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Interferon-gamma (IFN-gamma) exhibits various properties including antigrowth activity in neoplastic and normal cells and regulatory roles in immune responses and hematopoiesis, but studies of IFN-gamma effects on human megakaryopoiesis have been inconclusive. Recently we have used serum-free culture of purified CD34+ cells to demonstrate that IFN-gamma stimulates the proliferation of relatively mature megakaryocytic progenitors independently of thrombopoietin. It has been also shown that IFN-gamma stimulates the maturation of megakaryocytes, and has a significant synergism with stem cell factor in human megakaryopoiesis. Further studies are needed to clarify the in vivo effect of IFN-gamma on human megakaryopoiesis and the clinical relevance of IFN-gamma.

We recently showed that c-kit signal synergizes with glycoprotein (gp)130 signal mediated by a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R) to stimulate the expansion of human primitive hematopoietic progenitor cells and erythropoietin-independent erythropoiesis. In the present study, we examined the effect of a ligand for Flt3 (FL), whose receptor tyrosine kinase is closely related to c-kit, in combination with IL-6/sIL-6R on human hematopoiesis in vitro. In serum-containing methylcellulose clonal culture of cord blood CD34+ cells, whereas FL alone stimulated only granulocyte-macrophage (GM) colony formation, erythroid bursts and mixed colonies in addition to GM colonies were induced by FL with IL-6/sIL-6R, but not IL-6/sIL-6R alone. In suspension culture, CD34+ cells generated a small number of myeloid cells in the presence of FL or IL-6/sIL-6R alone. However, the addition of IL-6/sIL-6R to the culture with FL induced the generation of a significant number of erythroid cells and megakaryocytes in addition to myeloid cells. The combination of FL and IL-6/sIL-6R also induced a remarkable expansion of GM colony- and erythroid burst-forming cells and multipotential progenitors, although FL or IL-6/sIL-6R alone induced the generation of only a small number of progenitors for GM colonies. The synergistic effects of FL and IL-6/sIL-6R were confirmed in serum-free clonal and suspension cultures. In addition, the addition of anti-human gp130 monoclonal antibodies abrogated the synergistic action. These results indicate that Flt3 signal, as well as c-kit signal, synergizes with gp130 signal to stimulate human myelopoiesis, erythropoiesis and megakaryopoiesis, and the expansion of primitive multipotential hematopoietic progenitor cells.

We examined the effects of recombinant human thrombopoietin (TPO, c-Mpl ligand) on the proliferation and differentiation of human haemopoietic progenitors other than megakaryocytic progenitors using serum-free cultures. TPO alone supported the generation of not only megakaryocytic (MK) but also blast cell (blast) colonies from cord blood CD34+ cells. Delayed addition of a cytokine cocktail (cytokines; interleukin (IL)-3, IL-6, stem cell factor, erythropoietin, granulocyte-macrophage colony-stimulating factor, and TPO) to cultures with TPO alone on day 7 induced various colonies including granulocyte-macrophage (GM) colonies, erythroid bursts (E), granulocyte-erythrocyte-macrophage-megakaryocyte (GEMM) colonies. Replating experiments of blast colonies supported by TPO alone for culture with cytokines revealed that approximately 60% of the blast colonies contained various haemopoietic progenitors. Single cell cultures of clone-sorted CD34+ cells indicated that TPO supported the early proliferation and/or survival of both primitive and committed haemopoietic progenitors. In serum-free suspension cultures, TPO alone significantly stimulated the production of progenitors for MK, GM, E and GEMM colonies as well as long-term culture-initiating cells. These effects were completely abrogated by anti-TPO antibody. These results suggest that TPO is an important cytokine in the early proliferation of human primitive as well as committed haemopoietic progenitors, and in the ex vivo manipulation of human haemopoietic progenitors.

Group A streptococcal infection is associated with the occurrence of acute glomerulonephritis (AGN) and rheumatic fever (RF). A surveillance study in the Saga area, in northern Kyushu, Japan, showed a small variation in the reported number of group A streptococcal infections in the period 1988-94. However, of the AGN cases reported in this period, more than half were observed in 1992. In order to examine whether some change had occurred in the serotype distribution of Streptococcus pyogenes during the period, patients in the Saga area diagnosed as having group A streptococcal infection and patients with AGN or RF were analyzed. Serological T-typing of S. pyogenes was carried out for patients with group A streptococcal infections, and the association between the occurrence of AGN or RF and the distribution of each different T subtype was analyzed. M-typing of S. pyogenes was also carried out and the correlation between T and M types was examined. From 1988 to 1994, the annual number of patients with group A streptococcal infections in the Saga area showed a small variation, range 65-100 patients/year. Of the 42 patients with AGN and three with RF observed in this period, 27 with AGN (64%) and one with RF (33.3%) were detected in 1992. Only the T1 subtype increased in 1992; the other T subtypes showed little variation in incidence. The number of patients with the T1 subtype was significantly correlated with the occurrence of AGN by regression analysis (P < 0.01). Of the 170 subjects tested for both T and M subtypes, 44 of the 45 T1-typed subjects had the M1 protein. Our epidemiological study suggested that the T1 subtype of streptococcal infection was associated with an outbreak of AGN in 1992 in the Saga area.

Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.

gp130, a signal-transducing receptor component of interleukin 6 (IL-6), associates with an IL-6 and IL-6 receptor (IL-6) complex and transduces signals. To examine the role of gp130 signaling in the expansion of human hemopoietic progenitor cells, we tested the effects of a recombinant soluble human IL-6 receptor (sIL-6R) and/or IL-6 in combination with other cytokines on purified human umbilical cord blood CD34+ cells, using methylcellulose clonal assay and suspension culture in the presence or absence of serum. A combination of sIL-6R and IL-6 (sIL-6R/IL-6), but not sIL-6R or IL-6 alone, was found to dramatically stimulate expansion of hemopoietic progenitor cells as well as CD34+ cells in the presence of stem cell factor. Significant generation of multipotential hemopoietic progenitors over a period of 3 weeks in suspension culture and efficient formation of colonies, especially multilineage and blast cell colonies, in methylcellulose assay supplemented with a combination of sIL-6R/IL-6 together with stem cell factor were observed in serum-containing and serum-free culture. Addition of anti-gp130 monoclonal antibodies or anti-IL-6R monoclonal antibodies to the above cultures dose-dependently inhibited the expansion of progenitor cells in suspension culture and also completely blocked the formation of multilineage colonies in methylcellulose culture. These findings demonstrated that the significant expansion of human primitive hemopoietic progenitors could be achieved with the gp130 and c-Kit signalings initiated by the sIL-6R/IL-6 complex in the presence of stem cell factor and suggested the possible application of this method for ex vivo expansion of CD34+ cells for bone marrow transplantation.

Afibrinogenaemia usually induces a bleeding tendency during infancy, whereas protein C deficiency increases susceptibility to thrombosis in children or adolescence. Mutations of these genes have been, therefore, established as independent risk factors for coagulation disorders. We describe the homozygous mutation of the fibrinogen alpha chain gene and additional heterozygous mutation of the protein C gene in a male infant who showed prolonged umbilical bleeding after birth. On examination, the plasma fibrinogen was undetectable, and the activity and antigen level of protein C were reduced. The patient showed no fibrinogen Aalpha chain as well as Bbeta and gamma chains by Western blotting. The sequencing analysis showed the homozygous deletion of 1238 bases from intron 3 at position 2008 to intron 4 at position 3245 in the fibrinogen alpha chain gene. Both parents were heterozygous carriers of this mutation. In this patient, an additional mutation was also detected in the protein C gene: the heterozygous deletion of exon 7 at position 6161-6163 or 6164-6166, resulting the deletion of one amino acid (Lys150 or 151). His mother was also a carrier of this mutation. As the simultaneous mutation of the fibrinogen alpha chain and protein C genes has not been previously reported, the influence of the interaction between these two mutations on the clinical manifestations of this patient should be carefully monitored for a long period.

To assess the abnormal T-cell expansion in chronic active Epstein-Barr virus infection (CAEBV), T-cell antigen receptor (TCR) repertoire was analyzed in four patients with the disease. All fulfilled the diagnostic criteria of CAEBV, presenting with fever, hepatosplenomegaly, cytopenia, abnormal high titers of anti EBV-antibodies, and positive EBV genome of unknown cause. Southern blotting probed with EBV-terminal repeats and TCR Cbeta gene indicated clonal expansion of the infected cells in 3 and 2 patients, respectively. The number of CD4+ HLA-DR+ cells appreciably increased in patients 1 (59%) and 2 (24%), who had a coronary aneurysm and central nervous system involvement, respectively. TCR gene expression examined by the inverse polymerase chain reaction methods revealed that Vbeta gene usages were preferential in all patients (Vbeta7 and Vbeta12: patient 1, Vbeta4: patient 2, Vbeta13: patients 3 and 4), compared with those in healthy controls. Valpha18 gene expression was remarkably high in patients 1 and 2. Moreover, Jbeta gene expression was skewing in the reigning Vbeta clones in all patients. Vbeta4-Jbeta1.5 and Vbeta13-Jbeta1.5 genes were clonally expressed in patients 2 and 4, respectively. These results suggest that CAEBV is associated with the restricted diversity of T-cells, which may stem from the sustained expansion of oligoclonal T-cells possibly driven by conventional viral antigens, but not, superantigens. Although the study is limited by the small number of patients, the unbalanced T-cell repertoire might contribute to the evolution of T-lymphoproliferative disease, otherwise, imply the innate defective immunity to EBV in CAEBV patients.

We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34(+) cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34(+) cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34(+) cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34(+) cells were subfractionated into two populations of IL-6R-negative (CD34(+) IL-6R-) and IL-6R-positive (CD34(+) IL-6R+) cells by fluorescence-activated cell sorting. The CD34(+) IL-6R- cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34(+) IL-6R+ cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.

Flow cytometric study revealed that almost all CD34+ cells in human umbilical cord blood expressed interferon-gamma receptor (IFN-gammaR). To clarify the precise functional roles of IFN-gammaR in human CD34+ cells, we examined the effect of IFN-gamma alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34+ cells using a serum-free clonal culture. Surprisingly, IFN-gamma alone supported only megakaryocyte (MK) colonies in a dose-dependent manner with a plateau level at 1000 U/ml of IFN-gamma. IFN-gamma at 1000 U/ml induced 10 +/- 1.2 MK colonies from 1 x 10(3) CD34+ cells, whereas thrombopoietin (TPO), interleukin (IL)-3, stem cell factor (SCF) or IL-6 alone induced 22 +/- 4.0, 22 +/- 4.2, 4 +/- 0.6 and 0 MK colonies, respectively. The addition of anti-IFN-gamma monoclonal antibody (mAb) to the IFN-gamma culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO-dependent production of MK colonies. In contrast, although anti-TPO polyclonal Ab almost completely blocked TPO-dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN-gamma, suggesting that the observed effect of IFN-gamma on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN-gamma to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL-3-dependent MK colony formation. Additionally, IFN-gamma induced the increase of DNA content of cultured glycoprotein IIb/IIIa-positive megakaryocytes. These results suggest that IFN-gamma may have regulatory roles in human megakaryocytopoiesis.

Congenital amegakaryocytic thrombocytopenia (CAMT) is an uncommon disorder in newborns and infants, characterized by isolated thrombocytopenia and megakaryocytopenia in the first year without physical anomalies. The defect of thrombopoiesis is not well understood. Recently, thrombopoietin (TPO), the ligand for the c-mpl receptor, was cloned. Accumulating evidence from in vitro and in vivo studies indicate that TPO plays a key role in the regulation of megakaryocytopoiesis. In this study we examined the effect of TPO on megakaryocyte colony formation from a patient with CAMT using a plasma-containing methylcellulose clonal culture. The in vitro results demonstrated a defective response to TPO in megakaryocyte colony formation from bone marrow mononuclear cells (MNC) of the patient. although interleukin-3 (IL-3) but not stem cell factor (SCF) induced only a small number of megakaryocyte colonies. These findings indicated that thrombocytopenia in CAMT could not be corrected by administration of TPO in vitro. Additionally, clonal cultures containing SCF, IL-3, IL-6 and erythropoietin showed decreased numbers of erythroid and myelocytic progenitors in the bone marrow of the patient. The serum TPO level measured by enzyme-linked immunosorbent assay was significantly higher than that in healthy controls. By PCR, marrow MNC from healthy children and from a patient with essential thrombocytosis expressed c-mpl mRNA, whereas no c-mpl mRNA was detected in marrow MNC from the patient with CAMT. There was no difference in the CD34 expression and c-kit mRNA between the CAMT patient and healthy children. The results of this study suggest that the pathophysiology in CAMT may be a defective response to TPO in haemopoietic cells through impaired expression of c-mpl mRNA.

We recently demonstrated that stimulation of gp130 by a combination of soluble interleukin 6 receptor (sIL-6R) and IL-6 but not IL-6 alone significantly stimulates the ex vivo expansion of primitive hematopoietic progenitors and the generation of erythroid cells from human CD34+ cells in the presence of stem cell factor (SCF). Here, we show that gp130 is found low positively on most CD34+ cells, whereas IL-6R is expressed on only 30-50% of these cells. Although most of the colonies generated from FACS-sorted CD34+IL-6R+ cells were granulocyte/macrophage (GM) colonies, CD34+IL-6R- cells gave rise to various types of colonies, including erythroid bursts, GM, megakaryocytes, and mixed colonies in methylcellulose culture with a combination of IL-6, sIL-6R, and SCF. Similar results were obtained in culture supplemented with a combination of IL-3, IL-6, SCF, granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. A limiting dilution analysis of long-term culture-initiating cells (LTC-IC) showed that the CD34+IL-6R- cells contained a larger number of LTC-IC than did the CD34+IL-6R+ cells. In a serum-free suspension of CD34+IL-6R- cells, the addition of sIL-6R to the combination of IL-6 and SCF dramatically increased the total and multipotential progenitors, whereas CD34+IL-6R+ cells failed to do so under the same conditions. These results indicate that most of the erythroid, megakaryocytic, and primitive human hematopoietic progenitors are included in the IL-6R- populations, and the activation of gp130 on these progenitors can be achieved by a complex of IL-6-sIL-6R, but not by IL-6 alone. The present culture system using IL-6, sIL-6R, and SCF may provide a novel approach for ex vivo expansion of human primitive hematopoietic progenitors.

Filgrastim (rHuG-CSF)-mobilized peripheral blood progenitor cells (PBPC) in healthy Japanese volunteers were characterized in detail using two clonal cell culture systems and double-colour flow cytometry to detect multilineage colony-forming cells and subsets of CD34+ cells. The kinetics of PBPC during the administration of filgrastim was studied, and possible differences in the character of progenitor cells relative to given doses of filgrastim were investigated. Filgrastim was administered subcutaneously to normal volunteers for 7 d at doses of 100, 200 or 400 microgram/m2 (10 per cohort). Treatment with 100 or 200 microgram/m2 filgrastim was well tolerated; however, the 400 microgram/m2 dose level was not completed because of bone pain and myalgia. The treatment strikingly mobilized various types of progenitor cells, including highly proliferative megakaryocytic colony-forming cells. The number of progenitor cells peaked on days 5 and 6. The fold increase of circulating progenitor cells from the baseline value in the volunteers treated with 200 microgram/m2 filgrastim was more pronounced than in those treated with 100 microgram/m2. Treatment with 200 microgram/m2 also released the less mature progenitor cells (i.e. mixed colony-forming cells CD34+/33- cells, and CD34+/HLA-DR-cells) into circulation better than the 100 microgram/m2 dose. These results suggest that daily subcutaneous injection with 200 microgram/m/2 filgrastim for 5 d will effectively mobilize, both qualitatively and quantitatively, PBPC in healthy donors.

Glycoprotein (gp) 130, a receptor component for interleukin 6 (IL-6), can associate with a soluble IL-6 receptor (sIL-6R) – IL-6 complex. To examine the role of gp130 signaling in human hematopoietic progenitor-cell proliferation and differentiation, we studied the effects of the sIL-6R – IL-6 complex in combination with other cytokines on human CD34+ cells in clonal and suspension cultures. The sIL-6R – IL-6 complex, but not sIL-6R or IL-6 alone, in the presence of stem-cell factor (SCF) produced dramatic increases in the populations of various cell lineages, including erythroid cells and various hematopoietic progenitors, in suspension culture. Significant numbers of colonies of (particularly) multilineage and blast cells were generated in methylcellulose culture supplemented with a combination of sIL-6R – IL-6 complex and SCF. Addition of anti-gp130 monoclonal antibodies (MAbs) and anti-IL-6R MAbs to the above-mentioned cultures dose-dependently inhibited the generation of cells of various lineages and of progenitor cells in suspension culture and completely blocked multilineage colony production in methylcellulose culture; an anti-erythropoietin antibody did not cause inhibition. These findings demonstrate that both proliferation and differentiation of hematopoietic progenitor cells can be induced through gp130 and c-Kit signaling, indicating that progenitor cells are responsive to the sIL-6R – IL-6 complex, even though they do not express IL-6R. Together with previous studies showing that detectable levels of sIL-6R, IL-6, and SCF are present in human serum, these results suggest that gp130 signaling may play an important role in human hematopoiesis in vivo.