Role Of Granulocyte-macrophage Colony-stimulating Factor Research Articles

GM-CSF mediates autoimmunity by enhancing IL-6–dependent Th17 cell development and survival

Granulocyte macrophage–colony stimulating factor (GM-CSF) is critically involved in development of organ-related autoimmune inflammatory diseases including experimental allergic encephalitis and collagen-induced arthritis. Roles of GM-CSF in the initiation and in the effector phase of the autoimmune response have been proposed. Our study was designed to investigate the mechanisms of GM-CSF in autoimmunity using a model of autoimmune heart inflammatory disease (myocarditis). The pathological sequel after immunization with heart myosin has been shown previously to depend on IL-1, IL-6, IL-23, and IL-17. We found that innate GM-CSF was critical for IL-6 and IL-23 responses by dendritic cells and generation of pathological Th17 cells in vivo. Moreover, GM-CSF promoted autoimmunity by enhancing IL-6–dependent survival of antigen specific CD4+ T cells. These results suggest a novel role for GM-CSF in promoting generation and maintenance of Th17 cells by regulation of IL-6 and IL-23 in vivo.

The role of GM-CSF in adipose tissue inflammation

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a proinflammatory cytokine that has a central action to reduce food intake and body weight. Consistent with this, GM-CSF knockout mice are more obese and hyperphagic than wild-type mice. However, in lung, GM-CSF is an important determinant of macrophage infiltration. Consequently, we sought to determine if GM-CSF might contribute to adipose tissue macrophage accumulation, insulin resistance, and low-grade inflammation that occurs when animals gain weight on a high-fat diet (HFD). We therefore determined how targeted genetic disruption of GM-CSF can affect adipose tissue macrophage and cytokine gene expression as well as glucose homeostasis by performing hyperinsulinemic-euglycemic clamps. The number of macrophages and CCR2 gene expression in adipose tissue of GM-CSF knockout mice was decreased relative to those in wild-type mice, and the adipocyte size of mesenteric fat was increased in GM-CSF knockout mice on a HFD compared with wild-type mice. The level of mRNA of the proinflammatory cytokines interleukin-1beta, tumor necrosis factor-alpha, and macrophage inflammatory protein-1alpha was significantly lower in mesenteric fat of GM-CSF knockout mice on the HFD than in wild-type mice. Using the hyperinsulinemic-euglycemic clamp technique, GM-CSF knockout mice had greater overall insulin sensitivity. This increase was due to enhanced peripheral uptake and utilization of glucose rather than to increased hepatic insulin sensitivity. Collectively, the data suggest that the GM-CSF knockout mutation ameliorates peripheral insulin resistance in spite of increased adiposity by reducing inflammation in adipose tissue in response to a HFD.

Granulocyte Macrophage‐Colony Stimulating Factor receptor expression on human cardiomyocytes from end‐stage heart failure patients

In remodelling ventricles, the progression of heart failure is associated with structural changes involving the extra-cellular matrix (ECM) and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death. The role of some cytokines and haematopoietic growth factors in the mechanism of both damage and regeneration of cardiac tissue during acute myocardial infarction has been demonstrated. Following heart damage, the development of scarred tissue was considered to be the only outcome, since myocytes were considered to be terminally differentiated cells. However, recent studies in animal models and adult human hearts have shown that myocytes can proliferate under the modulation of several factors. To assess Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) receptor expression in healthy and diseased human hearts, and to evaluate the possible role of GM-CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy. GM-CSFR expression in human cardiac tissue from explanted hearts of ten patients with end-stage heart failure and in cardiac biopsies from eight normal human hearts was studied by immunohistochemistry, and cellular and molecular biology assays. Our results demonstrated an increase in GM-CSFR in cardiomyocytes from end-stage heart failure tissues as compared to normal control tissues. We hypothesize that GM-CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium.

Soluble HLA-G Molecules Are Increased during Acute Leukemia, Especially in Subtypes Affecting Monocytic and Lymphoid Lineages'

Human leukocyte antigen G (HLA-G) molecules corresponding to nonclassic class I genes of the major histocompatibility complex exhibit immunomodulatory properties. They are either membrane-bound or solubly expressed during certain tumoral malignancies. Soluble human leukocyte antigen G (sHLA-G) molecules seem more frequently expressed than membranebound isoforms during hematologic malignancies, such as lymphoproliferative disorders. Assay of these molecules by enzyme-linked immunosorbent assay in patients suffering from another hematologic disorder (acute leukemia) highlights increased sHLA-G secretion. This increased secretion seems more marked in acute leukemia subtypes affecting monocytic and lymphoid lineages such as FABM4 and FABM5, as well as both B and T acute lymphoblastic leukemia (ALL). Moreover, this study uses in vitro cytokine stimulations and reveals the respective potential roles of granulocyte-macrophage colony-stimulating factor and interferon-γ in increasing this secretion in FABM4 and ALL. Correlations between sHLA-G plasma level and clinical biologic features suggest a link between elevated sHLA-G level and 1) the absence of anterior myelodysplasia and 2) high-level leukocytosis. All these findings suggest that sHLA-G molecules could be a factor in tumoral escape from immune survey during acute leukemia.

Redox State and O2•- Production in Neutrophils of Crohn's Disease Patients

The aim of this in vitro study was to evaluate the intracellular redox state and respiratory burst (RB) in neutrophils of patients with Crohn's disease (CD). The intracellular redox state and RB in neutrophils was assessed by the superoxide anion (O2*-) production induced in these cells after stimulation by various factors related to the molecular mechanisms that, if altered, may be responsible for an abnormal immune response. This can, in part, cause the onset of inflammation and tissue damage seen in CD. This study demonstrated a decreased glutathione/glutathione disulfide (GSH/GSSG) ratio index of an increased oxidative state in CD patient neutrophils. Moreover, our findings showed a decrease in tumor necrosis factor (TNF-alpha)- or phorbol 12-myristate 13-acetate (PMA)-induced O2*- production in CD patient neutrophils adherent to fibronectin as compared with controls. A decreased adhesion was also demonstrated. For this reason, the involvement of altered mechanisms of protein kinase C (PKC) and beta-integrin activation in CD patient neutrophils is suggested. These data also showed that the harmful effects of TNF-alpha cannot be caused by excessive reactive oxygen species (ROS) production induced by neutrophils. Decreased cell viability after a prolonged time of adhesion (20 hrs) was also measured in CD patient neutrophils. The findings of this study demonstrate, for the first time, that granulocyte-macrophage colony-stimulating factor (GM-CSF), a compound recently used in CD therapy, is able to activate the RB for a prolonged time both in control and CD patient neutrophils. Increased viability of CD patient neutrophils caused by GM-CSF stimulation was also observed. In conclusion, our results indicate that decreased O2*- production and adhesion, caused, in part, by an anomalous response to TNF-alpha, together with low GSH level and low cell viability, may be responsible for the defective neutrophil function found in CD patients. This can contribute to the chronic inflammation and relapses that characterize this pathology. A possible role of GM-CSF in inducing O2*- production and in restoring the defensive role of neutrophils in CD patients is suggested.

Activation of Vascular Endothelial Growth Factor Receptor-2 in Bone Marrow Leads to Accumulation of Myeloid Cells: Role of Granulocyte-Macrophage Colony-Stimulating Factor

Vascular endothelial growth factor (VEGF) is a secreted cytokine that plays a major role in the formation and maintenance of the hemopoietic and vascular compartments. VEGF and its receptors, VEGFR-1 and VEGFR-2, have been found to be expressed on subsets of normal and malignant hemopoietic cells, but the role of the individual receptors in hemopoiesis requires further study. Using a VEGFR-2 fusion protein that can be dimerized with a synthetic drug, we were able to specifically examine the effects of VEGFR-2 signaling in hemopoietic cells in vivo. Mice transplanted with bone marrow transduced with this inducible VEGFR-2 fusion protein demonstrated expansion of myeloid cells (Gr-1+, CD11b+). Levels of myeloid progenitors were also increased following VEGFR-2 activation, through autocrine and paracrine mechanisms, as measured by clonogenic progenitor assays. VEGFR-2 activation induced expression of GM-CSF and increased serum levels in vivo. Abrogation of GM-CSF activity, either with neutralizing Abs or by using GM-CSF-null hemopoietic cells, inhibited VEGFR-2-mediated myeloid progenitor activity. Our findings indicate that VEGF signaling through VEGFR-2 promotes myelopoiesis through GM-CSF-dependent and -independent mechanisms.

Pulmonary alveolar proteinosis associated with psoriasis and complicated by mycobacterial infection: Successful treatment with granulocyte‐macrophage colony stimulating factor after a partial response to whole lung lavage

Pulmonary alveolar proteinosis (PAP) is a rare lung disease. Although whole lung lavage is considered the most effective treatment, not every patient shows a complete response. The case ofa young man with PAP in association with psoriasis who underwent frequent whole lung lavage but only achieved remission following treatment with granulocyte-macrophage colony stimulating factor (GM-CSF) is reported. His lung problem was complicated by atypical mycobacterial infection,which resolved with treatment. The role of GM-CSF is discussed.

Nervous system injury: focus on the inflammatory cytokine ?granulocyte-macrophage colony stimulating factor?*1

Any lesion in the nervous system, be it infectious, immunopathological, ischemic or traumatic, is followed by an inflammatory process that induces rapid activation of glial cells and additional recruitment of granulocytes, T-cells and monocytes/macrophages from the blood stream. Neuroinflammation is a double-sided sword. It can cause neuronal damage and participate in neuropathic pain, but it also has neuroprotective and neurotrophic effects at some stages. Cytokines are the main molecular actors of this ‘network of inflammation’. Among them, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pro-inflammatory hematopoietic cytokine widely used in haematological disorders to stimulate proliferation and differentiation of neutrophilic, eosinophilic and monocytic lineages. GM-CSF and its receptor are expressed in the brain and the cytokine can cross the blood–brain barrier. It is thus likely to affect various nervous system functions. This review will focus on the role of GM-CSF in nervous system disorders and their experimental models with particular emphasis on its possible beneficial effect on axonal regeneration after PNS and CNS injury.

Nervous system injury: focus on the inflammatory cytokine ‘granulocyte-macrophage colony stimulating factor’

Any lesion in the nervous system, be it infectious, immunopathological, ischemic or traumatic, is followed by an inflammatory process that induces rapid activation of glial cells and additional recruitment of granulocytes, T-cells and monocytes/macrophages from the blood stream. Neuroinflammation is a double-sided sword. It can cause neuronal damage and participate in neuropathic pain, but it also has neuroprotective and neurotrophic effects at some stages. Cytokines are the main molecular actors of this ‘network of inflammation’. Among them, granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pro-inflammatory hematopoietic cytokine widely used in haematological disorders to stimulate proliferation and differentiation of neutrophilic, eosinophilic and monocytic lineages. GM-CSF and its receptor are expressed in the brain and the cytokine can cross the blood–brain barrier. It is thus likely to affect various nervous system functions. This review will focus on the role of GM-CSF in nervous system disorders and their experimental models with particular emphasis on its possible beneficial effect on axonal regeneration after PNS and CNS injury.

PU.1 regulation of human alveolar macrophage differentiation requires granulocyte-macrophage colony-stimulating factor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically implicated in lung homeostasis in the GM-CSF knockout mouse model. These animals develop an isolated lung lesion reminiscent of pulmonary alveolar proteinosis (PAP) seen in humans. The development of the adult form of human alveolar proteinosis is not due to the absence of a GM-CSF gene or receptor defect but to the development of an anti-GM-CSF autoimmunity. The role of GM-CSF in the development of PAP is unknown. Studies in the GM-CSF knockout mouse have shown that lack of PU.1 protein expression in alveolar macrophages is correlated with decreased maturation, differentiation, and surfactant catabolism. This study investigates PU.1 expression in vitro and in vivo in human PAP alveolar macrophages as well as the regulation of PU.1 by GM-CSF. We show for the first time that PU.1 mRNA expression in PAP bronchoalveolar lavage cells is deficient compared with healthy controls. PU.1-dependent terminal differentiation markers CD32 (FCgammaII), mannose receptor, and macrophage colony-stimulating factor receptor (M-CSFR) are decreased in PAP alveolar macrophages. In vitro studies demonstrate that exogenous GMCSF treatment upregulated PU.1 and M-CSFR gene expression in PAP alveolar macrophages. Finally, in vivo studies showed that PAP patients treated with GM-CSF therapy have higher levels of PU.1 and M-CSFR expression in alveolar macrophages compared with healthy control and PAP patients before GM-CSF therapy. These observations suggest that PU.1 is critical in the terminal differentiation of human alveolar macrophages.

Role of Granulocyte-Macrophage Colony-Stimulating Factor on Apoptosis Induced by Ischemia-Reperfusion in the Intestinal Epithelium

Background: To evaluate the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on ischemia-reperfusion-induced apoptosis in the intestinal epithelium. Methods: In this study, 50 male Wistar albino rats were used. After midline laparotomy superior mesenteric artery (SMA) was identified only in the sham group, while 60 min of ischemia and 2 h of reperfusion were performed in the control group. In the treatment groups, after 15, 30 and 60 min of ischemia, respectively, 1 µg/kg GM-CSF was administered subcutaneously, followed by 2 h of reperfusion. Malondialdehyde (MDA), campothecin (CAM), an indicator of DNA fragmentation, and histopathology were evaluated in the intestinal mucosa. Results: Tissue MDA levels were found significantly high in all groups at various times of ischemia and 2 h of reperfusion compared with the sham group (p < 0.001). Administration of GM-CSF following 60 min of ischemia caused a significant increase in the MDA levels compared with the control group (6,430 ± 725 vs. 4,174 ± 565 nmol/g protein for jejunum. 7,576 ± 618 vs. 4,938 ± 809 nmol/g protein for ileum, p < 0.05). Intestinal ischemia and reperfusion resulted in a significant increase in tissue CAM levels (p < 0.05). The highest CAM value was found in the group in which 60 min of ischemia and 2 h of reperfusion were performed (50 ± 3.2 ng/ml for jejunum, 52.8 ± 2.7 ng/mg for ileum). Compared with the control group, GM-CSF administration following 1 h of ischemia aggravated the tissue injury. Conclusions: Apoptosis was induced in the small intestine by ischemia-reperfusion. GM-CSF increased the apoptosis of intestinal epithelial cells and exacerbated mucosal injury due to ischemia-reperfusion.

Toxoplasma gondii induces granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor secretion by human fibroblasts: implications for neutrophil apoptosis.

Human neutrophils are rescued from apoptosis following incubation with once-washed, fibroblast-derived Toxoplasma gondii tachyzoites. Both infected and uninfected neutrophils are rescued, implicating a soluble mediator. In this study we investigated the origin and identity of this soluble mediator. Neutrophils were incubated either with purified tachyzoites or with conditioned medium derived from T. gondii-infected human fibroblasts. Conditioned medium was found to be a potent stimulus that delayed neutrophil apoptosis up to 72 h, whereas purified and extensively washed tachyzoites had no effect. Delayed apoptosis correlated with up-regulation of the neutrophil antiapoptotic protein, Mcl-1, and the neutrophil interleukin 3 receptor alpha subunit (IL-3Ralpha), suggesting a role for granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF and granulocyte colony-stimulating factor (G-CSF) were measurable in conditioned medium by enzyme-linked immunosorbent assay. Neutralizing antibodies to GM-CSF and G-CSF were additive in abrogating delayed neutrophil apoptosis induced by conditioned medium. Inhibitors of Src family tyrosine kinases, G(i) proteins, phosphatidylinositol 3-kinase, p44(erk1) and p42(erk2) mitogen-activated protein kinases, and Jak2 kinases partially attenuated the effect of conditioned medium, consistent with a role for G-CSF and/or GM-CSF. Hence, delayed neutrophil apoptosis is mediated by GM-CSF and G-CSF secreted by T. gondii-infected human fibroblasts. This enhanced neutrophil survival may contribute to the robust proinflammatory response elicited in the T. gondii-infected host.

Autoantibodies against granulocyte macrophage colony-stimulating factor are diagnostic for pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a rare disease characterized by the accumulation of phospholipids and surfactant proteins in the lung. The central role for granulocyte-macrophage colony-stimulating factor (GM-CSF) in surfactant homeostasis has been established in mice lacking the GM-CSF gene, which results in murine pulmonary alveolar proteinosis. No GM-CSF gene defect has been defined in adult patients with idiopathic PAP. Previous studies indicated that the human disease differs from the murine model by the presence of circulating, neutralizing autoantibodies against GM-CSF. Therefore, the final common pathway between the GM-CSF knockout and human PAP appears to be the deficiency of functionally active GM-CSF. In the present study, all patients with idiopathic PAP were found to have systemic and localized antibodies against GM-CSF. Anti-GM-CSF titers were a specific and sensitive marker for PAP. In addition, we present data showing that the absence of active GM-CSF is associated with enhanced levels of macrophage colony-stimulating factor, monocyte chemoattractant protein-1, and interleukin-8. These studies confirm and strengthen previous studies and support the concept that adult idiopathic PAP is an autoimmune disease defined by the presence of anti-GM-CSF. Further, using anti-GM-CSF as an indicator of pulmonary alveolar proteinosis may avoid the use of more invasive means of evaluating patients with pulmonary disease characterized by alveolar infiltrates.

In vivo evidence for the role of GM-CSF as a mediator in acute pancreatitis-associated lung injury.

Severe pancreatitis is frequently associated with acute lung injury (ALI) and the respiratory distress syndrome. The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mediating the ALI associated with secretagogue-induced experimental pancreatitis was evaluated with GM-CSF knockout mice (GM-CSF -/-). Pancreatitis was induced by hourly (12x) intraperitoneal injection of a supramaximally stimulating dose of the cholecystokinin analog caerulein. The resulting pancreatitis was similar in GM-CSF-sufficient (GM-CSF +/+) control animals and GM-CSF -/- mice. Lung injury, quantitated by measuring lung myeloperoxidase activity (an indicator of neutrophil sequestration), alveolar-capillary permeability, and alveolar membrane thickness was less severe in GM-CSF -/- than in GM-CSF +/+ mice. In GM-CSF +/+ mice, pancreas, lung and serum GM-CSF levels increase during pancreatitis. Lung levels of macrophage inflammatory protein (MIP)-2 are also increased during pancreatitis, but, in this case, the rise is less profound in GM-CSF -/- mice than in GM-CSF +/+ controls. Administration of anti-MIP-2 antibodies was found to reduce the severity of pancreatitis-associated ALI. Our findings indicate that GM-CSF plays a critical role in coupling pancreatitis to ALI and suggest that GM-CSF may act indirectly by regulating the release of other proinflammatory factors including MIP-2.

Granulocyte macrophage colony stimulating factor is elevated in alveolar macrophages from sheep naturally infected with maedi-visna virus and stimulates maedi-visna virus replication in macrophages in vitro.

Infection by maedi-visna virus, a lentivirus of sheep, leads to chronic inflammatory reactions of various tissues. In this report we have analysed the role of specific cytokines in the disease process. A significant increase in expression of interleukin-6, interleukin-10, granulocyte macrophage-colony stimulating factor (GM-CSF) and transforming growth factor-beta1 mRNA was observed in alveolar macrophages isolated from the lungs of naturally infected animals when compared with lungs of seronegative controls. Levels of GM-CSF mRNA expression in alveolar macrophages correlated with the presence of lung lesions, but there was no correlation of interleukin-10, interleukin-6, tumour necrosis factor-alpha and transforming growth factor-beta1 mRNA levels in alveolar macrophages from animals with pulmonary lesions. In vitro investigation showed that GM-CSF in the range 0.1-10 ng/ml induced a significant increase in viral p25 production after 7 days in acutely infected blood monocyte-derived macrophages. The production of p25 peaked between 7 and 14 days exposure to 10 ng/ml of GM-CSF. Quantitative polymerase chain reaction showed that the level of viral DNA in monocyte-derived macrophages was dose-dependent following GM-CSF treatment in the range 0.1-100 ng/ml after 7 days. Viral mRNA expression was also enhanced. These findings indicate a role for GM-CSF in the pathogenesis of lymphoid interstitial pneumonia in infected animals.

GM-CSF and M-CSF modulate beta-chemokine and HIV-1 expression in microglia.

Significant numbers of patients with acquired immunodeficiency syndrome (AIDS) develop CNS infection primarily in macrophages and microglial cells. Therefore, the regulation of human immunodeficiency virus type 1 (HIV-1) infection and activation of the brain mononuclear phagocytes subsequent to infection are important areas of investigation. In the current report, we studied the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage-CSF (M-CSF) in the expression of antiviral beta-chemokines and HIV-1 p24 in cultures of primary human fetal microglia. We found that stimulation with GM-CSF or M-CSF induced macrophage inflammatory proteins (MIP-1alpha and MIP-1beta) and augmented RANTES expression, after HIV-1 infection of microglia. This was not due to the effect of GM-CSF on viral expression because GM-CSF was neither necessary nor stimulatory for viral infection, nor did GM-CSF enhance the expression of env-pseudotyped reporter viruses. Blocking GM-CSF-induced microglial proliferation by nocodazole had no effect on beta-chemokine or p24 expression. The functional significance of the GM-CSF-induced beta-chemokines was suggested by the finding that, in the presence of GM-CSF, exogenous beta-chemokines lost their anti-HIV-1 effects. We further show that although HIV-1-infected microglia produced M-CSF, they failed to produce GM-CSF. In vivo, GM-CSF expression was localized to activated astrocytes and some inflammatory cells in HIV-1 encephalitis, suggesting paracrine activation of microglia through GM-CSF. Our results demonstrate a complex interplay between CSFs, chemokines, and virus in microglial cells and may have bearing on the interpretation of data derived in vivo and in vitro.

Penicillium marneffei causes osteopontin-mediated production of interleukin-12 by peripheral blood mononuclear cells.

We investigated the role of osteopontin (OPN) in interleukin-12 (IL-12) production from peripheral blood mononuclear cells (PBMCs) stimulated with Penicillium marneffei. Kinetic studies showed that OPN synthesis preceded that of IL-12 at both mRNA and protein levels when PBMCs were cocultured with P. marneffei. Treatment with anti-OPN monoclonal antibodies (MAb) significantly suppressed IL-12 secretion. Furthermore, native OPN induced a profound level of synthesis of IL-12 from noninfected PBMCs. The major cellular source of OPN was monocytes, because depletion of CD14(+) cells resulted in the abrogation of such production. We also examined the regulatory role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in OPN secretion from P. marneffei-stimulated PBMCs. Neutralizing anti-GM-CSF MAb significantly reduced OPN secretion, and treatment with this cytokine induced OPN production from both infected and noninfected PBMCs. Finally, antagonists against the mannose receptor but not the beta-glucan receptor almost completely abrogated the production of OPN. Our results demonstrated that OPN secreted from monocytes is involved in the production of IL-12 from PBMCs after stimulation with P. marneffei and that OPN production is regulated by GM-CSF. Our results also indicated the possible involvement of the mannose receptor as a signal-transducing receptor for triggering the secretion of OPN by P. marneffei-stimulated PBMCs.

Role of cytokines in the maturation and function of macrophages. Effect of GM-CSF and IL-4.

Macrophages and monocytes that infiltrate into chronic inflammatory sites might be influenced by cytokines such as GM-CSF, IL-3, IL-4 and IFNthat are abundant in these sites. Granulocyte Macrophage Colony Stimulating Factor (GM-CSF), is a glycoprotein which is characterized by its capacity to stimulate cell precursors in the bone marrow to undergo proliferation and differentiation into granulocytes and macrophages (Metcalf 1985, Golde and Gasson. 1988). Granulocyte-macrophage CSF is secreted by various cell types, and its production might be enhanced by bacterial products (Chodakewitz et al 1988), and by cytokines such as Interleukin 1 (Zucali et al 1986, Sieff et al 1987, Fibbe et al 1988, Herrmann et al 1988), Interferon (Piacibello et al 1985), and TNF(Lu et al 1988, Munker et al 1986). GM-CSF which is present in chronic inflammatory sites (Williamson et al 1988, Xu et al 1989) was implicated as a key cytokine in inflammatory joint disease (Campbell 1998). The role of GM-CSF as a proinflammatory cytokine was further substantiated by the findings that GM-CSF deficient mice exhibited an attenuated production of inflammatory cytokines i n response to lipopolysacchauide (LPS) (Basu 1997).

Enhancement of natural killer cell-mediated cytotoxicity by coexpression of GM-CSF/B70 in hepatoma

On investigating the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) and costimulatory molecule, B70, in antitumor immunity, we have found important effects of GM-CSF/B70 coexpression in the interaction with natural killer (NK) cells. We used the pLSN vector system to contain the neomycin-resistant gene and LTR promoter. The pLSNGM-CSF, pLSNB70 and pLSNB70/GM-CSF, pLSN vectors each containing GM-CSF, B70, and B70/GM-CSF cDNA, respectively, were constructed. They were transfected into human hepatocellular carcinoma cell (SK-HEP1), and stable cells (SK-pLSN, SK-GM, SK-B70 and SK-BG) were selected after neomycin treatment. According to enzyme-linked immunosorbent analysis and FACS, we showed that expression of GM-CSF was increased up to 23-fold in SK-GM and SK-BG cells, and also expression of B70 was induced at least 76–97% in SK-B70 and SK-BG cells. Expression of B70 was remarkably increased by autocrine effect of GM-CSF in SK-BG cells. Primary cytolytic ability of GM-CSF and B70 significantly increased almost 4-fold (effector/target ratio, 100:1) in SK-BG cells. In in vivo studies, SK-BG cells showed much less subcutaneous tumor formation in nude mice accompanying increased NK cell proliferation and cytotoxicity. Therefore, these results suggest that combining expression of GM-CSF and B70 may enhance NK-mediated cytotoxicity, and then induce the antitumor immunity in hepatoma transplanted into nude mice.

Granulocyte-macrophage colony-stimulating factor amplifies lipopolysaccharide-induced bronchoconstriction by a neutrophil- and cyclooxygenase 2-dependent mechanism.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is used to ameliorate neutropenia in patients after antineoplastic treatment. It has also been suggested as an adjunct treatment in septic patients; however, the recruitment and priming of leukocytes by GM-CSF bears the hazard of a hyperinflammatory response. In particular, the role of GM-CSF in pulmonary functions in septic lungs is still unclear. Therefore, we pretreated rats in vivo with GM-CSF (50 microg/kg, intravenous) and assessed the pulmonary functions of their subsequently prepared isolated perfused lungs when exposed to subtoxic concentrations of lipopolysaccharide (LPS, 2 microg/ml). These lungs showed enhanced expression of cyclooxygenase 2 (COX-2), a significant increase in thromboxane (TX) and tumor necrosis factor (TNF) release into the venous perfusate, and bronchoconstriction. COX-2 inhibition or blocking of the TX receptor abolished the GM-CSF/LPS-induced bronchoconstriction, but not the TNF release. Neutralizing antibodies against TNF did not prevent GM-CSF/LPS-induced bronchoconstriction. After GM-CSF pretreatment, massive neutrophil invasion into the lung occurred. Neutropenic rats were protected against GM-CSF/ LPS-induced lung injury. Similar results were obtained in rats pretreated with G-CSF instead of GM-CSF. We conclude that GM-CSF pretreatment exacerbates pulmonary injury by low-dose LPS via COX-2 expression, TX release, and bronchoconstriction by initiating neutrophil invasion and activation.