Concentrations Of M-CSF Research Articles

The expression pattern of inflammatory mediators in cerebrospinal fluid differentiates Guillain–Barré syndrome from chronic inflammatory demyelinating polyneuropathy

Introduction Guillain–Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP) share histopathological features but display different disease courses; we measured the concentration of 50 inflammatory mediators in the cerebrospinal fluid (CSF) of patients with either of these diseases. Patients and methods CSF samples were collected during a diagnostic lumbar puncture and stored at −30 °C. We analyzed the CSF of nine subjects with GBS; eight with CIDP; eight with diabetic polyneuropathy (DP) and seven with headache (controls). Fifty inflammatory mediators were simultaneously measured with a multiplex bead-based ELISA on a Suspension Array System. After Bonferroni’s correction for repeated measures, non-parametric variance and post hoc test were calculated. Results Thirty-two inflammatory mediators were expressed. The median concentration of IL-6, IL-9, IL-15, IL-18, CCL4, CXCL1, LIF, MIF, PDGFbb, IFN-γ2, IL-2ra, IL-12(p40), IL-16, SCGF-b, TRAIL, FGF, G-CSF, GM-CSF, and M-CSF was not different among groups (variance: n.s.). The median concentration of CCL2, CCL7, CCL27, CXCL9, CXCL10, CXCL12, ICAM-1, VCAM1 and VEGF was higher in CIDP and GBS compared with controls ( p < 0.002). The median concentration of IL-8 and IL-1ra was higher in GBS than CIDP or DP or controls, whereas stem cell factor (SCF) and hepatocyte growth factor (HGF) were higher in CIDP than GBS or DP or controls ( p < 0.002). Discussion Mediators of the recruitment and activation of lymphocytes and monocytes are expressed in the CSF of CIDP and GBS. IL-8 and IL-1ra are characteristic of GBS, whereas growth factors (SCF, HGF) of CIDP are possibly related to chronicity or to the survival/repair processes of neurons.

Reciprocal Synergistic Interactions of Leukemic Cells with Osteoclast Progenitors in the Bone Microenvironment

The bone marrow provides an essential regulatory microenvironment for adult hematopoiesis, however the relationship between the bone marrow microenvironment and malignant hematopoiesis remains poorly understood. To investigate the interactions between leukemia and the bone marrow microenvironment we utilized a mouse model of blast-crisis chronic myelogenous leukemia (BC-CML), in which primitive normal murine hematopoietic cells are modified to leukemic cells by expressing the translocation products BCR/ABL and Nup98/HoxA9. The presence of each translocation was confirmed by their co-expression of Green Fluorescent Protein (GFP) and Yellow Fluorescent Protein (YFP) respectively. Ten days after injection of GFP+/YFP+ leukemic cells into strain-matched immunocompetent, non-myeloablated recipient mice, 50% of the bone marrow was composed of leukemic cells as determined by flow cytometric analysis. Histologic analysis of the contralateral tibiae and femora demonstrated not only progressive replacement of the bone marrow by leukemic cells, but also a significant bone loss. Histomorphometric analysis confirmed 50% decreased trabecular bone volume in leukemic mice compared to control mice that were not injected with leukemic cells (bone volume/total volume (%): 12±2 vs 26±2 p=0.01). Interestingly, numerous multi-nucleated osteoclasts were observed in the bone marrow of leukemic mice and were localized adjacent to leukemic cells, suggesting that leukemic cells may affect osteoclastogenesis and result in massive bone loss. To test this hypothesis, we first measured the expression of known regulators of osteoclastogenesis, including RANKL, in our leukemic cells by quantitative RT-PCR analysis. Compared to GFP−/YFP− cells, GFP+/YFP+ cells have 3-fold increased expression of RANKL, a major osteoclastogenic cytokine. We then examined if leukemic cells can give rise to osteoclasts in the presence of RANKL and M-CSF in vitro and found that these cells were unable to differentiate into osteoclasts themselves. To determine if leukemic cells can induce osteoclastogenesis of normal osteoclast progenitors, we cocultured spleen-derived osteoclast precursors from wild-type mice with GFP+/YFP+ leukemic cells or GFP−/YFP− non-leukemic cells in osteoclastic differentiating media containing optimal concentrations of M-CSF and RANKL. As expected, there was abundant formation of mature osteoclasts, identified as TRAP+ multinucleated cells, in control cultures containing non-leukemic cells and osteoclast precursors. Leukemic cells significantly increased TRAP+ mono-nucleated osteoclast precursors (No. TRAP+ mononucleated cells/well: 34±3.3 vs 20±6.0 in non-leukemic cells, p=0.0136). Under this culture condition, we did not observe increased mature osteoclast formation by leukemic cells. Surprisingly, we found that osteoclast precursors strongly prolonged the survival of leukemia cells. In control cultures without a feeder layer of osteoclast precursors there were no viable leukemia cells present after 6 days in culture while in the co-culture system viable leukemia cells were still abundant after 6 days in culture, identifiable by their expression of GFP/YFP (No. GFP+/YFP+/high power field: 0 vs 142±6.4, p<0.01). In summary, in a murine model of BC-CML, there was a rapid loss of trabecular bone and an increase in the number of osteoclasts. Expression of osteoclast-regulating cytokines in leukemic cells favored osteoclastogenesis, however modified cells did not themselves give rise to osteoclasts, while increasing the population of normal immature osteoclasts. In turn, osteoclast progenitors prolonged survival of leukemic cells in vitro. Taken together, these data strongly suggest reciprocal synergistic interactions of leukemic cells with osteoclast progenitors in the bone microenvironment. These previously unrecognized interactions could be exploited to modify disease progression, providing a novel approach for leukemia treatment.

Peritoneal Fluid Cytokines and the Differential Diagnosis of Benign and Malignant Ovarian Tumors and Residual/Recurrent Disease Examination

This study aimed to assess the potential value of peritoneal fluid cytokine examination for the differential diagnosis of ovarian tumors and for evaluating residual or recurrent disease after treatment. The cytokines that are commonly elevated in ovarian cancer, VEGF, IL-6, bFGF, IL-8 and M-CSF, and a reference ovarian tumor marker, CA 125, were measured in peritoneal fluids of 53 previously untreated patients with epithelial ovarian cancer, 18 ovarian cancer patients after surgical treatment and chemotherapy, and 17 patients with benign epithelial ovarian tumors. Non-parametric statistical analysis of data was performed. Ovarian cancer peritoneal fluids, as compared to peritoneal fluids of patients with benign ovarian tumors, contained significantly higher concentrations of IL-6, VEGF and CA 125, and significantly lower concentrations of bFGF and M-CSF, but only the levels of IL-6 and VEGF were significantly higher in peritoneal fluids of stage I and II ovarian cancer patients than of patients with benign ovarian conditions. IL-6 at the cutoff level of 400 pg/mL discriminated benign and malignant ovarian tumors with 92% sensitivity and 60% specificity, while VEGF at the cutoff of 400 pg/mL had 90% sensitivity and 80% specificity. At the cutoff level of 1200 pg/mL, IL-6 had 84% sensitivity and 87% specificity. A radical decrease in local cytokine and CA 125 levels in patients after treatment was independent of therapy outcome. IL-6 and VEGF measurements in peritoneal fluids might be useful for the differential diagnosis of malignant and benign ovarian conditions, but not for residual or recurrent disease examination.

Peritoneal fluid cytokines and the differential diagnosis of benign and malignant ovarian tumors and residual/recurrent disease examination

This study aimed to assess the potential value of peritoneal fluid cytokine examination for the differential diagnosis of ovarian tumors and for evaluating residual or recurrent disease after treatment. The cytokines that are commonly elevated in ovarian cancer, VEGF, IL-6, bFGF, IL-8 and M-CSF, and a reference ovarian tumor marker, CA 125, were measured in peritoneal fluids of 53 previously untreated patients with epithelial ovarian cancer, 18 ovarian cancer patients after surgical treatment and chemotherapy, and 17 patients with benign epithelial ovarian tumors. Non-parametric statistical analysis of data was performed. Ovarian cancer peritoneal fluids, as compared to peritoneal fluids of patients with benign ovarian tumors, contained significantly higher concentrations of IL-6, VEGF and CA 125, and significantly lower concentrations of bFGF and M-CSF, but only the levels of IL-6 and VEGF were significantly higher in peritoneal fluids of stage I and II ovarian cancer patients than of patients with benign ovarian conditions. IL-6 at the cutoff level of 400 pg/mL discriminated benign and malignant ovarian tumors with 92% sensitivity and 60% specificity, while VEGF at the cutoff of 400 pg/mL had 90% sensitivity and 80% specificity. At the cutoff level of 1200 pg/mL, IL-6 had 84% sensitivity and 87% specificity. A radical decrease in local cytokine and CA 125 levels in patients after treatment was independent of therapy outcome. IL-6 and VEGF measurements in peritoneal fluids might be useful for the differential diagnosis of malignant and benign ovarian conditions, but not for residual or recurrent disease examination.

Hyperactivation of p21ras and PI3-K Cooperate To Alter Murine and Human NF1 Haploinsufficient Osteoclast Functions.

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations of the NF1 tumor suppressor gene that functions as a GTPase activating protein for Ras. Though nullizygous loss of NF1 is associated with the development of malignancies, haploinsufficient phenotypes are now being increasingly recognized to alter cell fates and functions in a number of tissues resulting in nonmalignant disease manifestations. Bone manifestations, including skeletal dysplasias, scoliosis, and osteoporosis occur in 30–60% of all NF1 patients and osteoporosis is an increasingly recognized health problem for women with NF1. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone resorbing cells of the skeleton. Using an established murine model of NF1 developed using homologous recombination, we found that Nf1+/− mice contain elevated numbers of multinucleated osteoclasts and osteoclast progenitors per femur in vivo. Both osteoclasts and osteoclast progenitors from Nf1+/− mice were hyperresponsive to limiting concentrations of M-CSF and RANKL, growth factors that are integral to osteoclast maturation and activation. M-CSF stimulated p21ras-GTP and Akt phosphorylation was elevated in Nf1+/− osteoclasts associated with gains in function in survival and proliferation. Bone resorption by osteoclasts is linked to the migration and adherence of these cells to a local bone surface. Purified populations of Nf1+/− osteoclasts were initially placed in the upper chamber of a transwell coated with vitronectin and haptotaxis to M-CSF was determined. Nf1+/− osteoclasts had a 2–3 fold increase in migration as compared to syngeneic wildtype cells. A similar increase in adhesion of Nf1+/− osteoclasts to the integrin avb3 was also observed. Following adhesion, osteoclasts form a specialized cell-extracellular matrix to initiate degradation of bone matrix by secreting proteinases. Nf1+/− osteoclasts had significantly increased bone resorption as measured by scoring the number and area of individual bone resorbing “pits” on dentine slices and by scoring the total area of resorption. These collective increases in osteoclast function were validated in vivo by the observation that serum TRAP5b activity, a sensitive measure of osteoclast lytic activity was 2.5 fold higher in Nf1+/− mice as compared to WT mice. Furthermore, we performed ovariectomy, an established model of osteoporosis associated with an increase in osteoclast function. In two independent experiments, we found that Nf1+/− mice had a significantly greater reduction in bone mineral density following ovariectomy as compared to syngeneic wildtype mice. We hypothesized that hyperactivation of class1A-PI3-K may contribute to these gains in cellular function. We found that intercrossing Nf1+/− and Class1A PI3-K deficient mice (p85a) restores elevated PI3-K activity, and Nf1+/− osteoclast functions to wildtype levels. Furthermore, in vitro differentiated osteoclasts from NF1 patients also display elevated Ras-PI3-K activity and increased lytic activity analogous to murine Nf1+/− osteoclasts. Collectively, we identify a novel cellular and biochemical NF1 haploinsufficient phenotype in osteoclasts that has potential implications in the pathogenesis of NF1 bone disease.

Hyperactivation of p21ras and PI3K cooperate to alter murine and human neurofibromatosis type 1–haploinsufficient osteoclast functions

Individuals with neurofibromatosis type 1 (NF1) have a high incidence of osteoporosis and osteopenia. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone-resorbing cells of the skeleton. We found that Nf1(+/-) mice contain elevated numbers of multinucleated osteoclasts. Both osteoclasts and osteoclast progenitors from Nf1(+/-) mice were hyperresponsive to limiting concentrations of M-CSF and receptor activator of NF-kappaB ligand (RANKL) levels. M-CSF-stimulated p21(ras)-GTP and Akt phosphorylation was elevated in Nf1(+/-) osteoclasts associated with gains of function in survival, proliferation, migration, adhesion, and lytic activity. These gains of function are associated with more severe bone loss following ovariectomy as compared with that in syngeneic WT mice. Intercrossing Nf1(+/-) mice and mice deficient in class 1(A) PI3K (p85alpha) restored elevated PI3K activity and Nf1(+/-) osteoclast functions to WT levels. Furthermore, in vitro-differentiated osteoclasts from NF1 patients also displayed elevated Ras/PI3K activity and increased lytic activity analogous to those in murine Nf1(+/-) osteoclasts. Collectively, our results identify a what we believe to be a novel cellular and biochemical NF1-haploinsufficient phenotype in osteoclasts that has potential implications for the pathogenesis of NF1 bone disease.

Elevated gelatinase activity in pulmonary alveolar proteinosis: role of macrophage-colony stimulating factor

Pulmonary alveolar proteinosis (PAP) is an anti-granulocyte macrophage-colony stimulating factor (GM-CSF) autoimmune disease resulting in the accumulation of phospholipids in the alveoli. GM-CSF knockout (KO) mice exhibit a strikingly similar lung pathology to patients with PAP. The lack of functionally active GM-CSF correlates with highly elevated concentrations of M-CSF in the lungs of PAP patients and GM-CSF KO mice. M-CSF has been associated with alternative macrophage activation, and in models of pulmonary fibrosis, M-CSF also contributes to tissue resorption and fibrosis. Matrix metalloproteinase-2 (MMP-2) and MMP-9 have been implicated in extracellular matrix degradation in animal models of fibrosis and asthma. We show for the first time that the lungs of PAP patients contain highly elevated levels of MMP-2 and MMP-9. PAP broncholaveolar lavage (BAL) cells but not bronchial epithelial cells expressed increased MMP-2 and MMP-9 mRNA relative to healthy controls. Both MMPs were detectable as pro and active proteins by gelatin zymography; and by fluorometric global assay, PAP-MMP activity was elevated. BAL cells/fluids from GM-CSF KO mice also demonstrated significantly elevated MMP-2 and MMP-9 gene expression, protein, and activity. Finally, PAP patients undergoing GM-CSF therapy exhibited significantly reduced MMPs and M-CSF. These data suggest that in the absence of GM-CSF, excess M-CSF in PAP may redirect alveolar macrophage activation, thus potentially contributing to elevated MMP expression in the lung.

Nf1 Plays an Essential Role in Osteoclasts Development Via Hyperactivation of Ras- PI3K Signaling.

Patients with neurofibromatosis type 1 (NF1) have mutations in the NF1 tumor-suppressor gene that functions as a GTPase activating protein (GAP) for p21ras. Individuals with NF1 display a variety of skeletal defects including lytic bone lesions. However, the effect of loss of NF1 on osteoclasts or osteoblasts, the two principal cells involved in bone remodeling, is not understood. Osteoclasts are specialized myeloid cells that adhere to bone matrix, and secrete lytic enzymes that degrade bone. Given that we have previously identified haploinsufficient phenotypes in Nf1 +/− mast cells, we defined the role of neurofibromin in regulating various osteoclast functions in vitro and in vivo in Nf1 +/− mice. Strikingly, histologic sections from femurs of Nf1 +/− mice, revealed large numbers of multinucleated osteoclasts. This phenotype is reminiscent of the osteoclasts in patients with Paget's disease and of osteoclasts isolated from SHIP deficient mice, which have elevated levels of PI-3K activity. Additionally, osteoclast progenitors from Nf1 +/− mice were hyperresponsive to limiting concentrations of M-CSF and RANKL and formed higher numbers of OCL progenitors compared to +/+ controls (P<0.05). M-CSF binding to its receptor (c-fms) causes a rapid increase in p21ras activity. Nf1 +/− osteoclasts had higher basal and M-CSF-stimulated p21ras-GTP levels compared to wild type cells. The Ras-PI3K- pathway has been implicated in controlling numerous osteoclast functions. An increase in basal and M-CSF stimulated Akt activation, a sensitive indicator of PI3-K activity, was observed in Nf1 +/− osteoclasts. Since a key initiating component of the resorptive process of osteoclasts is their ability to bind to αv B3 on the bone surface, we next examined whether haploinsufficiency of Nf1 altered adhesion by culturing osteoclasts on αv B3 coated plates. Nf1 +/− osteoclasts had a significant increase in adhesion to αv B3 compared to wildtype controls. Osteoclasts were then cultured onto bone slices and the number and size of the resorption pits were scored. Nf1 +/− osteoclasts have a higher frequency of forming pits as compared to wildtype controls and additionally the area of each pit was significantly greater. Further, a solid phase immunofixed-enzyme activity assay that measures bone resorption revealed that Nf1 +/− mice had a 2 fold increase in activity as compared to WT controls. Consistent with this activity, Nf1 +/− femurs required less stress to fracture in biomechanical testing. Further, utilizing a genetic intercross between Nf1+/− and Class1A PI3K deficient mice (p85α), we demonstrate that the increased number of osteoclast progenitors, as well as the increased hypersensitivity of Nf1 +/− osteoclast progenitors to M-CSF and RANKL were corrected to WT levels in progenitors that are mutant at both the Nf1 and the p85α loci. Additionally, the gain in function in the proliferation, migration, adhesion, and lytic activity of Nf1 +/− osteoclasts is mediated via the Ras-PI3K signaling axis. Collectively, these studies provide novel insights into the role of neurofibromin in regulating a lineage that may contribute to skeletal abnormalities in individuals with NF1.

Recent advances in osteoclast biology and pathological bone resorption.

The osteoclast is a bone-degrading polykaryon. Recent studies have clarified the differentiation of this cell and the biochemical mechanisms it uses to resorb bone. The osteoclast derives from a monocyte/macrophage precursor. Osteoclast formation requires permissive concentrations of M-CSF and is driven by contact with mesenchymal cells in bone that bear the TNF-family ligand RANKL. Osteoclast precursors express RANK, and the interaction between RANKL and RANK (which is inhibited by OPG) is the major determinant of osteoclast formation. Hormones, such as PTH/PTHrP, glucocorticoids and 1,25(OH)2D3, and humoral factors, including TNFalpha, interleukin-1, TGFss and prostaglandins, influence osteoclast formation by altering expression of these molecular factors. TNFalpha, IL-6 and IL-11 have also been shown to promote osteoclast formation by RANKL-independent processes. RANKL-dependent/independent osteoclast formation is likely to play an important role in conditions where there is pathological bone resorption such as inflammatory arthritis and malignant bone resorption. Osteoclast functional defects cause sclerotic bone disorders, many of which have recently been identified as specific genetic defects. Osteoclasts express specialized proteins including a vacuolar-type H+-ATPase that drives HCl secretion for dissolution of bone mineral. One v-ATPase component, the 116 kD V0 subunit, has several isoforms. Only one isoform, TCIRG1, is up-regulated in osteoclasts. Defects in TCIRG1 are common causes of osteopetrosis. HCl secretion is dependent on chloride channels; a chloride channel homologue, CLCN7, is another common defect in osteopetrosis. Humans who are deficient in carbonic anhydrase II or who have defects in phagocytosis also have variable defects in bone remodelling. Organic bone matrix is degraded by thiol proteinases, principally cathepsin K, and abnormalities in cathepsin K cause another sclerotic bone disorder, pycnodysostosis. Thus, bone turnover in normal subjects depends on relative expression of key cytokines, and defects in osteoclastic turnover usually reflect defects in specific ion transporters or enzymes that play essential roles in bone degradation.

Macrophage colony-stimulating factor restored chemotherapy-induced granulocyte dysfunctions: role of IL-8 production by monocytes

We evaluated the influence of M-CSF treatment on granulocyte functions in patients with ovarian cancer. Eighteen patients with ovarian cancer received two consecutive courses of chemotherapy (16 cases, CAP therapy and two cases, CP therapy) at 4-week intervals. M-CSF (8 million U/day) was infused for 7 days starting from the next day after chemotherapy. Superoxide anion production by isolated peripheral blood granulocytes, their phagocytosis, and expression of cell adhesion molecules such as CD11a, CD11b, and CD18 on granulocytes were measured by flow cytometry. Cytokine (IL-8, G-CSF, and GM-CSF) levels in peripheral blood monocyte (PBM) culture supernatants were measured by enzyme-linked immunosorbent assay in 5 out of 18 cases. The levels of CD11a, CD11b and CD18 expression on peripheral blood granulocytes and superoxide anion production by granulocytes were significantly suppressed by chemotherapy without CSF support. The levels of CD11a and CD18 expression on granulocytes were significantly enhanced by administration of M-CSF. When M-CSF was added to cultured PBM, the level of IL-8 in the supernatant increased with the concentration of M-CSF. When IL-8 was added to cultured granulocytes, the levels of CD18 expression on granulocytes and superoxide anion production by granulocytes were significantly increased. These observations suggest that M-CSF enhances the production of IL-8 from monocytes in vivo, thereby improving chemotherapy-induced granulocyte dysfunction.

Suppressor of Cytokine Signaling 1 Interacts with the Macrophage Colony-stimulating Factor Receptor and Negatively Regulates Its Proliferation Signal

Macrophage colony-stimulating factor receptor (M-CSF-R) is a tyrosine kinase that regulates proliferation, differentiation, and cell survival during monocytic lineage development. Upon activation, M-CSF-R dimerizes and autophosphorylates on specific tyrosines, creating binding sites for several cytoplasmic SH2-containing signaling molecules that relay and modulate the M-CSF signal. Here we show that M-CSF-R interacts with suppressor of cytokine signaling 1 (Socs1), a negative regulator of various cytokine and growth factor signaling pathways. Using the yeast two-hybrid system, in vitro glutathione S-transferase-M-CSF-R pull-down, and in vivo coimmunoprecipitation experiments, we demonstrated a direct interaction between the SH2 domain of Socs1 and phosphorylated tyrosines 697 or 721 of the M-CSF-R kinase insert region. Moreover, Socs1 is tyrosine-phosphorylated in response to M-CSF. Ectopic expression of Socs1 in FDC-P1/MAC and EML hematopoietic cell lines decreased their growth rates in the presence of limiting concentrations of M-CSF. However, Socs1 expression did not totally suppress long term cell growth in the presence of saturating M-CSF concentrations, in contrast to other cytokines such as stem cell factor and interleukin 3. Taken together, these results suggest that Socs1 is an M-CSF-R-binding partner involved in negative regulation of proliferation signaling and that it differentially affects cytokine receptor signals.

Hémophagocytose au cours des défaillances polyviscérales : implication du M-CSF ou réactivation virale ?

Objective: This study was aimed to assess the potential role of M-CSF and viral reactivation in the genesis of haemophagocytosis during the multiple organ failure (MOF) syndrome. Methods: Twenty-five patients (mean age : 60 ± 16 years ; Apache II : 23 ± 5) sustaining MOF with an unexplained thrombocytopenia were studied. In each patient, a bone marrow aspirate, serum M-CSF concentration, and a virological examination (Herpes viruses) were obtained on admission. In addition, 20 patients (mean age: 57 ± 15 years ; Apache II: 24 ± 7) with at least two organ failures but no thrombocytopenia constituted the control group. Circulating M-CSF levels and the frequency of virus reactivation were compared between groups. Results: Haemophagocytosis was diagnosed in 11/25 patients (44%). No viral reactivation was found. Serum M-CSF concentrations were higher in the presence of haemophagocytosis (699 ± 242 vs 438 ± 157 IU·mL –1 ; p < 0.05). Ferritin levels were also increased in the presence of a macrophage activation (3 258 ± 2 807 vs. 520 ± 280 mg·L –1 ; p < 0.0001). In contrast, both circulating M-CSF and ferritin levels were similar between thrombocytopenic patients with no hemophagocytosis and controls. Conclusions: This study confirmed the high incidence of haemophagocytosis in critically ill patients sustaining MOF. In this setting, circulating M-CSF levels were markedly elevated, whereas no Herpes viruses reactivation was found.

FLT3 Ligand induces functional effects through STAT5a activation and bcl-xl expression as evidenced through biochemical analysis and use of stat 5A −/− mice

Flt3 ligand (FL) is an early acting co-stimulatory cytokine that regulates proliferation, differentiation and survival of hematopoietic stem and progenitor cells. Its receptor, Flt3/Flk2, belongs to the type III receptor tyrosine kinases (RTK) that also include the receptors for M-CSF, Steel factor, and PDGF. Although some post-receptor signaling events and substrate specificities of the Flt3 receptor have been characterized, involvement of the Jak/Stat pathway in Flt3 signaling has not been clearly studied. We found that FL selectively activated Stat5a, without activation of Janus kinases in Baf3/Flt3 cells, a murine IL-3-dependent hematopoietic cell line which stably expresses full-length human Flt3 receptor. FL did not activate Stats 1,2,3,5b, or 6. Activation of Stat5a required the kinase activity of Flt3. We have also examined whether FL upregulates bcl-xL, a STAT5 target gene. Expression of bcl-xL was up-regulated at both the mRNA and protein levels after FL stimulation of Baf3/Flt3 cells. To evaluate physiological relevance of these observations, we assessed responsiveness of myeloid progenitor cells from Stat 5a −/− mice and their littermate controls (+/+) to stimulation by a number of growth factors and co-stimulating molecules, alone and in combination. CFU-GM from Stat 5a −/− and +/+ mice responded similarly to stimulation by various concentrations of GM-CSF, M-CSF, G-CSF, or steel factor (SLF) and to synergism of SLF plus either GM-CSF, M-CSF or G-CSF. In contrast, CFU-GM from control, but not Stat 5a −/−, mice responded to FL and to the synergistic effects of FL with either GM-CSF, M-CSF or G-CSF. In a preliminary experiment, levels of bcl-xl were greatly reduced in Stat 5a −/− compared to +/+ cells. These results demonstrate that Stat5a is essential for the costimulatory effects of FL and suggest that a mechanism for the functional activity of FL is to upregulate expression of bcl-xL through activation of Stat5a.

Enhancement of antibody dependent cellular cytotoxicity (ADCC) by combination of cytokines.

Monoclonal antibodies (MAb) specific for tumor-associated antigens (TAA) can induce an immunological cellular attack of tumor cells by a process termed antibody dependent cellular cytotoxicity (ADCC). Cytokines may augment ADCC by direct activation of immune cells or by enhancement of TAA on tumor cells. Thus, we investigated whether ADCC by MAb 17-1A and BR55-2, which recognize TAA on colorectal tumor cells, can be augmented by 3-day incubation with different concentrations of IL-2, IL-4, IL-6, IL-12, IFN-alpha, IFN-gamma, GM-CSF, M-CSF, and TNF-alpha. ADCC was assessed by a new flowcytometric cytotoxicity assay (Flieger et al. Immunol Methods 1995; 180:1-13) using PKH-2 labeled HT29 cells as targets and PKH-26 labeled peripheral blood mononuclear cells from three healthy volunteers as effector cells. We found three reaction patterns with the cytokines tested: (a) cytokines, which increase ADCC (IL-2, IL-12, IFN-alpha, and IFN-gamma, which represent Thl cytokines); (b) cytokines with no effect (GM-CSF, M-CSF, and TNF-alpha); and (c) cytokines, which decrease ADCC (IL-4 and IL-6, which represent Th2 cytokines). Then, we tested cytokines that increase ADCC in combination with the other cytokines. We found that the combinations IL-2/IFN-alpha, IL-2/IFN-gamma, IL-2/IL-12, and IL-12/IFN-alpha potentiated ADCC. By contrast, IL-4 reduced the IL-2, IL-12, and IFN-alpha-induced ADCC. Since the Thl response, cooperation of monocytes and CD4 cells is involved, we plan to elucidate by magnetic cell sorting (MACS) separation techniques, which cells are involved in cytokine-induced ADCC. Our results may be useful for finding combinations of cytokines and MAb for the locoregional treatment of colorectal cancer.

The Inhibitory Effect of RANTES on the Infection of Primary Macrophages by R5 Human Immunodeficiency Virus Type-1 Depends on the Macrophage Activation State

We investigated whether culture conditions could affect the RANTES antiviral effect on human immunodeficiency virus type 1 (HIV-1) infection of primary macrophages. Monocyte-derived macrophages (MDM) were obtained either as (1) the adherent cells of 5-day cultures of blood mononuclear cells (PBMC), followed by 2 days without nonadherent PBMC or added cytokines (MDM-5d), or (2) as the adherent cells recovered from 1-h incubation of PBMC, which were cultured for 7 days with macrophage colony-stimulating factor (M-CSF; MDM-MCSF). Infection of MDM-5d from different donors with HIV-1 R5 strains was reproducibly inhibited by RANTES (IC50≤ 10 nM), but infection of MDM-MCSF was not inhibited by ≥100 nM RANTES, even when added at initiation of cultures, although it was still inhibited by a CD4 antibody. RANTES had no antiviral effect when MDM-5d were treated with physiological concentrations of M-CSF or GM-CSF before infection. CCR5 and CXCR4 expression as well as that of other cell surface molecules, including adhesion molecules, was not affected by the cytokines. MDM-MCSF from Δ32CCR5 homozygous individuals did not render them permissive to HIV-1, suggesting that it is unlikely that the virus uses another coreceptor. RANTES binding to MDM was chondroitin sulfate, but not heparan sulfate, dependent, and RANTES bound more efficiently to MDM-5d than to MDM-MCSF. Chondroitin sulfate removal partially offset the RANTES antiviral effect for MDM-5d. Thus RANTES anti-HIV-1 activity for primary macrophages depends on culture conditions and their consequent activation status, which may lead to differences in proteoglycan surface expression. These data may be relevant for the development of chemokine-based therapy for HIV-1 infection.

Myelopoiesis and Myeloproliferative Disorders

Myeloid cells arise from a common stem cell whose development is regulated by stimulatory and inhibitory growth factors. Pluripotential hematopoietic stem cells are most influenced by IL-3, GM-CSF, and stem cell factor while committed progenitor cells are regulated by variable concentrations of GM-CSF, G-CSF, M-CSF, IL-5, Epo, and Tpo. As a result of their common origin, a key point to remember about myeloproliferative disorders is the involvement of multiple cell lines in dysplastic and neoplastic conditions. Dysplastic changes may signal early neoplastic changes with cases progressing to acute leukemia. Myelodysplastic syndrome (MDS) is associated with anemia or multiple cytopenias, normal to hypercellular bone marrow, ineffective hematopoiesis, and less than 30% blast cells of all nucleated cells in the bone marrow. Chronic myeloid leukemias also have less than 30% blast cells of all nucleated cells in the bone marrow and are distinguished from MDS by elevated cell counts of one or more cell lines with mature forms predominating. Acute myeloid leukemias, often the end result of all myeloproliferative disorders, are recognized by equal or greater 30% blast cells of all nucleated cells in the bone marrow. Additional diagnostic information from cytochemical stains, immunohistochemical staining, and cytogenetic analysis can influence the final diagnosis when morphology alone is equivocal. In conclusion, prognosis and response to treatment are best determined by application of a uniform set of standards in evaluating hematolymphatic neoplasia. Critical to diagnosis are complete blood and bone marrow evaluations including observation for dysplastic changes and blast cell quantitation. In addition, evidence for tissue infiltration identified through cytologic or histologic evaluations of lymph node, spleen, or liver is recommended.

Physiological neutrophilia is associated with elevated serum level of macrophage colony‐stimulating factor (M‐CSF)

American Journal of HematologyVolume 52, Issue 1 p. 66-66 Letters and CorrespondenceFree Access Physiological neutrophilia is associated with elevated serum level of macrophage colony-stimulating factor (M-CSF) Kiyohiko Hatake, Corresponding Author Kiyohiko Hatake Division of Hematology, Department of Medicine, Jichi Medical School, TochigiDivision of Hematology, Department of Medicine, Jichi Medical School, TochigiSearch for more papers by this authorYasusada Miura, Yasusada Miura Division of Hematology, Department of Medicine, Jichi Medical School, TochigiSearch for more papers by this authorKazuo Motoyoshi, Kazuo Motoyoshi Third Department of Internal Medicine, National Defence Medical College, Saitama, JapanSearch for more papers by this author Kiyohiko Hatake, Corresponding Author Kiyohiko Hatake Division of Hematology, Department of Medicine, Jichi Medical School, TochigiDivision of Hematology, Department of Medicine, Jichi Medical School, TochigiSearch for more papers by this authorYasusada Miura, Yasusada Miura Division of Hematology, Department of Medicine, Jichi Medical School, TochigiSearch for more papers by this authorKazuo Motoyoshi, Kazuo Motoyoshi Third Department of Internal Medicine, National Defence Medical College, Saitama, JapanSearch for more papers by this author First published: May 1996 https://doi.org/10.1002/(SICI)1096-8652(199605)52:1<66::AID-AJH17>3.0.CO;2-5Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. References 1 Cincotta R, Balloch A, Metz J, Layton JE, Lieschke GJ: Physiological neutrophilia of pregnancy is not associated with a rise in plasma granulocyte colony-stimulating factor (G-CSF) Am J Hematol 48: 288– 289, 1995. 2 Hanamura T, Motoyoshi K, Yoshida K, Saito M, Miura Y, Kawashima T, Nishida M, Takaku F: Quantitation and identification of human monocytic colony-stimulating factor in human serum by enzyme-linked immunosorbent assay. Blood 72: 886– 890, 1988. 3 Suzu S, Yanai N, Sato-Somoto J, Yamada M, Kawashima T, Hanamura T, Nagata N, Takaku F, Motoyoshi K: Characterization of macrophage colony-stimulating factor in body fluids by immunoblot analysis. Blood 77: 2160– 2164, 1991. 4 Praloran V, Coupey L, Donnard M, Berrada L, Naud MF: Elevation of serum MCSF concentrations during pregnancy and ovarian hyperstimulation. Br J Haematol 86: 675– 677, 1994. Citing Literature Volume52, Issue1May 1996Pages 66-66 ReferencesRelatedInformation

Endogenous haemopoietic growth factors in neutropenia and infection.

Haemopoietic growth factors (HGFs) are being administered to patients with neutropenic fever; however, little is known about the endogenous HGF response in these patients. Specific assays were used to study four HGFs, granulocyte (G-) CSF, granulocyte-macrophage (GM-) CSF, macrophage (M-) CSF and interleukin (IL-) 6 levels in the blood of patients with neutropenic fever (46 episodes). For comparison, levels were also measured in three control populations: normals (20), afebrile neutropenic (14), and bacteraemic but not neutropenic patients (20). In febrile patients, levels of G-CSF (median, range) (0.46, < 0.10-142 ng/ml). IL-6 (0.054, 0.005-24.3 ng/ml) and M-CSF (18.5, 9.9-79.1 ng/ml) were elevated compared with afebrile subjects (< 0.10, < 0.10-1.62 ng/ml). (0.008, 0.002-0.024 ng/ml) and (6.45, < 5.0-31.3 ng/ml) respectively. GM-CSF was not elevated (< 0.02, < 0.02-8.0 ng/ml) compared with afebrile subjects (0.021, < 0.02-0.20 ng/ml). Variables significantly associated (P < 0.05) with elevated cytokine levels were determined by multiple regression analyses. Factors associated with G-CSF elevation were fever, neutropenia, pathogen type and raised bilirubin and creatinine. In contrast, neutropenia was not associated with IL-6 elevation although there was an association between IL-6 elevation and fever, Gram-negative and fungal infections and raised creatinine and bilirubin. M-CSF elevation was associated with fever, renal impairment and known pathogen. Elevated G-CSF and IL-6 levels normalized rapidly (hours-days) with the resolution of infection, whereas M-CSF concentrations remained elevated for up to 10 d. Cytokine levels remained elevated in septic neutropenic patients who did not recover. In summary, G-CSF, IL-6 and M-CSF levels were significantly elevated in sepsis. In contrast, GM-CSF levels were not elevated. These studies should assist the development of therapeutic strategies using HGFs in the treatment of sepsis.

造血器疾患とマクロファージ機能

We report the results of in vitro experiments and in vivo studies to explore the possibility whether macrophage function might influence on the prognosis of human leukemia. At first, we noticed a transient but remarkable monocytosis during recovery after intensive chemotherapy in peripheral blood of patients with acute leukemia. Such remarkable monocytosis was observed only when they achieved a complete hematological remission. This may reflect the recovery of normal hematopoiesis, in addition, monocytes might contribute to the reduction of residual leukemic cells. To explore such possibility, we took advantage of monocyte-derived macrophages (Mφ). Mφ, pretreated with IFN-γ or LPS, lysed human leukemic cells, HL-60 and K562. When Mφ were pretreated with IFN-γ and LPS simultaneously, they lysed much more leukemic cells. Furthermore, activated Mφ could induce lysis of leukemic cells separated from Mφ by a microporous membrane. As to the mechanism of leukemic cell lysis by activated Mφ, TNF was found to be at least an important effector molecule. It was also suggested that TNF and other labile factor(s) might be involved in the leukemic cell lysis. Next, we measured the concentration of various cytokines in the blood of leukemia patients before chemotherapy, at nadir, and on recovery phase. Among them, the concentration of IL-6 was high on recovery phase. G-CSF was high at early recovery phase in the blood of some patients. In the patients with high grade fever the concentration of G-CSF, M-CSF, and IFN-γ was high. Elevated concentration of IL-6 may contribute for early recovery of normal blood cells.Monocyte-derived Mφ of patients with leukemia, obtained during monocytosis, lysed HL-60 to a similar extent as control Mφ. The finding that activated human Mφ could lyse human leukemic cells suggests a role of Mφ in the eradication of leukemic cells in vivo and supports the possibility of clinical application of activated Mφ.

Effects of Human Stem Cell Factor ( c-kit ligand ) on Proliferation of Myeloid Leukemia Cells: Heterogeneity in Response and Synergy With Other Hematopoietic Growth Factors

A novel hematopoietic growth factor, the stem cell factor (SCF), for primitive hematopoietic progenitor cells has recently been purified and its gene has been cloned. In this study we tested the mitogenic activity of recombinant human SCF on myeloid leukemia cells as well as the expression of its receptor. We have investigated the proliferation of 31 myeloid leukemia cell lines as well as fresh myeloid leukemic blasts from 17 patients in a 72-hour 3H-thymidine uptake assay in the presence of various concentrations of recombinant human (rh) SCF alone or in combination with saturating concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, interleukin-3 (IL-3), or erythropoietin (EPO). Only five of 31 lines, but fresh leukemic blasts from 12 of 17 patients with acute myeloid leukemia (AML), significantly responded to SCF. The responding cell lines were of the acute promyelocytic, chronic myeloid, megakaryoblastic, and erythroleukemia origin, the responding blast preparations of all French-American-British subtypes. Synergistic activities of SCF were found with G-CSF, GM-CSF, EPO, and IL-3. To determine the SCF binding sites on leukemic cells, we used 125I-radiolabeled SCF in Scatchard analysis and cross-linking studies. The leukemic cell lines responding to SCF expressed from 2,300 up to 29,000 binding sites per cell. The SCF receptor expression was downregulated in vitro by the presence of its ligand. Cross-linking studies demonstrated a 150-Kd SCF receptor on the surface of all responding myeloid leukemias. This study suggests that SCF may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor for other cytokines. Furthermore, using polymerase chain reaction analysis of total RNA from the myeloid leukemia lines, we found expression of SCF-mRNA in 17 of 30 lines, suggesting autocrine mechanisms in the growth of a subgroup of leukemic cells by coexpression of SCF and its receptor.