-treated mice showed EMH involving the myeloid, erythroid, and megakaryocytic lineages (Fig. 2C). In parallel with decreased BM harm in TNF-/- mice (Fig. 2A), EMH was much less comprehensive in livers of TMPD-treated TNF-/- mice (Fig. 2C). Constant using the BM hypocellularity, total red blood cell count, hemoglobin, and hematocrit all were lowered in TMPD-treated mice vs. untreated controls (Table 1). These abnormalities had been absent in TNF-/- mice. In contrast, TMPD did not alter leukocyte or platelet counts in wild-type mice (Table 1). As in SLE individuals, extensive cell death was apparent in BM from TMPD-treated wild-type mice by caspase-3 staining (Fig. 1H) and annexin V/7AAD staining (flow cytometry) (not shown). These similarities suggested that, as in human SLE, TNF also might be produced within the BM of mice with TMPD-lupus, prompting us to execute intracellular staining for TNF. TNF is made by BM neutrophils and monocytes and is TLR7-dependent We investigated TNF production in BM from B6 mice 2wks immediately after TMPD injection (Fig. 3). BM cells had been cultured with GolgiStop ahead of surface staining and intracellular TNF staining. Viable intracellular TNF+ BM cells have been visualized readily just after 4hr culture (Fig.Gabapentin 3A).Rivastigmine TNF was developed exclusively by CD11b+CD3-B220- cells, mainly Ly6G+ neutrophils, but also TNF+CD11b+Ly6G- monocytes/macrophages. Ly6G+TNF+ cells also had been located within the peritoneum, but were absent in spleen (Fig. 3B), indicating that TMPD-stimulated TNF production was a regional phenomenon.PMID:23671446 Induction of autoantibodies and nephritis by TMPD is abolished in TLR7-/- and IFNAR-/- mice (7). Unexpectedly, IFNAR-deficiency had small effect on BM hypocellularity (Fig.Arthritis Rheumatol. Author manuscript; available in PMC 2015 January 01.Zhuang et al.Page2A). In wild-type mice the BM and peritoneum both contained intracellular TNF+Ly6G+ neutrophils at the same time as TNF+Ly6G- monocyte/macrophages, even though in BM TNF was observed primarily in neutrophils (Fig. 3B). TNF-producing cells also have been located in TMPDtreated IFNAR-/- mice (Fig. 3C ). In contrast, though CD11b+Ly6G+ neutrophils and CD11b+Ly6G- monocytes were present within the BM and peritoneum of TLR7-/- mice, TNF generating cells had been absent (Fig. 3C ). Thus, TMPD-stimulated TNF production necessary TLR7 but not signaling by way of the IFNAR. TNF production by BM neutrophils needs B cells TNF expression in BM, but not spleen, following i.p. TMPD injection led us to examine no matter whether circulating immunoglobulin was involved. BM from TMPD-treated wild-type, but not t, mice contained TNF+ neutrophils (Fig. 4A). To evaluate no matter whether this was due to immunoglobulin secretion, t mice had been treated i.p. with TMPD followed 2wks later by i.v. injection of untreated wild-type mouse plasma or plasma from a wild-type mouse treated 2wks earlier with TMPD (Fig. 4B,C). TNF+ cells have been noticed in TMPD-treated t mice following plasma transfer from either TMPD-treated or untreated B6 donors. In untreated t mice, no TNF was developed following plasma transfer from untreated B6 donors. On the other hand, when plasma from TMPD-treated B6 donors was transferred to untreated t mice, the BM contained tiny numbers of TNF+ neutrophils. Local TNF production suppresses BM CXCL12 Hematopoietic stem cells are regulated by specialized microenvironments (“niches”) within the BM, 1 of that is formed by osteoblasts lining the bone surface (15). Other niches are localized near endothelial cells. CXCL12, a chemokine produced by mesenchymal-derived cel.