Spatial distribution of CD115+ and CD11b+ cells and their temporal activation during oxygen-induced retinopathy in mice.

PURPOSE: The model of oxygen-induced retinopathy (OIR) is widely used to analyze pathomechanisms in retinal neovascularization. Previous studies have shown that macrophages (MP) play a key role in vessel formation in OIR, the influence of microglia (MG) having been discussed. The aim of our study was to analyze the spatial and temporal distribution and activation of MP/MG expressing CD115 and CD11b during the process of neovascularization in OIR. METHODS: We used MacGreen mice expressing the green fluorescence protein (GFP) under the promoter for CD115. CD115+ cells were investigated in vivo by scanning laser ophthalmoscopy at postnatal days (P) 17 and 21 in MacGreen mice with OIR (75% oxygen from P7 to P12), and were compared to MacGreen room-air controls. In addition MP/MG were examined ex vivo using immunohistochemistry for CD11b+ detection on retinal flatmounts at P14, P17, and P21 of wild type mice with OIR. RESULTS: In-vivo imaging revealed the highest density of activated MP/MG in tuft areas at P17 of MacGreen mice with OIR. Tufts and regions with a high density of CD115+ cells were detected close to veins, rather to arteries. In peripheral, fully vascularized areas, the distribution of CD115+ cells in MacGreen mice with OIR was similar to MacGreen room-air controls. Correspondingly, immunohistochemical analyses of retinal flatmounts from wild type mice with OIR induction revealed that the number of CD11b+ cells significantly varies between vascular, avascular, and tuft areas as well as between the retinal layers. Activated CD11b+ cells were almost exclusively found in avascular areas and tufts of wild type mice with OIR induction; here, the proportion of activated cells related to the total number of CD11b+ cells remained stable over the course of time. CONCLUSIONS: Using two different approaches to monitor MP/MG cells, our findings demonstrated that MP/MG concentrate within pathologically vascularized areas during OIR. We were able to clarify that reactive changes of CD11b+ cell distribution to OIR primarily occur in the deep retinal layers. Furthermore, we found the highest proportion of activated CD11b+ cells in regions with pathologic neovascularization processes. Our findings support previous reports about activated MP/MG guiding revascularization in avascular areas and playing a key role in the formation and regression of neovascular tufts.

A novel TNF receptor-associated factor 6 binding domain mediates NF-kappa B signaling by the common cytokine receptor beta subunit.

GM-CSF, IL-3, and IL-5 are proinflammatory cytokines that control the production and function of myeloid and lymphoid cells. Their receptors are composed of a ligand-specific alpha subunit and a shared common signal-transducing beta subunit (beta common receptor or GM-CSFR beta [beta(c)]). The pleiotropic nature of biologic outcomes mediated by beta(c) and the presence of large, uncharacterized regions of its cytoplasmic domain suggest that much remains to be learned about its downstream signaling pathways. Although some previous work has attempted to link beta(c) with NF-kappaB activation, a definitive mechanism that mediates this pathway has not been described and, to date, it has not been clear whether the receptor can directly activate NF-kappaB. We demonstrate that NF-kappaB activation by beta(c) is dependent on TNFR-associated factor 6 (TRAF6) and that association of TRAF6 with beta(c) requires a consensus-binding motif found in other molecules known to interact with TRAF6. Furthermore, point mutation of this motif abrogated the ability of beta(c) to mediate NF-kappaB activation and reduced the viability of an IL-3-dependent hematopoietic cell line. Because this receptor plays a key role in hematopoiesis and the beta(c) cytoplasmic domain identified in this work mediates hematopoietic cell viability, this new pathway is likely to contribute to immune cell biology. This work is significant because it is the first description of a TRAF6-dependent signaling pathway associated with a type I cytokine receptor. It also suggests that TRAF6, a mediator of TNFR and TLR signaling, may be a common signaling intermediate in diverse cytokine receptor systems.

Is CSF1R Expression Localization Crucial for its Prognostic Value in Colorectal Cancer?

The colony-stimulating factor 1 receptor (CSF1R) is commonly known as a transmembrane receptor on tumor-associated macrophages, which are essential in the tumor microenvironment. However, the expression pattern and prognosis of CSF1R are still unknown in colorectal cancer (CRC) and are still controversial among various cancers. To clarify the expression pattern and prognosis of CSF1R in CRC, we performed immunohistochemistry on 332 CRC tissue and 283 nontumorous adjacent tissues. We explored the expression pattern of CSF1R and analyzed its relationship with clinical characteristics and prognosis. The positive expression ratio of CSF1R was much higher in nontumorous adjacent tissues, while the positive cytomembrane ratio of CSF1R was much higher in cancer tissue. Furthermore, we found that CSF1R expression, especially in the cytoplasm, acted as a protective factor. However, our findings indicated that CSF1R expression on the cytomembrane decreased its prognostic predictive value. These results emphasize the important role of the localization of CSF1R and may also explain the contrasting effects seen between various cancers.

miR-532-3p-CSF2RA Axis as a Key Regulator of Vulnerable Atherosclerotic Plaque Formation.

BACKGROUND: The most dangerous atherosclerotic plaques, referred to as "vulnerable," are most likely to trigger acute atherothrombotic events such as myocardial infarction (heart attack) and stroke. Our goal was to uncover the molecular drivers of vulnerable plaque formation. METHODS: To elucidate the functional gene modules that drive vulnerable plaque formation, we performed a weighted gene coexpression network analysis integrated with a protein-protein interaction network analysis in human atherosclerotic carotid samples, which identified the candidate gene granulocyte-macrophage colony-stimulating factor 2 (GM-CSF) receptor alpha subunit (CSF2RA). Follow-up in vitro experiments were performed to elucidate the regulatory relationship between CSF2RA and the microRNA miR-532-3p as well as modifiers of macrophagic miR-532-3p-CSF2RA axis expression. Microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) studies elucidated the effect of statins on carotid miR-532-3p-CSF2RA axis expression in patients with carotid atherosclerotic disease. Apoe-/-, Ldlr-/-, and Csf2ra mutant Apoe-/- mouse models of atherosclerosis were employed to assess the effects of agomiR-532-3p therapy in vivo. RESULTS: The integrated weighted gene coexpression network analysis/protein-protein interaction network analysis revealed that the macrophagic GM-CSF receptor CSF2RA is significantly upregulated in macrophage-rich vulnerable plaques. Follow-up analysis identified the miR-532-3p-CSF2RA axis, as miR-532-3p downregulates CSF2RA via binding to CSF2RA's 3'UTR. Macrophagic miR-532-3p-CSF2RA dysregulation was enhanced via modified low-density lipoprotein or tumor necrosis factor α exposure in vitro. Moreover, this miR-532-3p-CSF2RA dysregulation was observed in human vulnerable plaques and Apoe-/- mouse plaques, effects rescued by statin therapy. In vivo, agomiR-532-3p therapy suppressed murine plaque formation and promoted plaque stabilization in a Csf2ra-dependent manner. CONCLUSION: Macrophagic miR-532-3p-CSF2RA axis dysregulation is a key driver in vulnerable plaque formation.

Mammalian granulocyte-macrophage colony-stimulating factor receptor expressed in primary avian hematopoietic progenitors: lineage-specific regulation of proliferation and differentiation.

The cytokine Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) regulates proliferation, differentiation, and apoptosis during myelopoiesis and erythropoiesis. Structure-function relationships of GM-CSF interactions with its receptor (GM-R), the biochemistry of GM-R signal transduction, and GM-CSF action in vivo are relatively well understood. Much less is known, however, about GM-R function in primary hematopoietic cells. In this paper we show that expression of the human GM-R in a heterologous cell system (primary avian erythroid and myeloid cells) confirms respective results in murine or human cell lines, but also provides new insights how the GM-R regulates progenitor proliferation and differentiation. As expected, the hGM-CSF stimulated myeloid progenitor proliferation and differentiation and enhanced erythroid progenitor proliferation during terminal differentiation. In the latter cells, however, the hGM-R only partially substituted for the activities of the erythropoietin receptor (EpoR). It failed to replace the EpoR in its cooperation with c-Kit to induce long-term proliferation of erythroid progenitors. Furthermore, the hGM-R alpha chain specifically interfered with EpoR signaling, an activity neither seen for the betac subunit of the receptor complex alone, nor for the alpha chain of the closely related Interleukin-3 receptor. These results point to a novel role of the GM-R alpha chain in defining cell type-specific functions of the GM-R.

HIV-1-transgene expression in rats decreases alveolar macrophage zinc levels and phagocytosis.

HIV-1 infection impairs alveolar macrophage immune function and renders patients susceptible to pneumonia by poorly understood mechanisms. Alveolar macrophage maturation and function depends on granulocyte-macrophage colony-stimulating factor (GM-CSF), which is produced and secreted by the alveolar epithelium. Macrophages respond to GM-CSF through the GM-CSF receptor (GM-CSFR), which has a binding subunit (GM-CSFRalpha) and a signaling subunit (GM-CSFRbeta). In this study, we measured GM-CSFR expression and alveolar macrophage function in a transgene HIV-1 rat model (NL4-3Delta gag/pol); this construct bears a pro-virus with gag and pol deleted, but other HIV-1-related proteins, such as gp120 and Tat, are expressed, and the rats develop an AIDS-like phenotype as they age. We first determined that HIV-1-transgenic expression selectively decreased alveolar macrophage expression of GM-CSFRbeta and impaired bacterial phagocytosis in vitro. Next, we examined the role of zinc (Zn) deficiency as a potential mechanism underlying these effects, and determined that HIV-1-transgenic rats have significantly lower levels of Zn in the alveolar space and macrophages. To test the direct effect of Zn deficiency on macrophage dysfunction, we treated rat alveolar macrophage cell line with a Zn chelator, N,N,N',N'-tetrakis-(2-pyridyl-methyl) ethylenediamine, and this decreased GM-CSFRbeta expression and phagocytosis. In parallel, treatment with Zn acetate in vitro for 48 hours restored intracellular Zn levels and phagocytic function in alveolar macrophages from HIV-1-transgenic rats. Taken together, these data suggest that pulmonary Zn deficiency could be one of the mechanisms by which chronic HIV-1 infection impairs alveolar macrophage immune function and renders these individuals susceptible to serious lung infections.

A neuroprotective function for the hematopoietic protein granulocyte-macrophage colony stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine responsible for the proliferation, differentiation, and maturation of cells of the myeloid lineage, which was cloned more than 20 years ago. Here we uncovered a novel function of GM-CSF in the central nervous system (CNS). We identified the GM-CSF alpha-receptor as an upregulated gene in a screen for ischemia-induced genes in the cortex. This receptor is broadly expressed on neurons throughout the brain together with its ligand and induced by ischemic insults. In primary cortical neurons and human neuroblastoma cells, GM-CSF counteracts programmed cell death and induces BCL-2 and BCL-Xl expression in a dose- and time-dependent manner. Of the signaling pathways studied, GM-CSF most prominently induced the PI3K-Akt pathway, and inhibition of Akt strongly decreased antiapoptotic activity. Intravenously given GM-CSF passes the blood-brain barrier, and decreases infarct damage in two different experimental stroke models (middle cerebral artery occlusion (MCAO), and combined common carotid/distal MCA occlusion) concomitant with induction of BCL-Xl expression. Thus, GM-CSF acts as a neuroprotective protein in the CNS. This finding is remarkably reminiscent of the recently discovered functionality of two other hematopoietic factors, erythropoietin and granulocyte colony-stimulating factor in the CNS. The identification of a third hematopoietic factor acting as a neurotrophic factor in the CNS suggests a common principle in the functional evolution of these factors. Clinically, GM-CSF now broadens the repertoire of hematopoietic factors available as novel drug candidates for stroke and neurodegenerative diseases.

GM-CSF action in the CNS decreases food intake and body weight.

Many proinflammatory cytokines, such as leptin, play key roles in dynamic regulation of energy expenditure and food intake. The present work tested a role for the proinflammatory cytokine GM-CSF. Central but not peripheral administration of GM-CSF to adult rats significantly decreased food intake and body weight for at least 48 hours. Similar results were observed following central administration of GM-CSF in mice. GM-CSF receptor immunoreactivity was found on neurons within the paraventricular and arcuate nuclei of the hypothalamus. GM-CSF-deficient (GM-/-) mice weighed more and had significantly higher total body fat than wild-type (GM+/+) mice. Energy expenditure in GM-/- mice was decreased compared with that in GM+/+ mice. Taken together, these findings demonstrate that GM-CSF signaling in the CNS can regulate energy homeostasis.

Expression and role of granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) and granulocyte colony-stimulating factor receptor (G-CSFR) on Ph-positive acute B lymphoblastic leukemia.

OBJECTIVE: We observed that ph + ALL patients administrated with recombinant human G-CSF (rhG-CSF) after intense chemotherapy have presented a trend of disease relapse. Thus, we aim to thoroughly investigate the expression and role of GM-CSFR and G-CSFR on ph + ALL patients. METHOD: SUP-B15, BALL-1 and primary leukemia cells were used in this study. Transcript levels were analyzed by quantitative PCR while cell viability was measured using a CCK-8 assay. Flow cytometry was used to assess the different stages of cell cycle. RESULTS: We found that the mRNA expression levels of GM-CSFR and G-CSFR were higher in patients with ph + ALL, as well as in SUP-B15 cells. rhG-CSF was also observed to promote the viability of SUP-B15 cells while inversely inhibiting BALL-1 cell viability. In addition, we also determined that rhG-CSF (100 ng/ml) decreased the sensitivity of SUP-B15 cells to imatinib and nilotinib, while the results were exactly the contrary for dasatinib. CONCLUSION: We demonstrated high expression levels of GM-CSFR and G-CSFR, as well as their promotable role for viability in ph + ALL cells. We further found that rhG-CSF influenced the sensitivity of SUP-B15 cells to TKIs.

Elevated expression of granulocyte-macrophage colony-stimulating factor receptor in multiple sclerosis lesions.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease that disproportionately affects young adults, leading to disability and high costs to society. Infiltration of T cells and monocytes into the central nervous system (CNS) is critical for disease initiation and progression. However, despite a great deal of effort the molecular mechanisms by which immune cells initiate and perpetuate CNS damage in MS have not yet been elucidated. In experimental autoimmune encephalomyelitis (EAE), an animal model of MS, granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by pathogenic Th1 and Th17 cells is critical for the recruitment of monocytes into the CNS during the initial stage of disease. We and others have recently shown that, compared with healthy individuals, MS patients have greater numbers of CD4+ and CD8+ T cells that produce GM-CSF. Here, we describe the expression of GM-CSF and its receptor, GM-CSFR, in normal brain and MS lesions. Our data show that in acute and chronic MS lesions, microglia and astrocytes have upregulated expression of GM-CSFR; in addition, we show that GM-CSF-associated molecules are also upregulated in MS lesions. These findings further strengthen the argument that GM-CSF signaling contributes to MS pathogenesis.

JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.

A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.

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

BACKGROUND: 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. AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: We hypothesize that GM-CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium.

IL-4 prevents the blockade of dendritic cell differentiation induced by tumor cells.

Malignant cells may escape from the immune response in vivo because of a defective differentiation of professional antigen-presenting cells (APCs), i.e., dendritic cells (DCs). We recently reported that tumor cells release interleukin (IL)-6 and macrophage colony stimulating factor (M-CSF), which inhibit the differentiation of CD34+ cells into DCs and promote their commitment toward monocytic lineage with a poor APC function. The results presented here show that both IL-4 and IL-13 reverse the inhibitory effects of renal cell carcinoma conditioned media (RCC CM) or IL-6+M-CSF on the phenotypic and functional differentiation of CD34+ into DCs. IL-4 was found to act through a rapid blockade of the expression of M-CSF and the IL-6 receptor-transducing chain (gp130), along with a decrease of the secondary production of M-CSF, thereby preventing the loss of granulocyte macrophage colony stimulating factor (GM-CSF) receptor alpha chain expression on differentiating CD34+ cells. Consistent with these observations, the differentiation of DCs from monocytes cultured with GM-CSF and IL-4 was also impaired by RCC CM, but the minimal inhibitory concentrations of RCC CM were 10-fold higher than for CD34+ cells. In these conditions, monocytes cultured with GM-CSF and IL-4 also exhibited profound phenotypic changes (CD14+ D32+ CD86+ HLA-DR+ CD115(low) CD23(low) CD1a-) and a poor APC function. These alterations were overcome in a dose-dependent manner by IL-4 (5-500 IU/ml), although not beyond a 40% final concentration of RCC CM. The capacity of RCC CM to block DC differentiation from monocytes strongly correlated with IL-6 and M-CSF concentrations in medium. Taken together, these results demonstrate that IL-4 and IL-13 reverse the inhibitory effect of tumor cells on DC differentiation.

Transcriptional regulation of the c-fms proto-oncogene mediated by granulocyte/macrophage colony-stimulating factor (GM-CSF) in murine cell lines.

Differentiation of blood cells is paralleled by a timely ordered expression of cytokine receptor genes. We show here that the expression of the c-fms gene which encodes the lineage-specific receptor for macrophage colony-stimulating factor (M-CSF or CSF-1) is directly linked to ligand-mediated activation of the receptor for the granulocyte/macrophage colony-stimulating factor (GM-CSF). In interleukin-3 (IL-3) dependent multipotent progenitor cells, FDC-Pmix GMV#2 cells, GM-CSF treatment results in the rapid formation of full-length c-fms transcripts. Surprisingly, this upregulation of c-fms transcripts is also observed in mouse NIH3T3 fibroblasts stably transfected with genes coding for the alpha- and beta-subunits of the GM-CSF receptor. These results indicate a direct control by the GM-CSF receptor that takes place regardless of cell differentiation. Furthermore, a 2.1 kb genomic fragment containing the c-fms proximal promoter directs GM-CSF-inducible expression of a reporter gene, suggesting a regulation of c-fms gene expression on the transcriptional level.

Roles of JAK kinase in human GM-CSF receptor signals.

The IL-3 and GM-CSF (hGMR) receptors consist of two subunits, alpha and beta, both of which are members of the cytokine receptor superfamily. Phosphorylation of tyrosine residues of hGMR beta subunit and several cellular proteins are observed with hGM-CSF stimulation. We analyzed role of tyrosine residue of hGMR beta subunit and nature of tyrosine kinase, JAK2 in hGMR signals using several hGMR beta subunit mutants. In addition to box1 region, a membrane distal region (a.a. 544-589) of hGMR beta is required for c-fos activation. Only one tyrosine residue (Tyr577) exists within the region 544-589, and substitution of Tyr577 to phenylalanine in GMR beta 589 resulted in the loss of c-fos activation. In contrast, the same substitution in a wild type receptor did not affect GM-CSF-induced activities such as c-fos mRNA induction and proliferation but abolished Shc phosphorylation. These results suggest that the activation of Shc is not essential for c-fos activation and several tyrosine residues co-ordinate to activate c-fos activation. It is well documented that IL-3 or GM-CSF activates JAK2 in BA/F3 cells. However the role of JAK2 in IL-3/GM-CSF functions is largely unknown. We examined the role of JAK2 in GM-CSF-induced signaling pathways. Dominant negative JAK2 (delta JAK2) lacking the C-terminus kinase domain, suppressed IL-3/GM-CSF induced c-fos activation, c-myc activation and proliferation suggesting that JAK2 is involved in both signaling pathways. PTP1D and Shc are phosphorylated by IL-3/GM-CSF in BA/F3 cells, however these phosphorylation events were inhibited by expression of delta JAK2. Taken together, these results indicate that JAK2 is a primary kinase regulating all the known activities of GM-CSF. JAK2 mediates GM-CSF induced c-fos activation through receptor phosphorylation and Shc/PTP1D activation.

GM-CSF and its effects on replication of HIV-1 in cells of macrophage lineage.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hemopoetic growth factor that is a member of the four-helix bundle family of cytokines and growth factors. It regulates the proliferation and differentiation of granulocytes and cells of macrophage lineage from bone marrow progenitor cells, mediating these activities through binding to its receptor. Most studies examining the effects of GM-CSF on HIV-1 replication in primary monocytes and macrophages, and in related cell lines, have demonstrated augmentation of HIV-1 expression in vitro, although some reports have been at variance with these findings. These laboratory-based observations have been confirmed in limited clinical trials. This review outlines the details of these studies and considers mechanisms by which GM-CSF may exert its effects on cells of this lineage.

Primitive human hematopoietic progenitor cells express receptors for granulocyte-macrophage colony-stimulating factor.

Most cytokines act only synergistically in assays of primitive progenitor cell proliferation, and effects have usually been observed first after prolonged cell culture. Studies reporting that primitive progenitors lack receptors for a number of cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF), could indicate that several "synergistic" cytokines primarily affect cells that have differentiated in vitro. Here, however, we show that freshly isolated primitive progenitor cells (CD34hi CD38-) express receptors for GM-CSF at levels 20%-30% of granulo-monocytic progenitors. Although GM-CSF had minimal effects on the survival or proliferation of primitive progenitors when added alone, the cytokine enhanced stem cell factor (SCF) induced cell cycle entry in the first generation. The effect was not observed when cells were incubated sequentially with SCF and GM-CSF. The results suggest that the synergistic effects of GM-CSF are mediated directly on primitive progenitor cells and that the cytokine may be useful to enhance cell cycle entry of hematopoietic stem cells.

Regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors in a GM-CSF-dependent human myeloid leukemia cell line (AML-193) by interleukin-6.

AML-193 is a cytokine-dependent human leukemia cell line established from the bone marrow of an M5-type acute monocytic leukemia (AML) patient. The effect of recombinant human interleukin-6 (rhIL-6) on the proliferation of AML-193 cells was investigated. Both granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and rhIL-3 promoted the DNA synthesis and growth of AML-193 cells in vitro. rhIL-6 alone did not support the growth of AML-193 cells, yet pretreatment of AML-193 cells with rhIL-6 markedly enhanced their proliferative response to subsequent rhGM-CSF or rhIL-3 stimulation. The growth-promoting effect induced by rhIL-6 was attributable in part to the upregulation of GM-CSF receptors on AML-193 cells; treatment of AML-193 cells with rhIL-6 for 24 to 48 hours greatly increased their GM-CSF binding activity, which occurred in a dose-dependent manner. Both the growth-promoting and receptor-upregulating effects induced by rhIL-6 could be blocked by treating AML-193 cells with neutralizing anti-gp130 antibodies (GPX7). Treatment of AML-193 cells with anti-gp130 antibodies alone also led to a notable decline in GM-CSF binding activity, suggesting a possible role of gpl30 in regulating the expression of GM-CSF receptors. When AML-193 cells were starved in cytokine-free medium and then restimulated with rhGM-CSF, a rapid increase (5 minutes) in lyn kinase activity was observed. A similar upregulation of lyn kinase activity by rhIL-6 treatment also was noted in AML-193 cells, but only after a prolonged incubation of the cells with rhIL-6 (>24 hours). These findings show that the growth-promoting effects of rhIL-6 are mediated through the upregulation of GM-CSF receptors on AML-193 cells by mechanisms that appear to involve the activation of both gp130 and lyn kinase.

Developmental expression of Magmas in murine tissues and its co-expression with the GM-CSF receptor.

Magmas is a protein that is involved in GM-CSF signaling in a myeloid cell line. Its precise role in the signal transduction process is unclear. To accurately characterize Magmas expression in a variety of cells, mouse embryos and adult murine tissues were analyzed for both mRNA and protein content. Magmas expression was detected as early as the day 6.5 embryo. The level of expression was developmentally regulated. During embryogenesis, elevated Magmas was observed in several structures, including heart, liver, notochord, choroid plexus, cervical ganglion, and nasal mucosa. Muscle, pancreas, intestinal mucosa, and testes were among the adult tissues with high Magmas expression. Most cell types, including hepatocytes and skeletal, smooth, and cardiac myocytes, also expressed the GM-CSF receptor (GMR) but the relative tissue levels of GMR were not always proportional to Magmas. The expression patterns suggest that Magmas has a role in both developing and mature tissues.