技术资料

Polymorphonuclear neutrophils (PMNs) are critical for the formation,maintenance,and resolution of bacterial abscesses. However,the mechanisms that regulate PMN survival and proliferation during the evolution of an abscess are not well defined. Using a mouse model of Staphylococcus aureus abscess formation within a cutaneous wound,combined with real-time imaging of genetically tagged PMNs,we observed that a high bacterial burden elicited a sustained mobilization of PMNs from the bone marrow to the infected wound,where their lifespan was markedly extended. A continuous rise in wound PMN number,which was not accounted for by trafficking from the bone marrow or by prolonged survival,was correlated with the homing of c-kit(+)-progenitor cells from the blood to the wound,where they proliferated and formed mature PMNs. Furthermore,by blocking their recruitment with an antibody to c-kit,which severely limited the proliferation of mature PMNs in the wound and shortened mouse survival,we confirmed that progenitor cells are not only important contributors to PMN expansion in the wound,but are also functionally important for immune protection. We conclude that the abscess environment provides a niche capable of regulating PMN survival and local proliferation of bone marrow-derived c-kit(+)-progenitor cells. View Publication

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins,the widely expressed basic helix-loop-helix transcription factors,contribute to HSC and MPP activity,but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches,we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However,long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover,E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism,and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together,these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism,and demonstrate that E47 is not required for short-term myeloid differentiation. View Publication

Bone marrow (BM) hematopoietic cells are selectively sensitive to polycyclic aromatic hydrocarbons (PAH) in vivo. 7,12-Dimethylbenz(a)anthracene (DMBA),but not benzo(a)pyrene (BP),depletes BM hematopoietic cells in C57BL/6 mice. This difference is due to a BP-selective aryl hydrocarbon receptor (AhR)-mediated recovery. Colony-forming unit assays show suppression of lymphoid progenitors by each PAH within 6 h but a subsequent recovery,exclusively after BP treatment. Suppression of myeloid progenitors (6 h) occurs only for DMBA. Each progenitor responded equally to DMBA and BP in congenic mice expressing the PAH-resistant AhR (AhR(d)). AhR,therefore,mediates this BP recovery in each progenitor type. These PAH suppressions depend on Cyp1b1-mediated metabolism. Paradoxically,few genes responded to DMBA,whereas 12 times more responded to BP. Progenitor suppression by DMBA,therefore,occurs with minimal effects on the general BM population. Standard AhR-mediated stimulations (Cyp1a1,Cyp1b1,Ahrr) were similar for each PAH and for the specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin but were absent in AhR(d) mice. A group of 12 such AhR responses was sustained from 6 to 24 h. A second,larger set of BP responses (chemokines,cytokines,cyclooxygenase 2) differed in two respects; DMBA responses were low and BP responses declined extensively from 6 to 24 h. A third cluster exhibited BP-induced increases in protective genes (Nqo1,GST-mu) that appeared only after 12 h. Conversion of BP to quinones contributes oxidative signaling not seen with DMBA. We propose that genes in this second cluster,which share oxidative signaling and AhR activation,provide the AhR-dependent protection of hematopoietic progenitors seen for BP. View Publication

Pseudo-Pelger-Huët anomaly (PPHA) has been documented in association with transplant medications and other drugs. This iatrogenic neutrophilic dysplasia is reversible with cessation or adjustment of medications but is frequently confused with myelodysplastic syndrome (MDS) based on the conventional concept that PPHA is a marker for dysplasia. We investigated the clinicopathologic features in iatrogenic PPHA and compared them with MDS-related PPHA. The 13 cases studied included 5 bone marrow/stem cell transplantations,3 solid organ transplantations,1 autoimmune disease,3 chronic lymphocytic leukemias,and 1 breast carcinoma. For 12 cases,there was follow-up evaluation,and all demonstrated at least transient normalization of neutrophilic segmentation. All 9 cases of MDS demonstrated at least 2 of the following pathologic abnormalities on bone marrow biopsy: hypercellularity (8/9),morphologic dysplasia (8/9),clonal cytogenetic abnormality (7/9),and increased blasts (3/9),whereas these abnormalities were typically absent in iatrogenic PPHA. Iatrogenic PPHA displayed a higher proportion of circulating PPHA cells than in MDS (mean,47.4%; SD,31.6% vs mean,12.3%; SD,9.8; P textless .01). A diagnostic algorithm is proposed in which isolated PPHA is indicative of transient or benign PPHA unless proven otherwise. View Publication

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However,because most HES patients lack FIP1L1-PDGFRA,we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G,L507P,I562M,H570R,H650Q,N659S,L705P,R748G,and Y849S). When cloned into 32D cells,N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation,clonogenic growth,and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity,we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion,our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients. View Publication

Ecotropic viral integration site-1 (Evi-1) is a nuclear transcription factor that plays an essential role in the regulation of hematopoietic stem cells. Aberrant expression of Evi-1 has been reported in up to 10% of patients with acute myeloid leukemia and is a diagnostic marker that predicts a poor outcome. Although chromosomal rearrangement involving the Evi-1 gene is one of the major causes of Evi-1 activation,overexpression of Evi-1 is detected in a subgroup of acute myeloid leukemia patients without any chromosomal abnormalities,which indicates the presence of other mechanisms for Evi-1 activation. In this study,we found that Evi-1 is frequently up-regulated in bone marrow cells transformed by the mixed-lineage leukemia (MLL) chimeric genes MLL-ENL or MLL-AF9. Analysis of the Evi-1 gene promoter region revealed that MLL-ENL activates transcription of Evi-1. MLL-ENL-mediated up-regulation of Evi-1 occurs exclusively in the undifferentiated hematopoietic population,in which Evi-1 particularly contributes to the propagation of MLL-ENL-immortalized cells. Furthermore,gene-expression analysis of human acute myeloid leukemia cases demonstrated the stem cell-like gene-expression signature of MLL-rearranged leukemia with high levels of Evi-1. Our findings indicate that Evi-1 is one of the targets of MLL oncoproteins and is selectively activated in hematopoietic stem cell-derived MLL leukemic cells. View Publication

RARA (retinoic acid receptor alpha) haploinsufficiency is an invariable consequence of t(15;17)(q22;q21) translocations in acute promyelocytic leukemia (APL). Retinoids and RARA activity have been implicated in hematopoietic self-renewal and neutrophil maturation. We and others therefore predicted that RARA haploinsufficiency would contribute to APL pathogenesis. To test this hypothesis,we crossed Rara(+/-) mice with mice expressing PML (promyelocytic leukemia)-RARA from the cathepsin G locus (mCG-PR). We found that Rara haploinsufficiency cooperated with PML-RARA,but only modestly influenced the preleukemic and leukemic phenotype. Bone marrow from mCG-PR(+/-) × Rara(+/-) mice had decreased numbers of mature myeloid cells,increased ex vivo myeloid cell proliferation,and increased competitive advantage after transplantation. Rara haploinsufficiency did not alter mCG-PR-dependent leukemic latency or penetrance,but did influence the distribution of leukemic cells; leukemia in mCG-PR(+/-) × Rara(+/-) mice presented more commonly with low to normal white blood cell counts and with myeloid infiltration of lymph nodes. APL cells from these mice were responsive to all-trans retinoic acid and had virtually no differences in expression profiling compared with tumors arising in mCG-PR(+/-) × Rara(+/+) mice. These data show that Rara haploinsufficiency (like Pml haploinsufficiency and RARA-PML) can cooperate with PML-RARA to influence the pathogenesis of APL in mice,but that PML-RARA is the t(15;17) disease-initiating mutation. View Publication

Because lymphoid progenitors can give rise to natural killer (NK) cells,NK ontogeny has been considered to be exclusively lymphoid. Here,we show that rare human CD34(+) hematopoietic progenitors develop into NK cells in vitro in the presence of cytokines (interleukin-7,interleukin-15,stem cell factor,and fms-like tyrosine kinase-3 ligand). Adding hydrocortisone and stromal cells greatly increases the frequency of progenitor cells that give rise to NK cells through the recruitment of myeloid precursors,including common myeloid progenitors and granulocytic-monocytic precursors to the NK-cell lineage. WNT signaling was involved in this effect. Cells at more advanced stages of myeloid differentiation (with increasing expression of CD13 and macrophage colony-stimulating factor receptor [M-CSFR]) could also differentiate into NK cells in the presence of cytokines,stroma,and hydrocortisone. NK cells derived from myeloid precursors (CD56(-)CD117(+)M-CSFR(+)) showed more expression of killer immunoglobulin-like receptors,a fraction of killer immunoglobulin-like receptor-positive-expressing cells that lacked NKG2A,a higher cytotoxicity compared with CD56(-)CD117(+)M-CSFR(-) precursor-derived NK cells and thus resemble the CD56(dim) subset of NK cells. Collectively,these studies show that NK cells can be derived from the myeloid lineage. View Publication

The identity of T-cell progenitors that seed the thymus has remained controversial,largely because many studies differ over whether these progenitors retain myeloid potential. Contradictory reports diverge in their use of various in vitro and in vivo assays. To consolidate these discordant findings,we compared the myeloid potential of 2 putative thymus seeding populations,common lymphoid progenitors (CLPs) and multipotent progenitors (MPPs),and the earliest intrathymic progenitor (DN1),using 2 in vitro assays and in vivo readouts. These assays gave contradictory results: CLP and DN1 displayed surprisingly robust myeloid potential on OP9-DL1 in vitro stromal cocultures but displayed little myeloid potential in vivo,as well as in methylcellulose cultures. MPP,on the other hand,displayed robust myeloid potential in all settings. We conclude that stromal cocultures reveal cryptic,but nonphysiologic,myeloid potentials of lymphoid progenitors,providing an explanation for contradictory findings in the field and underscoring the importance of using in vivo assays for the determination of physiologic lineage potentials. View Publication

Although microbial infections can alter steady-state hematopoiesis,the mechanisms that drive such changes are not well understood. We addressed a role for IFN-γ signaling in infection-induced bone marrow suppression and anemia in a murine model of human monocytic ehrlichiosis,an emerging tick-borne disease. Within the bone marrow of Ehrlichia muris-infected C57BL/6 mice,we observed a reduction in myeloid progenitor cells,as defined both phenotypically and functionally. Infected mice exhibited a concomitant increase in developing myeloid cells within the bone marrow,an increase in the frequency of circulating monocytes,and an increase in splenic myeloid cells. The infection-induced changes in progenitor cell phenotype were critically dependent on IFN-γ,but not IFN-α,signaling. In mice deficient in the IFN-γ signaling pathway,we observed an increase in myeloid progenitor cells and CDllb(lo)Gr1(lo) promyelocytic cells within the bone marrow,as well as reduced frequencies of mature granulocytes and monocytes. Furthermore,E. muris-infected IFN-γR-deficient mice did not exhibit anemia or an increase in circulating monocytes,and they succumbed to infection. Gene transcription studies revealed that IFN-γR-deficient CDllb(lo)Gr1(lo) promyelocytes from E. muris-infected mice exhibited significantly reduced expression of irf-1 and irf-8,both key transcription factors that regulate the differentiation of granulocytes and monocytes. Finally,using mixed bone marrow chimeric mice,we show that IFN-γ-dependent infection-induced myelopoiesis occurs via the direct effect of the cytokine on developing myeloid cells. We propose that,in addition to its many other known roles,IFN-γ acts to control infection by directly promoting the differentiation of myeloid cells that contribute to host defense. View Publication

Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q-syndrome,a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction,but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14,the ribosomal protein gene deleted in the 5q-syndrome,or RPS19,the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect,whereas nutlin-3,a compound that activates p53 through inhibition of HDM2,selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome,we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53,providing a basis for the failure of erythropoiesis in the 5q-syndrome,DBA,and perhaps other bone marrow failure syndromes. View Publication

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia in the United States and globally. Despite the availability of pneumococcal capsular polysaccharide (PPS) and protein conjugate-based vaccines,the prevalence of antibiotic-resistant pneumococcal strains,serotype (ST) replacement in nonconjugate vaccine strains,and uncertainty as to whether the PPS vaccine that is used in adults protects against pneumonia emphasize the need for continued efforts to understand the nature of protective PPS antibody responses. In this study,we generated mouse monoclonal antibodies (MAbs) to a conjugate consisting of the PPS of serotype 8 (PPS8) S. pneumoniae and tetanus toxoid. Thirteen MAbs,including four IgMs that bound to PPS8 and phosphorylcholine (PC) and five IgMs and four IgG1s that bound to PPS8 but not PC,were produced,and their nucleotide sequences,epitope and fine specificity,and efficacy against lethal challenge with ST8 S. pneumoniae were determined. MAbs that bound to PPS8 exhibited gene use that was distinct from that exhibited by MAbs that bound to PC. Only PPS8-binding MAbs that did not bind PC were protective in mice. All 13 MAbs used germ line variable-region heavy (V(H)) and light (V(L)) chain genes,with no evidence of somatic hypermutation. Our data reveal a relationship between PPS specificity and V(H) gene use and MAb efficacy in mice. These findings provide insight into the relationship between antibody molecular structure and function and hold promise for the development of novel surrogates for pneumococcal vaccine efficacy. View Publication