiments. ‡, Below the level of detection.