Figure 4. IL-6, TNF-α, and CCL2 are responsible for hematopoietic defects in LPS-treated mice.
(A) The cytokine levels in the plasma of PBS- or LPS-treated mice at 2 or 72 hours after treatment (mean ± SD). (B) Diagram of experimental design. Six- to eight-week-old WT or Ccr2–/– mice receive LPS on days 0 and 3 (0.3 mg per mice). At both time points, the WT mice also received control mouse IgG, whereas the Ccr2–/– mice received equal amounts of mAbs specific for TNF-α and IL-6, respectively. Mice were analyzed on day 0, 3, and 7. aIL-6, anti–IL-6; aTNF-α, anti–TNF-α. (C) Involvement of inflammatory cytokines in bone marrow hypocellularity. Data shown are (mean ± SD) bone marrow cell numbers (n = 4). (D) The frequency (top) and absolute numbers (bottom) of HSCs in bone marrow after LPS treatment and cytokine blockade. WT mice were treated with control Ig, while Ccr2–/– mice received anti–IL-6 and anti-TNF-α mAbs. Mean ± SD. (E) Effect of cytokine blockade on apoptosis of HSCs at day 7. Data shown are FACS plots of DAPI and Annexin V staining and represent data from 4 mice per group. Numbers indicate the percentage of apoptotic (Annexin V+ DAPI–) and dead (Annexin V+ DAPI+) cells. (F) Role for inflammatory cytokines in LPS-induced HSC defects. WT or anti–IL-6 and anti–TNF-α–treated Ccr2–/– mice were treated with PBS or LPS twice. Four days after the second treatment, 5 × 105 bone marrow cells were mixed with equal numbers of recipient-type (CD45.1) bone marrow cells and were transplanted into lethally irradiated CD45.1 C57BL/6 recipients. Reconstitution ratios in the recipient peripheral blood by the donor cells were monitored at 4, 8, and 12 weeks after transplantation. Data shown are mean ± SD (n = 10). *P < 0.05; **P < 0.01; ***P < 0.001.