Search

Bone marrow (BM) and lymphocyte samples from aplastic anemia patients show up-regulated Fas and Fas-ligand (FasL) expression, respectively, supporting a relationship between immune-mediated BM destruction and the Fas apoptotic pathway. Mice with spontaneous lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) mutations exhibit abnormal expression of Fas and FasL, serving as potential models to elucidate underlying mechanisms of BM failure. We examined cellular and functional characteristics of lpr and gld mutants on the C57BL/6 (B6) background. Lymph node (LN) cells from lpr and gld mice produced less apoptosis when coincubated with C.B10-H2b/LilMcd (C.B10) BM cells in vitro. This functional difference was confirmed by infusing lpr, gld, and B6 LN cells into sublethally irradiated CB10 mice. All donor LN cells showed significant T cell expansion and activation, but only B6 LN cells caused severe BM destruction. Mice infused with gld LN cells developed mild to moderate BM failure despite receiving FasL-deficient effectors, thus suggesting the existence of alternative pathways or incomplete penetrance of the mutation. Paradoxically, mice that received Fas-deficient lpr LN cells also had reduced BM failure, likely due to down-regulation of proapoptotic genes, an effect that can be overcome by higher doses of lpr LN cells. Our model demonstrates that abnormal Fas or FasL expression interferes with the development of pancytopenia and marrow hypoplasia, validating a major role for the Fas/FasL cytotoxic pathway in immune-mediated BM failure, although disruption of this pathway does not completely abolish marrow destruction.

Objective To test function of hematopoietic stem cells (HSCs) in vivo in C57BL/6 (B6) and Trp53-deficient (Trp53 null) mice by using two HSC enrichment schemes. Materials and Methods Bone marrow (BM) Lin − CD41 − CD48 − CD150 + (signaling lymphocyte activation molecules [SLAM]), Lin − CD41 − CD48 − CD150 − (SLAM − ) and Lin − Sca1 + CD117 + (LSK) cells were defined by fluorescence-activated cell staining (FACS). Cellular reactive oxygen species (ROS) level was also analyzed by FACS. Sorted SLAM, SLAM − , and LSK cells were tested in vivo in the competitive repopulation (CR) and serial transplantation assays. Results SLAM cell fraction was 0.0078% ± 0.0010% and 0.0135% ± 0.0010% of total BM cells in B6 and Trp53 null mice, and was highly correlated ( R 2 = 0.7116) with LSK cells. CD150 + BM cells also contained more ROS low cells than did CD150 − cells. B6 SLAM cells repopulated recipients much better than B6 SLAM − cells, showing high HSC enrichment. B6 SLAM cells also engrafted recipients better than Trp53 null SLAM cells in the CR and the follow-up serial transplantation assays. Similarly, LSK cells from B6 donors also had higher repopulating ability than those from Trp53 null donors. However, whole BM cells from the same B6 and Trp53 null donors showed the opposite functional trend in recipient engraftment. Conclusion Both SLAM and LSK marker sets can enrich HSCs from B6 and Trp53 mice. Deficiency of Trp53 upregulates HSC self-renewal but causes no gain of HSC function.

Fas-Fas ligand and perforin-granzyme are two important cell death pathways associated with cytotoxic T cell induced target cell apoptosis. In patients with immune-mediated aplastic anemia, the development of bone marrow (BM) failure is associated with up-regulation in Fas ligand expression on effector cytotoxic T cells and elevated Fas expression on target BM cells. In some aplastic anemia patients, peripheral blood T lymphocytes also carry polymorphisms in the perforin gene which have been associated with familial hemophagocytosis. These findings suggested that Fas ligand/Fas might be the key signaling molecules mediating cell destruction while perforin might also play a role in the development of BM failure in patients with aplastic anemia. We have modeled immune-mediated BM failure in the mouse by infusing allogeneic lymph node (LN) cells from C57BL/6 (B6) donors into sublethally-irradiated CByB6F1 and C.B10 recipients that are mismatched at either major histocompatibility (MHC) or minor histocompatibility (minor-H) loci. Expansion and activation of allogeneic T cells results in increased production of the inflammatory cytokines gamma interferon and tissue necrosis factor alpha in recipient BM, massive BM cell destruction, severe marrow hypoplasia, and fatal pancytopenia. In the current study, we directly tested the roles of Fas, Fas ligand and perforin in the development of BM failure by using murine models with spontaneous mutations at the lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) loci, or with germline deletion of the gene perforin (prf−/−). Fas and Fas ligand-deficient lpr and gld mutant mice had no evidence of hematopoietic deficiency despite their autoimmune environment and marked lymphoproliferation. LN cells from lpr and gld mice caused significantly less apoptosis to minor-H mismatched C.B10 BM cells when co-cultured in a cytotoxicity assay in vitro, in comparison to LN cells from wild-type B6 mice. Infusion of lpr, gld, and B6 donor LN cells into sub-lethally irradiated CB10 recipients all caused massive T cell expansion in recipient BM with high level expression of CD11a, indicative of T cell activation, but only B6 LN cells caused severe BM destruction. In contrast, recipients of lpr and gld LN cells had only mild to moderate pancytopenia and marrow hypocellularity. We inferred from these results that disruption of the Fas ligand/Fas signaling pathway effectively abrogated immune mediated marrow destruction. To test the role of perforin in BM failure, we first analyzed prf−/−- mice and found no obvious change in cellular composition in lymphohematopoietic tissues in comparison to wild-type B6 controls. LN cells from prf−/− mice showed reduced ability to induce C.B10 BM cell apoptosis in an in vitro cytotoxicity assay when compared to wild-type B6 LN cells. Infusion of 5–10 million prf−/− LN cells into CByB6F1 and C.B10 recipients produced obvious BM failure in both recipient types with pancytopenia and marrow hypoplasia about 80–90% as severe as in control recipients of 5 million B6 LN cells. In both CByB6F1 and C.B10 recipients, infused prf−/− LN cells resulted in less T cell expansion, a similar level of T cell activation, higher proportions of T cells containing gamma-interferon and tissue necrosis factor-alpha, and a higher proportion of T cells expressing the Fas ligand CD178, in comparison to the infused B6 LN cells. We conclude that Fas-Fas ligand-mediated transmembrane signaling provides the major cell death pathway, while perforin-granzyme-mediated exocytosis plays a minor role, in BM cell destruction in animal models of immune-mediated BM failure.

Key cellular events in the development of aplastic anemia are activation and expansion of T cells that are destructive of hematopoietic tissue. Human immune-mediated bone marrow failure has been modeled in mice by infusion of lymph node (LN) cells into major- or minor-histocompatibility antigen mismatched recipients; oligoclonal T cell expansion causes indiscriminant hematopoietic cell destruction, marked marrow hypoplasia, and fatal pancytopenia. We have utilized this simple model in order to dissect the molecular events responsible for T cell mediated, selective organ destruction. In mice with spontaneous mutations: lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld), animals have an abnormal increase in lymphocytes and develop clinical manifestations similar to those seen in patients with systemic lupus erythematosus (SLE). In contrast to human SLE, the lpr and gld bone marrow appear unaffected by this typically self-destructive process. We examined C57BL/6 (B6) mice that carry the lpr and gld mutations along with normal B6 mice to define cellular and functional differences as they relate to BM damage. In our initial analyses of peripheral blood (PB), we found a reduced CD4 T cell proportion in lpr and gld mice (5.5±1.5%, 4.3±0.6%, 9.9±0.9%) and a reduced CD8 T cell proportion in gld mice when compared to B6 controls (13.4±1.9%, 7.9±0.9%, 12.7±1.8%). In the BM, both CD4 (1.5±0.3%, 2.3±0.3%, 1.0±0.1%) and CD8 (2.9±0.3%, 3.1±0.7%, 2.6±0.2%) T cell proportions were slightly increased in lpr and gld mice, while total number of BM cells was not significantly different among the three genotypes (438±48, 324±18, 350±27, millions). These findings indicated no PB lymphocyte accumulation and no BM destruction in lpr or gld mice. We then tested the hypothesis that lpr and gld BM cells were more resistant to immune attack by infusing 5 x 106 lymph node (LN) cells from minor-histocompatibility antigen-mismatched C.B10 mice into sublethally-irradiated (5 Gys) lpr, gld, and B6 recipients. At two weeks, all recipients infused with CB10 LN cells had reduced blood neutrophils, and gld and B6 recipients also had reduced RBCs, as compared to mice that did not receive LN cell infusions. This result suggests that lpr and gld hematopoietic cells, like those from B6 mice, are susceptible to immune-mediated destruction. We then examined lymphocyte function by co-incubating lpr, gld and B6 LN cells (effectors) with C.B10 BM cells (targets) in a cytotoxilux assay in vitro. B6 effectors produced 51–78% more apoptotic targets than did gld and lpr effectors. This difference was confirmed by the infusion of lpr, gld (15 x 106 cells/recipient) and B6 (5 x 106 cells/recipient) LN cells into sub-lethally irradiated CB10 mice in vivo. At three weeks, enumeration of blood neutrophils (0.69±0.60, 1.27±0.83, 0.08±0.03), RBCs (3.58±0.42, 4.80±1.20, 4.76±0.36), platelets (274± 94, 400±156, 298±91) and total BM cells (87± 23, 195±78, 82±6, millions) demonstrated that, despite massive lymphoproliferation, lymphocytes from lpr and gld mice were less effective in mediating BM cell destruction. Our results provide evidence supporting the role of Fas and Fas-ligand mediated programmed cell death in immune-mediated BM destruction. The ready availability of genetically modified mice with well characterized functional defects should allow a full description of the molecular pathways in immune-mediated marrow failure in this model.

CD150 is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors which can be used as a positive selection marker for hematopoietic stem cells (HSCs) from mouse bone marrow (BM) and fetal liver (Kiel et al., Cell 2005; Yilmaz et al., Blood 2006; Kim et al., Blood 2006). In the current study, we combined CD150 with a set of lineage-specific markers to enrich HSCs, comparing normal C57BL/6 (B6) mice with B6-Trp53-deficient (Trp53 null) mice which were previously shown to have elevated HSC activity. Using an anti-mouse CD150 antibody (Clone TC 15-12F 12.2, BioLegend), we defined a population of Lin−CD41−CD48−CD150+(SLAM) cells that is 0.0078 ± 0.0010% and 0.0135 ± 0.0010% of total BM cells from B6 and Trp53 null mice. The size of the SLAM cell fraction was strongly correlated (R2= 0.7116, P