Figure 6.
Granulocyte BM distribution, migratory response and CXCR4 expression in Dnm2-defective mouse granulocytes. (A-F) Immunohistochemical staining for mature granulocytes with Ly6G in the BM of representative CTRL (panels A-B) and Dnm2het female mice (panels C-F). Fewer Ly6G positive cells (granulocytes) were present in the BM of Dnm2het mice when comparing groups involving all ages (N = 30 and above). Histology slide images were captured using the Aperio AT2 slide scanner at 20X or the CRI Nuance Spectral Imaging microscope, unless specified otherwise. (G) A linear pattern of distribution of Ly6G positive cells (granulocytes) was observed in fewer mice among the Dnm2het group than in the CTRL group, indicating that the migration pattern within the BM is altered in the Dnm2het group (N = 30 and above). (Panel F) The BM from 1 Dnm2het mouse was completely effaced by immature myeloid precursors. (H) ANC in the peripheral blood 1 day after intraperitoneal alum injection in old female Dnm2het and CTRL mice. Older Dnm2het female mice (>52 weeks, N = 3) failed to show a rise of ANC, indicating a loss of rapid egress of neutrophils from the BM reserve. (I) Transwell migration assay of BM neutrophils from wild-type young female adults (aged 12-24 weeks, N = 3) treated with the DNM inhibitor DNSR, compared with CTRL neutrophils. Wild-type mouse neutrophils separated from the BM and treated with DNM-GTPase inhibitor, DNSR have reduced migration toward n-formylmethionyl-leucyl-phenylalanine, a chemoattractant for neutrophils, indicating that DNM function is necessary for this chemotactic response. (J) CXCR4 surface expression analyzed by flow cytometry in DNSR-treated and in CTRL BM Ly6G-high granulocytes from wild-type young female adults (aged 12-24 weeks, N = 4), when exposed for varying time periods to chemoattractant SDF1a (100 ng/mL). After 30 minutes of SDF1a exposure, surface CXCR4 was expressed to a greater extent among DNSR-treated neutrophils than in CTRL neutrophils. (Panels H-J) Each graph summarizes at minimum 3 to 4 independent experiments. Error bars indicate SEM. Only marked, group-wise comparisons are significant. ∗P < .05, t-test.

Granulocyte BM distribution, migratory response and CXCR4 expression in Dnm2-defective mouse granulocytes. (A-F) Immunohistochemical staining for mature granulocytes with Ly6G in the BM of representative CTRL (panels A-B) and Dnm2het female mice (panels C-F). Fewer Ly6G positive cells (granulocytes) were present in the BM of Dnm2het mice when comparing groups involving all ages (N = 30 and above). Histology slide images were captured using the Aperio AT2 slide scanner at 20X or the CRI Nuance Spectral Imaging microscope, unless specified otherwise. (G) A linear pattern of distribution of Ly6G positive cells (granulocytes) was observed in fewer mice among the Dnm2het group than in the CTRL group, indicating that the migration pattern within the BM is altered in the Dnm2het group (N = 30 and above). (Panel F) The BM from 1 Dnm2het mouse was completely effaced by immature myeloid precursors. (H) ANC in the peripheral blood 1 day after intraperitoneal alum injection in old female Dnm2het and CTRL mice. Older Dnm2het female mice (>52 weeks, N = 3) failed to show a rise of ANC, indicating a loss of rapid egress of neutrophils from the BM reserve. (I) Transwell migration assay of BM neutrophils from wild-type young female adults (aged 12-24 weeks, N = 3) treated with the DNM inhibitor DNSR, compared with CTRL neutrophils. Wild-type mouse neutrophils separated from the BM and treated with DNM-GTPase inhibitor, DNSR have reduced migration toward n-formylmethionyl-leucyl-phenylalanine, a chemoattractant for neutrophils, indicating that DNM function is necessary for this chemotactic response. (J) CXCR4 surface expression analyzed by flow cytometry in DNSR-treated and in CTRL BM Ly6G-high granulocytes from wild-type young female adults (aged 12-24 weeks, N = 4), when exposed for varying time periods to chemoattractant SDF1a (100 ng/mL). After 30 minutes of SDF1a exposure, surface CXCR4 was expressed to a greater extent among DNSR-treated neutrophils than in CTRL neutrophils. (Panels H-J) Each graph summarizes at minimum 3 to 4 independent experiments. Error bars indicate SEM. Only marked, group-wise comparisons are significant. ∗P < .05, t-test.

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