Mice treated with PBS were used as controls

Mice treated with PBS were used as controls. of the glucocorticoid receptor in CD19-Cre loxP Nr3c1 mice attenuated lymphocytopenia after tMCA. In twenty patients with acute stroke, increased cortisol levels inversely correlated with blood lymphocyte numbers. Conclusions: Our data demonstrate that this hypothalamicCpituitaryCadrenal axis mediates B lymphopoiesis defects after ischemic stroke. < 0.05, **< 0.01, and ***< 0.001, ns, not significant, Students test. CHMFL-ABL-039 The observed post-stroke loss of bone marrow B cell progenitors could be a result of either cell mobilization, a block of progenitor differentiation or induction of apoptosis in these cells. Because the spleen is the main site where bone marrow-generated immature B220int IgM+ B cells mature into IgD+ mature B cells in constant state26, and inflammatory signals may enhance B cell precursor mobilization to the spleen27, we looked for evidence of B cell trafficking early after stroke. In the first three days after tMCAO, Lin? B220int CD93+ developing B cells gradually declined as mice joined the subacute phase. However, B cell progenitor cells were barely detectable in the blood and spleen (Physique 2A), suggesting that mobilization into the blood did not cause the cells decline in the marrow. Enumerating circulating and splenic CD19+ B cells revealed severe peripheral lymphopenia and splenic atrophy, consistent with previous Mouse monoclonal to Dynamin-2 reports5,12 (Physique 2B-C). In addition, and concomitant with fewer early B cells, we also found a significantly enlarged pool of mature B220high IgD+ B cells in the bone marrow following stroke (Physique 3A). Due to absence of dsRed+ B cell precursor-derived cells in the bone marrow after stroke (Physique 1D), we speculated that increased mature IgD+ B cell numbers could be the result of their accumulation from the periphery rather than from enhanced B cell maturation directly from upstream progenitors within the bone marrow compartment28,29. Mature B cells had not incorporated BrdU 24 hours after injection, indicating that their increased number does not arise from upstream progenitors proliferation (Physique 3B). To confirm this hypothesis, we adoptively transferred dsRed+ B lymphocytes isolated from the spleen into wild-type mice prior to stroke induction. Intravital microscopy of the skull revealed a significant accumulation of dsRed+ cells in extravascular bone marrow spaces of animals with stroke when compared to sham-operated controls (Physique 3C-D), which was confirmed by flow cytometry analyses (Physique 3E). We next wondered if the decline of B cell production in the bone marrow was the result of the crosstalk between accumulating peripheral mature B cells and their progenitors as previously reported in the context of aging30. However, we found that bone marrow loss of early B cells still occurred in Rag-deficient mice with stroke (Physique 3F). Therefore, our data indicate that in addition to the B lymphopoietic defects, stroke also alters peripheral mature B cell trafficking by triggering their accumulation in CHMFL-ABL-039 the bone marrow. Open in a separate window Figure 2. B cell progenitor loss in the bone marrow does not result in mobilization.A, Representative FACS staining of early B cell progenitors (lineage? B220int CD93+ cells) in bone marrow, blood, and spleen at indicated time points after tMCAO. B, FACS-based enumeration of CD19+ B-lymphocytes in blood and spleen at indicated days after tMCAO induction. CHMFL-ABL-039 C, Bar graph shows total numbers of cells in the spleen over a three-day period after stroke in mice (n= 6 per group). Mean s.e.m; *< 0.05, **< 0.01, ***< 0.001. ns, not significant. Open in a separate window Figure 3. Stroke affects mature B cell trafficking.A, Quantification of mature lineage? B220high CD93? CD19+ IgD+ B cells by flow cytometry in bone marrow from mice with sham surgery versus tMCAO (n = 8-11 CHMFL-ABL-039 per group). B, 5-bromodeoxyuridine (BrdU) incorporation by mature IgD+ B cells in the bone CHMFL-ABL-039 marrow after stroke (n = 3 per group). C, C57BL/6 mice were transplanted with 5106 B cells from the spleen of dsRed donor animals one week prior to stroke or sham (n = 3 per group). Intravital microscopy of the calvarium bone marrow cavities was performed three days after surgery. Osteosense-750 highlights the endosteal surface (blue); blood vessels are visualized following in vivo labeling of endothelial cells.