Figure 4.
CFUs in BM of Il6−/− and LeprCre;Il6fl/fl mice upon chronic repetitive LPS treatment and evidence for direct IL-6 action on HSPCs. (A) Experimental outline illustrating chronic repetitive LPS treatment of Il6−/−, Lepr-Cre;Il6fl/fl, and control mice followed by collection of whole BM cells that were subjected to CFU assays. This schedule is applicable to experimental data depicted in panels B and C. (B-C) CFU activity shown as absolute CFU number per 1 hind leg in the BM of Il6−/−, Lepr-Cre;Il6fl/fl and respective control mice after chronic repetitive in vivo LPS treatment according to panel A. (D) Left panel shows a representative flow cytometric analysis for CD126 (IL-6R α chain, blue histograms) expression in LT-HSC, HPC, common myeloid progenitor (CMP), GMP, megakaryocyte-erythrocyte progenitor (MEP), CD11b+Gr1high, and CD11b+Gr1low cells in steady-state WT mice compared with isotype-matched controls (gray histograms). Right panel shows the delta mean fluorescent intensities (ΔMFI) as calculation of MFI testor – MFI isotype control from 3 independent experiments. (E) Left panel shows a representative flow cytometric analysis for CD130 (gp130, red histograms) expression in LT-HSC, HPC, CMP, GMP, MEP, CD11b+Gr1high, and CD11b+Gr1low cells in steady-state WT mice compared with appropriate isotype controls (gray histograms). Right panel shows the ΔMFI calculated as described earlier. Results from 3 independent experiments are shown. (F) CFU activity in c-Kit–enriched BM cells isolated from steady-state WT mice and cultured in the presence or absence of IL-6 but in otherwise fully cytokine-supplemented methylcellulose medium (initial plating 0°). After 7 days in culture, colonies were scored, cells harvested, and 3000 cells were re-plated (1° re-plating) and grown under the same conditions (ie, ±IL-6) for another 7 days. Upon the second re-plating (2°) no colony growth could be observed. *P < .05, **P < .01, ***P < .001, ****P < .0001. BFU-E, burst-forming unit erythrocyte; CFU-G, CFU granulocyte; CFU-GEMM, CFU granulocyte/erythrocyte/macrophage/megakaryocyte; CFU-GM, CFU granulocyte/macrophage; CFU-M, CFU macrophage; i.p., intraperitoneal; ns, nonsignificant.

CFUs in BM of Il6−/− and LeprCre;Il6fl/fl mice upon chronic repetitive LPS treatment and evidence for direct IL-6 action on HSPCs. (A) Experimental outline illustrating chronic repetitive LPS treatment of Il6−/−, Lepr-Cre;Il6fl/fl, and control mice followed by collection of whole BM cells that were subjected to CFU assays. This schedule is applicable to experimental data depicted in panels B and C. (B-C) CFU activity shown as absolute CFU number per 1 hind leg in the BM of Il6−/−, Lepr-Cre;Il6fl/fl and respective control mice after chronic repetitive in vivo LPS treatment according to panel A. (D) Left panel shows a representative flow cytometric analysis for CD126 (IL-6R α chain, blue histograms) expression in LT-HSC, HPC, common myeloid progenitor (CMP), GMP, megakaryocyte-erythrocyte progenitor (MEP), CD11b+Gr1high, and CD11b+Gr1low cells in steady-state WT mice compared with isotype-matched controls (gray histograms). Right panel shows the delta mean fluorescent intensities (ΔMFI) as calculation of MFI testor – MFI isotype control from 3 independent experiments. (E) Left panel shows a representative flow cytometric analysis for CD130 (gp130, red histograms) expression in LT-HSC, HPC, CMP, GMP, MEP, CD11b+Gr1high, and CD11b+Gr1low cells in steady-state WT mice compared with appropriate isotype controls (gray histograms). Right panel shows the ΔMFI calculated as described earlier. Results from 3 independent experiments are shown. (F) CFU activity in c-Kit–enriched BM cells isolated from steady-state WT mice and cultured in the presence or absence of IL-6 but in otherwise fully cytokine-supplemented methylcellulose medium (initial plating 0°). After 7 days in culture, colonies were scored, cells harvested, and 3000 cells were re-plated (1° re-plating) and grown under the same conditions (ie, ±IL-6) for another 7 days. Upon the second re-plating (2°) no colony growth could be observed. *P < .05, **P < .01, ***P < .001, ****P < .0001. BFU-E, burst-forming unit erythrocyte; CFU-G, CFU granulocyte; CFU-GEMM, CFU granulocyte/erythrocyte/macrophage/megakaryocyte; CFU-GM, CFU granulocyte/macrophage; CFU-M, CFU macrophage; i.p., intraperitoneal; ns, nonsignificant.

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