The development of bone changes induced in rats by recombinant human granulocyte colony-stimulating factor is suppressed by bisphosphonate.

We have previously demonstrated that high doses of recombinant human granulocyte colony-stimulating factor (rhG-CSF) induce bone changes characterized by osteoclastic bone resorption and osteogenesis due to intramembranous ossification in rats. In this communication we examined the effects of a pretreatment with 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP), which is a powerful inhibitor of osteoclastic bone resorption, on bone changes induced by rhG-CSF in order to investigate the relation between osteoclastic bone resorption and osteogenesis. AHPrBP (5 mg/kg/day) was subcutaneously given to 6-week-old rats for 2 days. From the following day of the final injection of AHPrBP, rats received a subcutaneous injection of rhG-CSF (1,000 micrograms/kg/day) for 14 days, and the femur and tibia were evaluated histopathologically. By the analysis of peripheral blood leukocyte counts, spleen weights and bone marrow findings, the pretreatment with AHPrBP had no effect on the activation of hematopoiesis related to the major pharmacological activity of rhG-CSF. In the rats treated with rhG-CSF alone, accelerated osteoclastic bone resorption and osteogenesis due to intramembranous ossification were observed in the trabeculae of metaphyseal spongiosa. The accelerated osteoclastic bone resorption induced by rhG-CSF was suppressed by the pharmacological activity of AHPrBP. Furthermore, the osteogenesis induced by rhG-CSF was also suppressed by AHPrBP. These results suggest that the osteogenesis induced by rhG-CSF is a sequential reaction of accelerated osteoclastic bone resorption, and moreover that the main action of rhG-CSF on bone is an acceleration of osteoclastic bone resorption.     

Granulocyte-macrophage colony-stimulating factor is required for bronchial eosinophilia in a murine model of allergic airway inflammation

GM-CSF plays an important role in inflammation by promoting the production, activation, and survival of granulocytes and macrophages. In this study, GM-CSF knockout (GM-CSF(-/-)) mice were used to investigate the role of GM-CSF in a model of allergic airway inflammation. In allergic GM-CSF(-/-) mice, eosinophil recruitment to the airways showed a striking pattern, with eosinophils present in perivascular areas, but almost completely absent in peribronchial areas, whereas in wild-type mice, eosinophil infiltration appeared in both areas. In the GM-CSF(-/-) mice, mucus production in the airways was also reduced, and eosinophil numbers were markedly reduced in the bronchoalveolar lavage (BAL)(3) fluid. IL-5 production was reduced in the lung tissue and BAL fluid of GM-CSF(-/-) mice, but IL-4 and IL-13 production, airway hyperresponsiveness, and serum IgE levels were not affected. The presence of eosinophils in perivascular but not peribronchial regions was suggestive of a cell migration defect in the airways of GM-CSF(-/-) mice. The CCR3 agonists CCL5 (RANTES) and CCL11 (eotaxin-1) were expressed at similar levels in GM-CSF(-/-) and wild-type mice. However, IFN-gamma mRNA and protein were increased in the lung tissue and BAL fluid in GM-CSF(-/-) mice, as were mRNA levels of the IFN-gamma-inducible chemokines CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-Tac). Interestingly, these IFN-gamma-inducible chemokines are natural antagonists of CCR3, suggesting that their overproduction in GM-CSF(-/-) mice contributes to the lack of airway eosinophils. These findings demonstrate distinctive abnormalities to a model of allergic asthma in the absence of GM-CSF.     

Gene expression-based detection of radiation exposure in mice after treatment with granulocyte colony-stimulating factor and lipopolysaccharide

In a large-scale nuclear incident, many thousands of people may be exposed to a wide range of radiation doses. Rapid biological dosimetry will be required on an individualized basis to estimate the exposures and to make treatment decisions. To ameliorate the adverse effects of exposure, victims may be treated with one or more cytokine growth factors, including granulocyte colony-stimulating factor (G-CSF), which has therapeutic efficacy for treating radiation-induced bone marrow ablation by stimulating granulopoiesis. The existence of infections and the administration of G-CSF each may confound the ability to achieve reliable dosimetry by gene expression analysis. In this study, C57BL/6 mice were used to determine the extent to which G-CSF and lipopolysaccharide (LPS, which simulates infection by gram-negative bacteria) alter the expression of genes that are either radiation-responsive or non-responsive, i.e., show potential for use as endogenous controls. Mice were acutely exposed to (60)Co γ rays at either 0 Gy or 6 Gy. Two hours later the animals were injected with either 0.1 mg/kg of G-CSF or 0.3 mg/kg of LPS. Expression levels of 96 different gene targets were evaluated in peripheral blood after an additional 4 or 24 h using real-time quantitative PCR. The results indicate that the expression levels of some genes are altered by LPS, but altered expression after G-CSF treatment was generally not observed. The expression levels of many genes therefore retain utility for biological dosimetry or as endogenous controls. These data suggest that PCR-based quantitative gene expression analyses may have utility in radiation biodosimetry in humans even in the presence of an infection or after treatment with G-CSF.     

Cell surface lectins of human granulocytes: their expression is modulated by mononuclear cells and granulocyte/macrophage colony-stimulating factor

This paper presents the characterization of a sugar-specific receptor on the surface of human circulating polymorphonuclear cells. With the help of fluorescent neoglycoproteins and flow cytometry, a receptor was identified as being specific for alpha-L-rhamnosyl residues. The number of receptors was 55,000/cell and their affinity reached 2 x 10(8) l mol-1. This number changed as a function of the biological state of the cells. Indeed, receptor expression was modulated by the presence of other cells. T cells and B cells increased the number of receptors on the granulocyte surface. Expression of the alpha-L-rhamnose-specific lectin was dependent on lymphocyte derived soluble factor(s), which induce(s) growth and differentiation of polymorphonuclear phagocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) specifically produced a significant increase in the number of receptors for alpha-L-rhamnose (2-10-fold/cell). This modulation was independent of protein kinase C activators such as phorbol ester, which produced no effect on alpha-L-rhamnose receptor expression. These findings demonstrate that GM-CSF may stimulate post differentiation functions and properties of mature granulocytes.     

Restoration of the acute phase response after infection in cyclophosphamide-treated mice by granulocyte colony-stimulating factor

The therapeutic effect of granulocyte colony-stimulating factor (G-CSF) against intramuscular infection withPseudomonas aeruginosa in cyclophosphamide (CY)-treated mice was analyzed by measuring plasma levels of amyloid P-component (APC) and proinflammatory cytokine levels. CY (100mg/kg) treatment of mice significantly suppressed plasma concentrations of APC and tumor-necrosis factor- (TNF-) following infection withP. aeruginosa, in associated with enhanced susceptibility of the treated mice to this bacterium. A 4-day treatment of CY-treated mice with recombinant human G-CSF (rhG-CSF) increased resistance of CY-treated mice, together with the marked restoration of APC and TNF- productions. The capacity to produce interleukin 1- and TNF- of peritoneal macrophages and also that to produce IL-6 of spleen cells were significantly enhanced by thein vivo administration of rhG-CSF in CY-treated mice. These results indicate that G-CSF may increase the functions of monocytes/macrophages directly or indirectlyin vivo. Therefore, the therapeutic effect of rhG-CSF seems to consist of not only increases in the number and functions of neutrophills but also enhancement of monocyte/macrophage functions.Abbreviations rhG-CSF recombinant human granulocyte-colony stimulating factor - PMNs polymorphonuclear leukocytes - CY cyclophosphamide - HBSS Hanks' balanced salt solution - APC amyloid P-component - IEP immunoelectrophoresis - CFU colony-forming units - TNF- tumor-necrosis factor- - d IL interleukin     

A novel differentiation antigen on human monocytes that is specifically induced by granulocyte-macrophage colony-stimulating factor or IL-3

A mouse mAb (TOMS-1) was generated against human blood monocytes that had been cultured for 4 days in medium with recombinant human granulocyte-macrophage CSF (GM-CSF). TOMS-1 (IgG1) detected a unique cell surface Ag with a molecular mass of about 43 kDa under both reducing and nonreducing conditions. TOMS-1Ag was expressed on monocytes treated with GM-CSF, but not on fresh or untreated monocytes. This Ag was induced dose dependently during culture of monocytes with GM-CSF for more than 24 h, reaching a maximum level in 3 or 4 days. Treatment of monocytes with cycloheximide in the presence of GM-CSF blocked TOMS-1Ag induction completely, indicating that de novo protein synthesis was required for its expression. TOMS-1Ag was also induced by treatment of monocytes with IL-3, but not with other cytokines such as macrophage-CSF, IL-4, and IFN-gamma or stimulators including LPS, desmethyl muramyl dipeptide, and PMA. TOMS-1Ag expression induced by GM-CSF was up-regulated by IL-4, but down-regulated by IFN-gamma. TOMS-1Ag was not induced on lymphocytes, granulocytes, or AM by GM-CSF or appropriate stimuli. TOMS-1Ag was also not expressed on any cell lines of human leukemias or solid tumors examined. Thus, TOMS-1Ag is a monocyte-specific differentiation Ag induced by GM-CSF or IL-3. These results suggest that TOMS-1 should be useful for monitoring the process of monocyte differentiation by GM-CSF or IL-3.     

Primary listerial infections are exacerbated in mice administered neutralizing antibody to macrophage colony-stimulating factor.

The serum and tissue levels of macrophage colony-stimulating factor (M-CSF) are elevated in mice during a primary immunologic response to infection by Listeria monocytogenes. Experiments were performed to determine the specific role of M-CSF in the resolution of listerial infections. The bulk of Listeria injected into a mouse i.v. is deposited in the liver. The expression of M-CSF mRNA in the liver increased markedly within 2 h postinfection. Maximum expression was dependent upon the dose of Listeria inoculated. The administration of anti-M-CSF mAb reduced the percentage of Mac-1+ mononuclear phagocytes subsequently found in the livers of infected animals. This reduction correlated inversely with an increase in the number of Listeria associated with both the parenchymal and NPC populations. These results suggest that M-CSF may play an important role in the primary immunologic response to Listeria in the liver by stimulating the production, mobilization, and/or biologic activity of Mac-1+ mononuclear phagocytes.     

A novel functional epitope formed by domains 1 and 4 of the human common beta-subunit is involved in receptor activation by granulocyte macrophage colony-stimulating factor and interleukin 5

The receptors for human interleukins 3 and 5 and granulocyte macrophage colony-stimulating factor are composed of ligand-specific alpha-subunits and a common beta-subunit (betac), the major signaling entity. The way in which betac interacts with ligands in the respective activation complexes has remained poorly understood. The recently determined crystal structure of the extracellular domain of betac revealed a possible ligand-binding interface composed of domain 1 of one chain of the betac dimer and the adjacent domain 4 of the symmetry-related chain. We have used site-directed mutagenesis, in conjunction with ligand binding and proliferation studies, to demonstrate the critical requirement of the domain 1 residues, Tyr(15) (A-B loop) and Phe(79) (E-F loop), in high affinity complex formation and receptor activation. The novel ligand-receptor interface formed between domains 1 and 4 represents the first example of a class I cytokine receptor interface to be composed of two noncontiguous fibronectin III domains.     

Recombinant human granulocyte colony-stimulating factor protects against MPTP-induced dopaminergic cell death in mice by altering Bcl-2/Bax expression levels

Granulocyte colony-stimulating factor (G-CSF) has been used for the treatment of neutropenia in hematologic disorders. The neuroprotective effects of G-CSF were reported in neurological disease models. In the present study, we examined whether G-CSF can protect dopaminergic neurons against MPTP-induced cell death in a mouse model of Parkinson's disease. Mice of one group were injected intraperitoneally with MPTP for five consecutive days, those of another group with MPTP and intraperitoneal G-CSF at 2 days and 1 day before the first MPTP injection, and 30 min before each MPTP injection, while control mice received saline injections. Immunohistochemistry, western blotting analysis, and HPLC were performed to evaluate damage of substantia nigra dopaminergic neurons and expression of Bcl-2 and Bax protein. MPTP induced dopaminergic cell death in the substantia nigra. G-CSF significantly prevented MPTP-induced loss of tyrosine hydroxylase-positive neurons (p < 0.05), increased Bcl-2 protein and decreased Bax protein expression. Our findings indicate that G-CSF provides neuroprotection against MPTP-induced cell death and this effect is mediated by increasing Bcl-2 expression levels and decreasing Bax expression levels in C57BL/6 mice.     

Prenatal testosterone exposure leads to hypertension that is gonadal hormone-dependent in adult rat male and female offspring

Prenatal testosterone exposure impacts postnatal reproductive and endocrine function, leading to alterations in sex steroid levels. Because gonadal steroids are key regulators of cardiovascular function, it is possible that alteration in sex steroid hormones may contribute to development of hypertension in prenatally testosterone-exposed adults. The objectives of this study were to evaluate whether prenatal testosterone exposure leads to development of hypertension in adult males and females and to assess the influence of gonadal hormones on arterial pressure in these animals. Offspring of pregnant rats treated with testosterone propionate or its vehicle (controls) were examined. Subsets of male and female offspring were gonadectomized at 7 wk of age, and some offspring from age 7 to 24 wk received hormone replacement, while others did not. Testosterone exposure during prenatal life significantly increased arterial pressure in both male and female adult offspring; however, the effect was greater in males. Prenatal androgen-exposed males and females had more circulating testosterone during adult life, with no change in estradiol levels. Gonadectomy prevented hyperandrogenism and also reversed hypertension in these rats. Testosterone replacement in orchiectomized males restored hypertension, while estradiol replacement in ovariectomized females was without effect. Steroidal changes were associated with defective expression of gonadal steroidogenic genes, with Star, Sf1, and Hsd17b1 upregulation in testes. In ovaries, Star and Cyp11a1 genes were upregulated, while Cyp19 was downregulated. This study showed that prenatal testosterone exposure led to development of gonad-dependent hypertension during adult life. Defective steroidogenesis may contribute in part to the observed steroidal changes.     

Role of protein kinase C and the Na+/H+ antiporter in suppression of apoptosis by granulocyte macrophage colony-stimulating factor and interleukin-3.

Granulocyte macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) suppress apoptosis in hemopoietic cells, a process of active cell death characterized by the degradation of genomic DNA into oligonucleosomic fragments. The present study was therefore initiated with the view that the two growth factors may trigger the same early events in the cell, leading to suppression of apoptosis. We provide evidence here for a role of protein kinase C and of the Na+/H+ antiporter in the signal transduction pathways activated by binding of GM-CSF or IL-3 to their respective receptors, resulting in suppression of apoptosis in target cells. First, kinetic studies indicate that the process is irreversible after two hours of deprivation. The suppression of apoptosis by GM-CSF and IL-3 is dose-dependent, with half-efficient concentrations that are in the range of the dissociation constants of the high affinity GM-CSF or IL-3 receptor, respectively. Second, the use of three inhibitors of protein kinase C (PKC), H7, staurosporine, and sphingosine, in concentrations that are below their toxicity limits, revert the suppression of apoptosis by IL-3 and GM-CSF. Conversely, the use of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, allows a bypass of receptor activation in suppression of apoptosis. Western blotting of cytosolic and membrane proteins indicate that exposure of the cells to GM-CSF, IL-3, or TPA results in translocation of PKC to the cell membrane. Our data, therefore, indicate that the activation of PKC is important in suppression of apoptosis by GM-CSF and IL-3. Third, the two amiloride derivatives 5-(N,N-hexamethylene) and 5-(N-ethyl-N-isopropyl)amiloride that specifically block the function of the Na+/H+ antiport also revert the protective effect of GM-CSF, IL-3, and TPA on MO7-E cells. Further, exposure of the cells to GM-CSF, IL-3, or TPA results in sustained pHi alkalinizatio, which is abrogated when the cells are preincubated with 5-(N-ethyl-N-isopropyl)amiloride, a specific inhibitor of the antiport. Preincubation of the cells with staurosporine, a PKC inhibitor, also significantly reduces the effect of GM-CSF or IL-3 on pHi. Taken together, our data indicate that a functional antiport is required in suppression of apoptosis by GM-CSF, IL-3, or TPA. Furthermore, our results are consistent with the view that GM-CSF or IL-3 receptor activation initiates the sequential activation of PKC and of the Na+/H+ antiporter, resulting in suppression of apoptosis in target cells.     

Proliferation of microglial cells induced by 1-methyl-4-phenylpyridinium in mesencephalic cultures results from an astrocyte-dependent mechanism: role of granulocyte macrophage colony-stimulating factor

There is evidence that an inflammatory microglial reaction participates in the pathophysiology of dopaminergic neuronal death in Parkinson's disease and in animal models of the disease. However, this phenomenon remains incompletely characterized. Using an in vitro model of neuronal/glial mesencephalic cultures, we show that the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+) stimulates the proliferation of microglial cells at concentrations that selectively reduce the survival of DA neurones. The mitogenic action of MPP+ was not the mere consequence of neuronal cell demise as the toxin produced the same effect in a model system of neuronal/glial cortical cultures, where target DA neurones are absent. Consistent with this observation, the proliferative effect of MPP+ was also detectable in neurone-free microglial/astroglial cultures. It disappeared, however, when MPP+ was added to pure microglial cell cultures suggesting that astrocytes played a key role in the mitogenic mechanism. Accordingly, the proliferation of microglial cells in response to MPP+ treatment was mimicked by granulocyte macrophage colony-stimulating factor (GM-CSF), a proinflammatory cytokine produced by astrocytes and was blocked by a neutralizing antibody to GM-CSF. Thus, we conclude that the microglial reaction observed following MPP+ exposure depends on astrocytic factors, e.g. GM-CSF, a finding that may have therapeutic implications.     

Immune response induced by airway sensitization after influenza A virus infection depends on timing of antigen exposure in mice

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.     

Production of a factor inhibiting macrophage migration to the Candida albicans antigen in mice and their resistance to this microorganism

Resistance to C. albicans, an opportunistic microorganism, has been studied in CBA and C57BL/6 mice, oppositely responsive in the production of the factor inhibiting migration of macrophages to antigen obtained from this fungus. The study has shown that CBA mice, highly responsive in the macrophage migration inhibiting factor, are less resistant to C. albicans, while C57BL/6 mice with low response to this antigen are more resistant to this infective agent. Macrophages play, probably, a certain role in the generalization of the process because not all phagocytized C. albicans cells are digested.     

MCP-1 is induced by receptor activator of nuclear factor-{kappa}B ligand, promotes human osteoclast fusion, and rescues granulocyte macrophage colony-stimulating factor suppression of osteoclast formation

Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-kappaB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant.     

Eosinophilia is induced in the colon of Th2-sensitized mice upon exposure to locally expressed antigen

Eosinophilic inflammation is a feature of a variety of gastrointestinal (GI) disorders including eosinophil-associated GI disorder, allergy, inflammatory bowel disease, and parasite infection. Elucidating the mechanisms of eosinophil infiltration into the GI tract is important to the understanding of multiple disease processes. We hypothesize that eosinophilia in the large intestine (colon) can be induced by an antigen in a host that is associated with Th2-skewed antigen-specific immune responses. To investigate the importance of antigenic triggering, we established polarized antigen-specific Th2 type responses in BALB/c mice, using ovalbumin in conjunction with aluminum hydroxide. Upon challenge at the colonic mucosa through transient (3-4 days) expression of the antigen gene encoded in an adenovirus vector, sensitized animals developed significant subepithelial colonic inflammation, characterized by marked eosinophilic infiltration, and the presence of enlarged and increased numbers of lymphoid follicles. The alterations peaked around day 5 and resolved over the next 5-10 days, and no epithelial cell damage was detected through the entire course. Administration of a control (empty) adenovirus vector did not lead to any pathological changes. These data suggest that colonic eosinophilia can be induced by exposure to an antigen associated with preexisting Th2-skewed responses. Thus the model established here may provide a useful tool to study GI and, in particular, colonic inflammation with respect to underlying mechanisms involved in the recruitment and the immediate function of eosinophils.     

Expression of macrophage migration inhibitory factor by neuroblastoma leads to the inhibition of antitumor T cell reactivity in vivo

Neuroblastomas and many other solid tumors produce high amounts of macrophage migration inhibitory factor (MIF), which appears to play a role in tumor progression. We found that MIF expression in neuroblastoma inhibits T cell proliferation in vitro, raising the possibility that MIF promotes tumorigenesis, in part, by suppressing antitumor immunity. To examine whether tumor-derived MIF leads to suppression of T cell immunity in vivo, we generated MIF-deficient neuroblastoma cell lines using short hairpin small interfering RNAs (siRNA). The MIF knockdown (MIFKD) AGN2a neuroblastoma cells were more effectively rejected in immune-competent mice than control siRNA-transduced or wild-type AGN2a. However, the increased rejection of MIFKD AGN2a was not observed in T cell-depleted mice. MIFKD tumors had increased infiltration of CD8(+) and CD4(+) T cells, as well as increased numbers of macrophages, dendritic cells, and B cells. Immunization with MIFKD AGN2a cells significantly increased protection against tumor challenge as compared with immunization with wild-type AGN2a, and the increased protection correlated with elevated frequencies of tumor-reactive CD8(+) T cells in the lymphoid tissue of treated animals. Increased numbers of infiltrating tumor-reactive CD8(+) T cells were also observed at the site of tumor vaccination. In vitro, treatment of AGN2a-derived culture supernatants with neutralizing MIF-specific Ab failed to reverse T cell suppressive activity, suggesting that MIF is not directly responsible for the immune suppression in vivo. This supports a model whereby MIF expression in neuroblastoma initiates a pathway that leads to the suppression of T cell immunity in vivo.