Erythropoietin is a major regulator of thrombopoiesis in thrombopoietin-dependent and -independent contexts.

Thrombopoietin (TPO), through activation of its cognate receptor Mpl, is the major regulator of platelet production. However, residual platelets observed in TPO- and Mpl-loss-of-function (LOF) mice suggest the existence of an additional factor to TPO in platelet production. As erythropoietin (EPO) exhibited both in vitro megakaryocytic potential, in association with other early-acting cytokines, and in vivo platelet activation activity, we sought to investigate its role in this setting. Here, we used multiple LOF models to decipher the reciprocal role of EPO and TPO in the regulation of platelet production in TPO-LOF and Mpl-LOF mice and of platelet size heterogeneity in wild-type mice. We first identified EPO as the major thrombopoietic factor in the absence of the TPO-Mpl pathway. Based on the study of several mouse models we found that the EPO-EPO receptor pathway acts on late-stage megakaryopoiesis and is responsible for large-sized platelet production, while the TPO-Mpl pathway promotes small-sized platelet production. On the basis of our data, EPO might be used for thrombocytopenia supportive therapy in congenital amegakaryocytopoiesis. Furthermore, as a distribution skewed toward large platelets is an independent risk factor and a poor prognosis indicator in atherothrombosis, the characterization of EPO's role in the production of large-sized platelets, if confirmed in humans, may open new perspectives in the understanding of the role of EPO-induced platelets in atherothrombosis.

Hypermucoviscous Klebsiella pneumoniae infections induce platelet aggregation and apoptosis and inhibit maturation of megakaryocytes.

INTRODUCTION: Different Klebsiella pneumoniae strains carry different virulence factors and antibiotic resistance and may cause thrombocytopenia. This study aimed to investigate the effects of different infections caused by K. pneumoniae on platelets. METHODS: Two hypermucoviscous K. pneumoniae strains and two classic strains were collected from clinical blood culture, and in both groups, there was a carbapenem-resistant strain and a carbapenem-sensitive strain. Mouse infection models were constructed by intraperitoneally injecting different strains, and mice injected with phosphate-buffered saline served as a control. Count, aggregation rate and apoptosis proportion of platelets within 12 h were examined. CD41 expression was measured in bone marrow cells to determine the maturation of megakaryocytes. The concentrations of lipopolysaccharides and related signaling molecules were also measured. RESULTS: The platelet aggregation rate was much significantly higher in the two hypermucoviscous groups, while it showed no difference in the classic groups compared to the control group. All infections induced apoptosis of platelets, among which the highest apoptosis proportions were observed in infections caused by the hypermucoviscous carbapenem-sensitive strain. In both hypermucoviscous groups the CD41 mean fluorescence intensity was much lower than that in the control group, indicating that the maturation of megakaryocytes in the hypermucoviscous groups was significantly inhibited. Lipopolysaccharides were significantly higher and TLR4/Myd88 and JNK/MAPK pathways were strongly activated in hypermucoviscous groups. CONCLUSIONS: The results indicate that hypermucoviscous K. pneumoniae can reduce platelet count by several pathways. Although antibiotic resistance is rapidly emerging worldwide, it has little influence on the decrease in platelets.

Anaplasma phagocytophilum infection modulates expression of megakaryocyte cell cycle genes through phosphatidylinositol-3-kinase signaling.

Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis infects neutrophils and other cells from hematopoietic origin. Using human megakaryocytic cell line, MEG-01, we show that expression of cell cycle genes in these cells are altered upon A. phagocytophilum infection. Expression of several cell cycle genes in MEG-01 cells was significantly up regulated at early and then down regulated at later stages of A. phagocytophilum infection. Lactate dehydrogenase (LDH) assays revealed reduced cellular cytotoxicity in MEG-01 cells upon A. phagocytophilum infection. The levels of both PI3KCA (p110 alpha, catalytic subunit) and PI3KR1 (p85, regulatory subunit) of Class I PI3 kinases and phosphorylated protein kinase B (Akt/PKB) and inhibitory kappa B (IκB) were elevated at both early and late stages of A. phagocytophilum infection. Inhibition of PI3 kinases with LY294002 treatment resulted in significant reduction in the expression of tested cell cycle genes, A. phagocytophilum burden and phosphorylated Akt levels in these MEG-01 cells. Collectively, these results suggest a role for PI3K-Akt-NF-κB signaling pathway in the modulation of megakaryocyte cell cycle genes upon A. phagocytophilum infection.

Anaplasma platys in bone marrow megakaryocytes of young dogs.

Anaplasma platys is an obligate intracellular rickettsial pathogen that infects platelets of dogs, forming basophilic intracellular morulae. In the present report, cellular inclusions were documented in bone marrow thrombocyte precursors of two young naturally infected dogs, indicating that A. platys can infect megakaryocytes and promegakaryocytes.

Anaplasma phagocytophilum infects cells of the megakaryocytic lineage through sialylated ligands but fails to alter platelet production.

Anaplasma phagocytophilum is an obligate intracellular bacterial pathogen that principally inhabits neutrophils. However, infection with A. phagocytophilum results in a moderate to marked thrombocytopenia. In host neutrophils, A. phagocytophilum uses sialylated ligands, primarily P-selectin glycoprotein ligand-1 (PSGL-1), to enter its host cell. PSGL-1 is expressed on a wide array of haematopoietic cells, including megakaryocytes. In this study, it was hypothesized that (i) cells of the megakaryocytic lineage (MEG-01 cells) would be susceptible to A. phagocytophilum infection and (ii) infection may induce alterations in platelet production contributing to infection-induced thrombocytopenia. It was found that MEG-01 cells are susceptible to infection. MEG-01 cells expressing abundant sialylated ligands were the most susceptible to infection, and the absence of sialylation, or blocking of PSGL-1, limited infection susceptibility. However, infected MEG-01 cells produced proplatelets and platelet-like particles comparable to uninfected cells. These results highlight a novel target of pathogen infection and suggest that the pathogen may utilize similar strategies to gain access to megakaryocytes. Direct pathogen modification of platelet production may not play a role in infection-induced thrombocytopenia.

Identification of a novel human immunodeficiency virus strain cytopathic to megakaryocytic cells.

Impaired megakaryocytopoiesis may be a contributing factor to thrombocytopenia associated with human immunodeficiency virus (HIV) infection. Because HIV isolates differ in their host range and pathogenicity, we investigated whether HIV strains with demonstrable cell tropism and increased cytopathicity for megakaryocytes could be derived from the blood of thrombocytopenic HIV-infected individuals. We derived a strain, HIV-WW, from the peripheral blood of an individual with severe thrombocytopenia and found the virus to be highly and specifically cytotoxic to CMK and DAMI megakaryocytic cells. CMK and DAMI cells were not permissive for the virus and HIV-WW induced cytopathicity for these megakaryocytic cells did not depend on viral replication. The CD4 N-terminus-binding domain of the HIV gp120 envelope protein did not appear to be involved in determining the cytopathic phenomenon. HIV may impair megakaryocytopoiesis through interactions at the cell surface in some cases rather than through viral entry and intracellular replication.

Human megakaryocytes have a CD4 molecule capable of binding human immunodeficiency virus-1.

Most human megakaryocytes (MGKs) express the CD4 antigen on their surface. Approximately 25% have a CD4 receptor density comparable to that of CD4+ T cells (Basch et al, Proc Natl Acad Sci USA 87:8085, 1990). In these studies, we show: (1) the presence of mRNA for CD4 in human MGKs; (2) the binding of human immunodeficiency virus-1 (HIV-1) to human MGKs; (3) the inhibition of binding by anti-CD4 (Leu3a) antibody or rCD4; (4) the infection of a human MGK line, CHRF-288 with HIV-1; and (5) inhibition of infection with anti-CD4. Human MGKs have mRNA for CD4 as shown by in situ hybridization with an RNA probe synthesized from a 3-kb cDNA sequence of plasmid pSP65.T4.8 containing the full-length CD4 sequence. MGKs (23% +/- 17%) bound HIV-1, as determined by anti-gp120 and anti-CD41 staining. Binding to human MGKs could be inhibited 55% to 75% with anti-CD4 or rCD4, respectively. Infection of a CD4+ MGK line (CHRF-288) could be accomplished with HIV-1, as determined by proviral DNA polymerase chain reaction and p24 production. Preincubation with anti-CD4 inhibited apparent proviral DNA infection by 100% and p24 production by 65% to 70%. Thus, human MGKs have a CD4 receptor capable of binding HIV-1. Using this receptor, HIV-1 can infect cells representative of the MGK lineage.

Characterization of 17 human immunodeficiency virus-1 carrier cell lines with T cell, myelomonocyte, or megakaryocyte lineages.

Of 29 hematopoietic cell lines tested for susceptibility to human immunodeficiency virus (HIV)-1HTLV-IIIB infection, all CD4+ cell lines became infected. Continuous culturing of infected cell lines resulted in nine HIV-1 carrier cell lines, including, for the first time, an HIV-1 carrier megakaryoblastic cell line, MEG-01/HIV. The immunophenotypic profiles of a total of 17 HIV-1 carrier cell lines (nine newly and eight previously established cell lines) were compared with their respective parental noninfected cell lines. Except for total absence of CD4 expression, the expression of other antigens was variable among the 17 HIV-1 carrier cell lines. Persistent and consistent replication of infectious HIV-1 was detected in all of them in varying quantities. The great variability observed in both the altered marker expression, with respect to that of the noninfected parental cell lines, and in the quantities of persistently produced infectious HIV-1 was, nevertheless, specific to the individual cell lines. Furthermore, the present study demonstrates that there is no apparent correlation in the quantity of HIV-1 produced to either T cell, myelomonocytic cell, or megakaryocytic cell types. Instead, the results suggest that a particular interaction between HIV-1 and individual clonal cell lines may provide insight into the extremely complex immune dysregulation associated with the pathogenesis of acquired immune deficiency syndrome. Thus, the 17 HIV-1 carrier cell lines of diverse origin presented here provide valuable and unique models for further understanding acquired immune deficiency syndrome pathogenesis at the cellular and molecular levels.

Detection of feline immunodeficiency virus infection in bone marrow of cats.

Natural or experimental feline immunodeficiency virus (FIV) infection in cats is often associated with hematologic abnormalities which are similar to those observed in human immunodeficiency virus (HIV) infected patients. To determine if cells in bone marrow are infected with FIV and whether severity of hematopoietic disorder is correlated with the level of viral infection, bone marrow tissues from ten experimentally and two naturally FIV infected cats were examined by in situ hybridization for presence of FIV RNA. Seven of the 12 FIV infected cats were also naturally or experimentally coinfected with feline leukemia virus (FeLV). FIV RNA was detected mainly in megakaryocytes and unidentified mononuclear cells in the bone marrow of cats that were sick and had marrow hypercellularity and immaturity. These included all cats in the acute phase of FIV infection and two of seven long term FIV infected cats. One long term FIV infected cat with lymphosarcoma was also positive for FIV RNA in bone marrow cells. The other four long term FIV infected cats were relatively healthy, with normal bone marrow morphology, and were negative for FIV infected cells. Bone marrow from three non-infected and two cats infected with FeLV alone were also negative for FIV RNA by in situ hybridization. We concluded that megakaryocytes and mononuclear cells were targets of the viral infection and that the presence of FIV RNA in cells of the bone marrow correlated with marrow hypercellularity and immaturity, and severity of illness.

In situ localization of PCR-amplified human and viral cDNAs.

We describe a technique, called reverse transcriptase (RT) in situ PCR, whereby RNA may be nonisotopically detected in fixed cells when amplified by PCR after cDNA synthesis by RT. RT in situ PCR using primers specific for the measles virus generated an intense signal in most measles-infected HeLa cells, as compared to the weak signal generated in few cells using standard in situ hybridization analysis. The viral RNA that localized to the nucleus spared the nucleoli, was most evident when the RT step used the primer complementary to the negative genomic strand, and was demonstrated in all multinucleated cells and the majority of uninucleate cells. A hybridization signal was evident with standard RNA in situ hybridization using the human megakaryocyte cell line Dami and a probe for glycoprotein IIB (GIIB) mRNA but not a probe for amyloid precursor protein (APP) or gelsolin (GEL) mRNA. After RT in situ PCR, signals were evident for each target localizing to the nucleolus for APP and to perinucleolar and cytoplasmic locations for GEL and GIIB. The latter findings suggest that mRNAs may follow different geographic pathways as they progress from premessage to transcriptionally active message.

Productive human immunodeficiency virus-1 infection of megakaryocytic cells is enhanced by tumor necrosis factor-alpha.

Recent findings have indicated that megakaryocytes may be susceptible to human immunodeficiency virus (HIV) infection, suggesting a potential role for megakaryocytes as viral reservoirs in HIV-infected patients. We report that the megakaryocytic cell line Dami could be productively infected with the HTLV III-B strain of HIV-1, in 26 different experiments (results of 16 experiments are reported); productive infection lasted up to 30 weeks. Despite a lack of detectable surface expression of the CD4 molecule and very low levels of CD4 mRNA, between 40% and 60% of megakaryocytic cells produced viral proteins after contact with HIV-1. Neither cytopathogenic effects nor syncytial formation was observed. Production of high levels of functional viral particles was indicated by analysis of p24 protein levels, reverse transcriptase activity, ultrastructural studies, and the capacity of supernatants from infected Dami cells to infect the Molt-4 T-lymphocytic cell line. HIV-1 RNA and protein levels in infected Dami cells were enhanced by treatment with tumor necrosis factor-alpha (TNF-alpha), and decreased by treatment with interferon-alpha (IFN-alpha) and IFN-gamma. Transient transfection of the megakaryocytic cells with various constructs of the HIV-1 promoter (LTR) linked to the luciferase reporter gene suggested that the effect of TNF-alpha was related, as in monocytic and T-cell lines, to transactivation of the enhancer region of the HIV-1 LTR. These findings indicate that signals provided by the immune system may modulate HIV-1 expression in cells of the megakaryocytic lineage.

Fungi in megakaryocytes. An unusual manifestation of fungal infection of the bone marrow.

When fungi infect the bone marrow, typically they are associated with granuloma formation and/or necrosis, and the fungi are found within histiocytes or admixed with necrotic debris. Recently two bone marrow biopsy specimens were encountered in which fungi were confined to the cytoplasm of megakaryocytes, a finding not previously reported in the literature. The first case was that of a 46-year-old man with pulmonary histoplasmosis and no known immunodeficiency. The second was that of a 38-year-old man with the acquired immune deficiency syndrome and cryptococcal meningitis. In the first case, many megakaryocytes contained fungal forms consistent with Histoplasma. In the second, one small cluster of megakaryocytes contained several budding yeast consistent with Cryptococcus. Neither marrow biopsy specimen had necrosis, granulomas, or histiocytic infiltration. In both cases, because of the unusual localization of the fungi, they were initially overlooked. The bone marrow may contain fungi even in the absence of abnormalities suggesting fungal infection on routinely stained sections. A silver stain or a periodic acid--Schiff stain should be performed on all marrow biopsy specimens in cases of known or suspected fungal infection outside the marrow. The phenomenon of megakaryocyte emperipolesis is well known, and this process may be responsible for the apparent ability of megakaryocytes to internalize fungi.

The platelet volume-number relationship in patients infected with the human immunodeficiency virus.

The pathophysiology of thrombocytopenia in the acquired immune deficiency syndrome has not been elucidated completely. Many findings in these patients are identical to those with immune thrombocytopenic purpura. However, recent findings in acquired immune deficiency syndrome patients including the effect of zidovudine on platelet count and the demonstration of ultrastructural changes and viral RNA in megakaryocytes, have suggested that the human immunodeficiency virus may directly infect megakaryocytes, and play a role in acquired immune deficiency syndrome-related thrombocytopenia. To investigate further the mechanism of decreased platelet counts in human immunodeficiency virus-infected patients, the platelet volume-number relationship and corresponding bone marrow findings in 34 patients infected with human immunodeficiency virus were studied. Parameters evaluated included platelet count and mean platelet volume; bone marrow cellularity, megakaryocyte number, and number and percentage of denuded megakaryocyte nuclei. Two thirds of the platelet counts were low, and of these 92% had an inappropriately low mean platelet volume. These individuals had a platelet-volume number relationship that is very similar to that seen in myelosuppressive disorders. In addition, more than 90% of the bone marrows from thrombocytopenic patients had either normal or decreased numbers of megakaryocytes. These observations provide additional evidence to support the hypothesis that the pathophysiology of human immunodeficiency virus-associated thrombocytopenia may be due, at least in part, to a direct effect on the megakaryocytes.

Infection of megakaryocytes by human immunodeficiency virus in seropositive patients with immune thrombocytopenic purpura.

Twenty-one human immunodeficiency virus (HIV)-positive patients, including 11 acquired immunodeficiency syndrome (AIDS)-free patients with immune thrombocytopenic purpura (ITP), were studied to determine whether the megakaryocytic/platelet lineage was infected by HIV. Because purification of platelets did not reach a level sufficient for unequivocal results by the polymerase chain reaction, in situ hybridization was thus performed. Purified marrow megakaryocytes (MK) from 10 HIV-infected ITP patients were studied using a 35S HIV riboprobe, antisense of an HIV ENV sequence. HIV transcripts were clearly detected in MK from five of these 10 patients, although heterogeneity among MK was observed. In three of these five cases, small amounts of HIV glycoproteins were detected in MK by means of immunofluorescence. In addition anti-HIV antibodies could be eluted from platelets of all patients. In contrast, HIV transcripts were not detected in MK derived from colony-forming units-MK (CFU-MK) cultured in suspension, suggesting either that MK are infected by HIV during terminal differentiation or that HIV-infected CFU-MK are unable to differentiate in vitro. In conclusion, this study suggests that HIV infection of MK may be implicated in the pathogenesis of thrombocytopenia of HIV-positive patients.

Human immunodeficiency virus infection of megakaryocytic cells.

The human immunodeficiency virus (HIV) is capable of infecting certain cells of hematopoietic lineage, particularly monocyte-macrophages and T lymphocytes. Recently, the possibility that cells of megakaryocytic lineage are susceptible to HIV infection has been raised. We have characterized infection of the permanent megakaryocytic cell line CMK by HIV in vitro. CMK cells were easily infected by HIV type 2 (HIV-2), producing significant amounts of virus in culture. Infection appeared to be mediated by the CD4 surface antigen on CMK cells. Three different strains of HIV-1 were able to minimally infect CMK cells, suggesting there may be isolates of HIV tropic for megakaryocytes. Infection of CMK cells led to downregulation of the CD4 surface antigen but no discernable change in expression of megakaryocyte-associated proteins glycoprotein Ib and glycoprotein IIb/IIIa. These observations support the likelihood that megakaryocytes are susceptible to HIV infection, and cell lines of megakaryocytic origin may provide a useful model to study effects of the retrovirus on megakaryocyte function.

Parvovirus B19-induced perturbation of human megakaryocytopoiesis in vitro.

Parvovirus B19 infection leads to transient aplastic crises in individuals with chronic hemolytic anemias or immunodeficiency states. An additional unexplained sequela of B19 infection is thrombocytopenia. Because B19 is known to have a remarkable tropism for human erythropoietic elements, and is not known to replicate in nonerythroid cells, the etiology of this thrombocytopenia is uncertain. We sought to define the pathobiology of B19-associated thrombocytopenia by examining the role of B19 on in vitro megakaryocytopoiesis. B19 infection of normal human bone marrow cells significantly suppressed megakaryocyte (MK) colony formation compared with mock-infected cells. No such inhibition was observed with a nonpathogenic human parvovirus, the adeno-associated virus 2 (AAV). The B19-MK cell interaction was also studied at the molecular level. Whereas low-density bone marrow cells containing erythroid precursor cells supported B19 DNA replication, no viral DNA replication was observed in B19-infected MK-enriched fractions as determined by the presence of viral DNA replicative intermediates on Southern blots. However, analysis of total cytoplasmic RNA isolated from B19-infected MK fractions showed a low-level expression of the B19 genome as detected by quantitative RNA dot blots as well as by Northern analysis. Furthermore, a frame-shift mutation in a recombinant AAV-B19 hybrid genome segment that encodes the viral nonstructural (NS1) protein significantly reduced the observed inhibition of MK colony formation. These studies indicate tissue-tropism of B19 beyond the erythroid progenitor cell, and lend support to the hypothesis that B19 genome expression may be toxic to cell populations that are nonpermissive for viral DNA replication.

Internalization of human immunodeficiency virus type I and other retroviruses by megakaryocytes and platelets.

Direct infection of megakaryocytes and platelets by human immunodeficiency virus type I (HIV-I) or other retroviruses has not been demonstrated. To determine whether this could occur, murine bone marrow was co-cultivated with the amphotropic retrovirus-producing cell line PA317-N2, and freshly isolated normal human bone marrow and platelets were co-cultivated with HIV-infected H9 cells. In each case, ultrastructural analyses showed viruses within megakaryocytes and platelets. In murine specimens, the uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent. These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIV-associated disease. In addition, these observations suggest that cells of the megakaryocyte lineage could serve as target cells in gene transfer experiments using retroviral-based vectors.

Megakaryocytopoiesis and granulopoiesis after murine cytomegalovirus infection.

Thrombopoiesis and granulopoiesis following murine cytomegalovirus infection were investigated by studying changes in megakaryocytes, megakaryocyte and granulocyte-macrophage progenitor cells, and spleen colony-forming cells. The soft gel in vitro culture system was used to assay for megakaryocyte and granulocyte-macrophage progenitor cells in marrow and spleen. Murine cytomegalovirus produced a mild thrombocytopenia to 90% of control values 1 day after infection at a time when marrow megakaryocyte levels were normal, suggesting a mild direct toxic effect of the virus on platelets. A reduction of megakaryocytes, megakaryocyte and granulocyte-macrophage progenitor cells, and spleen colony-forming cells to 40% to 60% of control values occurred within 24 to 48 hours of infection in association with an additional decrease in platelets to 58% of control levels on day 4. In vitro inoculation of marrow cell cultures with murine cytomegalovirus also resulted in a reduction of megakaryocyte- and granulocyte-macrophage colony-forming cells within 24 to 48 hours, suggesting that murine cytomegalovirus-induced thrombocytopenia and granulocytopenia may be in part caused by direct infection of precursor cells. The recovery of cells in the spleen was followed by a striking seven- to 10-fold increase in spleen colony-forming cells and megakaryocyte and granulocyte-macrophage progenitor cells in the spleen. These marked increases followed significant increases in spleen cell production of colony-stimulating activities within 2 days of murine cytomegalovirus infection, suggesting that hematopoietic cell recovery is mediated by increased local production of colony-stimulating activities in the spleen.