Inhibiting microglia proliferation after spinal cord injury improves recovery in mice and nonhuman primates.

No curative treatment is available for any deficits induced by spinal cord injury (SCI). Following injury, microglia undergo highly diverse activation processes, including proliferation, and play a critical role on functional recovery. In a translational objective, we investigated whether a transient pharmacological reduction of microglia proliferation after injury is beneficial for functional recovery after SCI in mice and nonhuman primates. Methods: The colony stimulating factor-1 receptor (CSF1R) regulates proliferation, differentiation, and survival of microglia. We orally administrated GW2580, a CSF1R inhibitor that inhibits microglia proliferation. In mice and nonhuman primates, we then analyzed treatment outcomes on locomotor function and spinal cord pathology. Finally, we used cell-specific transcriptomic analysis to uncover GW2580-induced molecular changes in microglia. Results: First, transient post-injury GW2580 administration in mice improves motor function recovery, promotes tissue preservation and/or reorganization (identified by coherent anti-stokes Raman scattering microscopy), and modulates glial reactivity. Second, post-injury GW2580-treatment in nonhuman primates reduces microglia proliferation, improves motor function recovery, and promotes tissue protection. Finally, GW2580-treatment in mice induced down-regulation of proliferation-associated transcripts and inflammatory associated genes in microglia that may account for reduced neuroinflammation and improved functional recovery following SCI. Conclusion: Thus, a transient oral GW2580 treatment post-injury may provide a promising therapeutic strategy for SCI patients and may also be extended to other central nervous system disorders displaying microglia activation.

Granulocyte-Macrophage Colony Stimulating Factor As an Indirect Mediator of Nociceptor Activation and Pain.

The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.

A critical role for microglia in maintaining vascular integrity in the hypoxic spinal cord.

Hypoxic preconditioning reduces disease severity in a mouse model of multiple sclerosis (MS), in part by enhancing the barrier properties of spinal cord blood vessels. Because other studies have shown that similar levels of hypoxia transiently increase permeability of central nervous system (CNS) blood vessels, the goal of this study was to define the impact of chronic mild hypoxia (CMH, 8% O2) on the integrity of spinal cord blood vessels and the responses of neighboring glial cells. Using extravascular fibrinogen as a marker of vascular disruption, we found that CMH triggered transient vascular leak in spinal cord blood vessels, particularly in white matter, which was associated with clustering and activation of Mac-1-positive microglia around disrupted vessels. Microglial depletion with the colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX5622, while having no effect under normoxic conditions, profoundly increased vascular leak in both white and gray matter during CMH, and this was associated with disruption of astrocyte-vascular coupling and enhanced loss of tight junction proteins. Microglial repair of leaky blood vessels was blocked by a peptide that inhibits the interaction between fibrinogen and its Mac-1 integrin receptor. These findings highlight an important role for microglia in maintaining vascular integrity in the hypoxic spinal cord and suggest that a fibrinogen-Mac-1 interaction underpins this response. As relative hypoxia is experienced in many situations including high altitude, lung disease, obstructive sleep apnea, and age-related CNS ischemia/hypoxia, our findings have important implications regarding the critical role of microglia in maintaining vascular integrity in the CNS.

Effect of granulocyte macrophage-colony stimulating factor on extracellular matrix deposition by dermal fibroblasts from patients with scleroderma.

OBJECTIVE: . To investigate the in vitro effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on the deposition of extracellular matrix (ECM) in fibroblasts obtained from the skin of patients with systemic sclerosis (Ssc), compared to healthy controls. METHODS: Dermal fibroblasts obtained from 14 patients with SSc (7 with the diffuse form and 7 with CREST syndrome) and from 7 controls were studied. Both SSc and normal skin fibroblast cultures were stimulated for 4 and 8 days with 100 ng/ml GM-CFS. GM-CSF receptor (GM-CSFR) expression was determined by Western blot of cell lysates. Immunofluorescence was used to determine GM-CSFR expression and to investigate the deposition of ECM (type I collagen, fibronectin, and tenascin). Quantitative analysis of ECM was performed by ELISA. Expression of type I collagen and metalloproteinase 1 (MMP-1) mRNA was determined by real-time quantitative PCR. RESULTS: Deposition of ECM by normal fibroblasts appeared not to be influenced by stimulation with GM-CSF; in contrast, after stimulation with GM-CSF SSc fibroblasts showed increased deposition of fibronectin and tenascin, while type I collagen production was decreased; these results were found with both immunofluorescence and ELISA. Quantitative PCR revealed that GM-CSF inhibited the expression of mRNA type I collagen in SSc fibroblasts but not in normal fibroblasts, whereas levels of the main collagenolytic enzyme, MMP-1, were not affected. CONCLUSION: These results suggest that in SSc fibroblasts GM-CSF exerts a blocking effect on the deposition of type I collagen, through an inhibitory action on mRNA, while the production of other components of ECM such as fibronectin and tenascin is increased by stimulation with this cytokine.

Role of 4-hydroxynonenal in the hemozoin-mediated inhibition of differentiation of human monocytes to dendritic cells induced by GM-CSF/IL-4.

In falciparum malaria, rupture of parasitized RBC liberates hemozoin (HZ), polymerized heme that contains and generates lipoperoxidation products. In HZ and HZ-loaded monocytes 4-HNE attained approx. 50 and 15 microM, respectively. In malaria, HZ-loaded monocytes are precursors of dendritic cells (DC). Here, the role of 4-HNE as inhibitor of DC differentiation was examined. 4-HNE in HZ was quantified after derivatization by HPLC. DC were differentiated in vitro from human monocytes supplemented with GM-CSF/IL-4 and analyzed for surface antigens and 4-HNE-adducts by FACScan after labelling with specific antibodies. HZ-loading, or treatment with 4-HNE induced large numbers of 4-HNE-protein-adducts on the monocyte membrane. As low as 10 nM 4-HNE inhibited up-regulation of functionally important DC differentiation markers. 1 microM 4-HNE elicited inhibition of up-regulation of DC differentiation markers as follows: MHC-class I and II, -29% and -40%; CD1a, -16%; CD40, -25%; CD54, -27%; and CD83 (the most important DC differentiation marker), -45%, with no signs of apoptosis. The sequence of additions was important, as the inhibitory effect was reduced when 4-HNE was added after GM-CSF/IL-4, indicating that GM-CSF/IL-4 receptors could be modified by 4-HNE. In conclusion, inhibition of DC differentiation by 4-HNE may play a role in malaria immunodepression.

Diphtheria toxin-murine granulocyte-macrophage colony-stimulating factor-induced hepatotoxicity is mediated by Kupffer cells.

DT388GMCSF, a fusion toxin composed of the NH2-terminal region of diphtheria toxin (DT) fused to human granulocyte-macrophage colony-stimulating factor (GMCSF) has shown efficacy in the treatment of acute myeloid leukemia. However, the primary dose-limiting side effect is liver toxicity. We have reproduced liver toxicity in rats using the rodent cell-tropic DT-murine GMCSF (DT390mGMCSF). Serum aspartate aminotransferase and alanine aminotransferase were elevated 15- and 4-fold, respectively, in DT390mGMCSF-treated rats relative to controls. Histologic analysis revealed hepatocyte swelling; however, this did not lead to hepatic necrosis or overt histopathologic changes in the liver. Immunohistochemical staining showed apoptotic cells in the sinusoids, and depletion of cells expressing the monocyte/macrophage markers, ED1 and ED2, indicating that Kupffer cells (KC) are targets of DT390mGMCSF. In contrast, sinusoidal endothelial cells seemed intact. In vitro, DT390mGMCSF was directly cytotoxic to primary KC but not hepatocytes. Two related fusion toxins, DT388GMCSF, which targets the human GMCSF receptor, and DT390mIL-3, which targets the rodent IL-3 receptor, induced a less than 2-fold elevation in serum transaminases and did not deplete KC in vivo. In addition, DTU2mGMCSF, a modified form of DT390mGMCSF with enhanced tumor cell specificity, was not hepatotoxic and was significantly less toxic to KC in vivo and in vitro. These results show that DT390mGMCSF causes liver toxicity by targeting KC, and establish a model for studying how this leads to hepatocyte injury. Furthermore, alternative fusion toxins with potentially reduced hepatotoxicity are presented.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the osteoblastic differentiation of the human osteosarcoma cell line SaOS-2.

The Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) is a hematopoietic growth factor that regulates the in vitro and in vivo proliferation and differentiation of hematopoietic cells through the interaction with a specific heterodimeric receptor complex (GM-CSFR), consisting of an alpha and a beta chain with molecular weights of 80 and 120 KDa, respectively. We have studied the expression of the GM-CSFR (alpha chain) on the surface of the human osteosarcoma cell line SaOS-2 and the in vitro effects of different concentrations (10, 100, and 200 ng/ml) of GM-CSF on GM-CSFR expression and the biological activity of SaOS-2 cells. Our data show that SaOS-2 cells express GM-CSFR and that GM-CSF can down-regulate the expression of its own receptor on these cells. Furthermore, to evaluate the biological effects of GM-CSF on SaOS-2 cells, we have investigated cell proliferation and differentiation of these cells treated with different doses of the growth factor through: (1) a morphological analysis of typical osteoblast differentiation markers such as osteopontin and BSP-II; (2) measurement of alkaline phosphatase (ALP) activity; (3) production of bone ECM components (collagen I, fibronectin, tenascin, and laminin); (4) production of interleukin-6 (IL-6) and osteocalcin in the culture medium. The results show that the in vitro treatment of SaOS-2 cells with recombinant human GM-CSF causes a decreased cell proliferation and an increased production of osteopontin, BSP-II, ALP, IL-6, and most but not all ECM components. These findings suggest that GM-CSF can regulate proliferation and differentiation of osteoblast-like SaOS-2 cells and could also play an unexpected role in the maturation of bone tissue.

Human GM-CSF induces HIV-1 LTR by multiple signalling pathways.

Human immunodeficiency virus type-1 (HIV-1) gene expression is known to be affected by numerous cytokines or growth factors. However, the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on long terminal repeat (LTR)-mediated transcription of HIV-1 still remains unknown. By transient transfection experiments with HIV-1 LTR reporter constructs, we showed that strong LTR-mediated activation was induced by GM-CSF in mouse Ba/F3 cells expressing human GM-CSF receptors (GM-CSFR). Mutational analysis of the HIV-1 LTR reporters revealed that both NF-kappaB and Sp1 binding sites play important roles as positive regulatory elements. Analysis of various mutants of the cytoplasmic region of GM-CSFR indicated that both the conserved membrane proximal region and tyrosine residues located in the distal part of the beta subunit were required for HIV-1 LTR activation. Possible involvement of MAPK and PI3-K signalling pathways was suggested by the partial inhibition by wortmannin, a specific inhibitor of the PI3-K pathway, and enhancement by constitutively active MEK1, of HIV-1 LTR activation. However, the MEK1 pathway is not essential since MEK1 inhibitor PD98059 did not suppress GM-CSF-induced HIV-1-LTR activation. Further analyses of GM-CSFR mutants suggested that some other unknown signalling pathway also participates in GM-CSF-induced HIV-1 LTR activation. Taken together, the data suggest that GM-CSF could upregulate the LTR-driven transcription of HIV-1 through modulation of NF-kappaB and SP1 by multiple signalling pathways.

Structure-based design of mimetics for granulocyte-macrophage colony stimulating factor (GM-CSF).

Granulocyte-macrophage colony stimulating factor (GM-CSF) activity has been linked to pro-inflammatory effects in autoimmune syndromes, such as rheumatoid arthritis. Thus GM-CSF mimetics with antagonist activity might play a therapeutic role in these diseases. The human GM-CSF core structure consists of a four alpha-helix bundle, and GM-CSF activity is controlled by its binding to a two-subunit receptor. A number of residues located on the B and C helices of GM-CSF are postulated to interact with the alpha chain of the GM-CSF receptor (GM-CSFR). Several approaches have been successfully utilized to develop peptide mimetics of this site, including peptides from the native sequence, a peptide derived from a recombinant antibody (rAb) light chain which mimicked GM-CSF receptor binding activity, and structurally guided de novo design. Analysis of the rAb light chain had suggested mimicry of GM-CSF with residues mostly contributed by the CDR I region. Key residues involved in CDR I peptide/GM-CSFR binding were identified by truncation and alteration of individual residues, while the structural elements required to antagonize the biological action of GM-CSF were separately tested in binding and inhibitory activity assays of multiple cyclic analogues. A peptide designed to retain the loop conformation of the CDR I region of the rAb light chain competed with GM-CSF for both antibody and receptor binding, but the role of specific residues in antibody versus receptor binding differed markedly. These studies suggest that structural analysis of peptide mimetics can reveal differences in receptor and antibody binding, perhaps including key interactions that impact binding kinetics. Peptide mimetics of other four-helix bundle cytokines are reviewed, including helical and reverse turn mimetics of helical structures. Use of peptide mimetics coupled with structural and kinetic analysis provides a powerful approach to identifying important receptor-ligand interactions, which implications for rational design of novel therapeutics.

Identification of primary structural features that define the differential actions of IL-3 and GM-CSF receptors.

Activation of human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors, ectopically expressed in FDCP-mix multipotent cells, stimulates self-renewal or myeloid differentiation, respectively. These receptors are composed of unique alpha subunits that interact with common beta(c) subunits. A chimeric receptor (hGM/beta(c)), comprising the extracellular domain of the hGM-CSF receptor alpha subunit (hGM Ralpha) fused to the intracellular domain of hbeta(c), was generated to determine whether hbeta(c) activation is alone sufficient to promote differentiation. hGM-CSF activation of hGM/beta(c), expressed in the presence and absence of the hbeta(c) subunit, promoted maintenance of primitive phenotype. This indicates that the cytosolic domain of the hGM Ralpha chain is required for differentiation mediated by activation of the hGM Ralpha, beta(c) receptor complex. We have previously demonstrated that the alpha cytosolic domain confers signal specificity for IL-3 and GM-CSF receptors. Bioinformatic analysis of the IL-3 Ralpha and GM Ralpha subunits identified a tripeptide sequence, adjacent to the conserved proline-rich domain, which was potentially a key difference between them. Cross-exchange of the equivalent tripeptides between the alpha subunits altered receptor function compared to the wild-type receptors. Both the mutant and the corresponding wild-type receptors promoted survival and proliferation in the short-term but had distinct effects on developmental outcome. The mutated hGM Ralpha promoted long-term proliferation and maintenance of primitive cell morphology, whereas cytokine activation of the corresponding hIL-3 Ralpha mutant promoted myeloid differentiation. We have thus identified a region of the alpha cytosolic domain that is of critical importance for defining receptor specificity.

Down-regulated IL-5 receptor expression on peripheral blood eosinophils from budesonide-treated children with asthma.

BACKGROUND: The expression and function of cytokine receptors on peripheral blood eosinophils (PBE) from healthy and asthmatic children are poorly characterized. METHODS: The PBE count and expression of IL-5 receptor (R) and GM-CSFR positive PBE was analyzed in nonsteroid-treated asthmatic children (n = 13), budesonide-treated asthmatic children (n = 24) and healthy children (n = 16) by flow cytometry. Alterations in intracellular EG2-epitope expression were used to measure the in vitro responsiveness of PBE to recombinant IL-5 and GM-CSF. RESULTS: The PBE count was increased (P < 0.05) in both asthmatic groups, independent of treatment, as compared to healthy children. The IL-5R expression on PBE, as well as the in vitro responsiveness of PBE to recombinant IL-5, was reduced (P < 0.05), in budesonide-treated asthmatic children compared to nonsteroid-treated asthmatic children and healthy children. The proportion of GM-CSFR positive PBE and in vitro responsiveness of PBE to recombinant GM-CSF were not different between the groups. In vitro treatment with budesonide did not down-regulate the proportion of IL-5R positive PBE. CONCLUSIONS: Budesonide-treatment of asthmatic children induces a selectively reduced IL-5R expression on PBE, concomitant with a reduced in vitro responsiveness of PBE to IL-5. We suggest that this budesonide-related down-regulation of the IL-5R might be a mechanism by which steroid treatment inhibits the action of IL-5 on eosinophil accumulation and activation in vivo.

Perverted responses of the human granulocyte-macrophage colony-stimulating factor receptor in mouse cell lines due to cross-species beta-subunit association.

Transfected murine cell lines are commonly used to study the function of many human cytokine or receptor mutants. This study reports the inappropriate activation of the human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor by the human GM-CSF antagonist, E21R, when the human receptor is introduced into the murine cell line BaF-B03. E21R-induced proliferation of the BaF-B03 cells is dependent on transfection with both hGM-CSF receptor alpha and beta(c) subunits. Studies on the underlying mechanism revealed constitutive association between human and mouse beta(c) and GM-CSF receptor-alpha, tyrosine phosphorylation of mouse and human beta(c), and association of phosphorylated mouse beta(c) into an activated human GM-CSF receptor complex in response to E21R and GM-CSF. This interspecies receptor cross-talk of receptor signaling subunits may produce misleading results and emphasizes the need to use cell lines devoid of the cognate endogenous receptors for functional analysis of ligand and receptor mutants.

Structural and functional hot spots in cytokine receptors.

The activation of cytokine receptors is a stepwise process that depends on their specific interaction with cognate cytokines, the formation of oligomeric receptor complexes, and the initiation of cytoplasmic phosphorylation events. The recent determination of the structure of extracellular domains of several cytokine receptors allows comparison of their cytokine-binding surfaces. This comparison reveals a common structural framework that supports considerable diversity and adaptability of the binding surfaces that determine both the specificity and the orientation of subunits in the active receptor complex. These regions of the cytokine receptors have been targeted for the development of specific agonists and antagonists. The physical coupling of signaling intermediates to the intracellular domains of their receptors plays a major role in determining biological responses to cytokines. In this review, we focus principally on the receptors for cytokines of the granulocyte-macrophage colony-stimulating factor (GM-CSF) family and, where appropriate, compare them with related cytokine receptors. Several paradigms are beginning to emerge that focus on the ability of the extracellular portion of the cytokine receptor to recognize the appropriate cytokine and on a phosphorylated motif in the intracellular region of the GM-CSF receptor that couples to a specific signaling pathway.

Hormonal modulation of GM-CSF receptor alpha-chain in in vitro models of endometrial cancer.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the proliferation and differentiation of bone marrow progenitors. Moreover, the presence and activity of GM-CSF and its receptor (GM-CSF-r) has been documented on tissues and cell lines of a non-hemopoietic origin. In this paper we studied the expression and putative role of GM-CSF and GM-CSF-r in endometrial cancer. The modulation of GM-CSF-r alpha-chain upon progesterone treatment suggests a role for GM-CSF and its receptor in the pathogenesis and development of endometrial cancer.

Analysis of mechanisms involved in the prevention of gamma irradiation-induced apoptosis by hGM-CSF.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) induces proliferation and sustains viability of the mouse interleukin (IL)-3 dependent lymphoid cell line BA/F3 expressing the hGM-CSF receptor. Caspase-3 like enzyme activity and DNA fragmentation were augmented by depletion of this factor from the cell, and exposure to gamma irradiation accelerated kinetics of these events. Anti gamma irradiation-induced apoptosis occurred through various mutant GM-CSF receptors and only the box1 region was essential while the C terminal region, including tyrosine residues which are required for MAPK cascade activation, was dispensable. Consistent with this notion, the addition of PD98059 had no effect on this activity thereby indicating that activation of MAPK is not essential for the activity. As expected, gamma irradiation increased p53 protein and bax mRNA levels and the presence of hGM-CSF dramatically modulated bax/bcl-X(L) ratio. The PI-3K specific inhibitor wortmannin did not affect hGM-CSF dependent anti gamma irradiation induced apoptosis nor bcl-X(L) induction, thus bcl-X(L) but not PI-3K pathway seems to be involved in hGM-CSF dependent anti gamma irradiation-induced apoptosis. It is well documented that the boxl region is essential for GM-CSF dependent activation of JAK2 and JAK2 specific inhibitor AG490 suppressed anti gamma, irradiation-induced apoptosis by hGM-CSF. An artificial JAK2 activating molecule in which extracellular and the transmembrane of beta(c) fused with whole JAK2 can sustain BA/F3 cells survival and proliferation mIL-3 independently, but these cells are susceptible to gamma irradiation. Furthermore GyrB/Jak2, which can activate STAT5 but not the MAPK cascade nor survival of BA/F3 cells, also could not prevent gamma irradiation-induced apoptosis. Although JAK2 is essential for hGM-CSF dependent anti gamma irradiation-induced apoptosis, it appeared that JAK2 does not seem sufficient for the activity.

Simultaneous antagonism of interleukin-5, granulocyte-macrophage colony-stimulating factor, and interleukin-3 stimulation of human eosinophils by targetting the common cytokine binding site of their receptors.

Human interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific alpha chain and a shared subunit (beta(c)). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor alpha chains is the first step in receptor activation, it is the recruitment of beta(c) that allows high-affinity binding and signal transduction to proceed. Thus, beta(c) is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of beta(c). BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of (125)I-IL-5, (125)I-GM-CSF, and (125)I-IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of beta(c). Interestingly, epitope analysis using several beta(c) mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of beta(c), suggesting that ligand contact with beta(c) is a prerequisite for recruitment of beta(c), receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.

Close association between clearance of recombinant human granulocyte colony-stimulating factor (G-CSF) and G-CSF receptor on neutrophils in cancer patients.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is used to counter chemotherapy-induced neutropenia. Our previous study showed an inverse correlation between serum rhG-CSF levels and the number of circulating neutrophils in cancer patients (H. Takatani, H. Soda, M. Fukuda, M. Watanabe, A. Kinoshita, T. Nakamura, and M. Oka, Antimicrob. Agents Chemother. 40:988-991, 1996). The aim of this study was to clarify the relationship between rhG-CSF clearance and G-CSF receptors on circulating neutrophils. In five cancer patients receiving chemotherapy, a bolus dose of rhG-CSF (5 microg/kg) was injected intravenously during defined phases of posttreatment neutropenia and neutrophilia. Serum rhG-CSF levels were measured by a chemiluminescence enzyme immunoassay and analyzed by moment analysis. G-CSF receptors on neutrophils were detected by flow cytometry with biotinylated rhG-CSF. rhG-CSF clearance was significantly higher at neutrophilia than at neutropenia (1,497 +/- 132 versus 995 +/- 266 ml/h; P < 0.01). The percentage of G-CSF receptor-positive neutrophils, reflecting the number of G-CSF receptors per cell, was low at neutropenia without rhG-CSF therapy (44.5% +/- 22.1%) and high at neutrophilia with rhG-CSF therapy (73. 0% +/- 11.4%; P < 0.01). rhG-CSF clearance closely correlated with the percentage of G-CSF receptor-positive neutrophils (r2 = 0.91; P < 0.0001) and neutrophil count (r2 = 0.72; P < 0.005). Our results indicate that, in cancer patients receiving chemotherapy, rhG-CSF increases the number of G-CSF receptors per cell as well as circulating neutrophil counts, resulting in modulation of its own clearance.

Interleukin-4, interleukin-5, and granulocyte-macrophage colony-stimulating factor receptor expression in chronic sinusitis and response to topical steroids.

Chronic sinusitis and its associated eosinophilic infiltrate are believed to be mediated, at least in part, by the upregulation of Th-2 cytokines, including interleukin-4, interleukin-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interleukin-4 is involved in IgE production and in eosinophil recruitment through upregulation of vascular cell adhesion molecule-1. Interleukin-5 and GM-CSF are involved in eosinophil growth and survival. The aim of this study was to investigate the expression of receptors for these cytokines in the sinus mucosa of subjects with chronic sinusitis. Using the technique of in situ hybridization to detect specific cytokine receptor messenger RNA, we studied the sinus mucosa of subjects with nonallergic chronic sinusitis, subjects with allergic chronic sinusitis, subjects with allergic chronic sinusitis treated with topical steroids, and normal controls. Our data demonstrate higher expression of interleukin-4 receptor in subjects with allergic chronic sinusitis than in controls (p < 0.001) and higher expression of interleukin-5 receptor in both subjects with nonallergic chronic sinusitis and subjects with allergic chronic sinusitis than in controls (p < 0.001, p < 0.001). The expression of interleukin-4 receptor and interleukin-5 receptor was higher in subjects with allergic chronic sinusitis than in subjects with nonallergic chronic sinusitis (p < 0.001). GM-CSF receptor expression was also found to be higher in subjects with allergic chronic sinusitis and subjects with nonallergic chronic sinusitis than in controls (p < 0.001, p < 0.001). In contrast to interleukin-4 receptor and interleukin-5 receptor, however, expression of GM-CSF receptor was higher in subjects with nonallergic chronic sinusitis than in subjects with allergic chronic sinusitis (p < 0.001). In subjects with allergic chronic sinusitis treated with topical corticosteroids, the expression of interleukin-4 receptor and interleukin-5 receptor messenger RNA levels was significantly lower than levels in patients with allergic chronic sinusitis who were not taking topical steroids (p < 0.001, p < 0.001). Steroid treatment had no effect on GM-CSF receptor messenger RNA expression. In conclusion, our data support a role for Th-2 cytokine receptors in the pathophysiology of chronic sinusitis. Further, our data lend support to the theory that differential activation of distinct cytokine pathways mediates inflammation in chronic sinusitis depending on whether there is associated allergy. Finally, treatment with topical corticosteroids has been demonstrated in chronic sinusitis to downregulate receptors for interleukin-4 and interleukin-5.

The human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor exists as a preformed receptor complex that can be activated by GM-CSF, interleukin-3, or interleukin-5.

The granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor is expressed on normal and malignant hematopoietic cells as well as on cells from other organs in which it transduces a variety of functions. Despite the widespread expression and pleiotropic nature of the GM-CSF receptor, little is known about its assembly and activation mechanism. Using a combination of biochemical and functional approaches, we have found that the human GM-CSF receptor exists as an inducible complex, analogous to the interleukin-3 (IL-3) receptor, and also as a preformed complex, unlike the IL-3 receptor or indeed other members of the cytokine receptor superfamily. We found that monoclonal antibodies to the GM-CSF receptor alpha chain (GMR alpha) and to the common beta chain of the GM-CSF, IL-3, and IL-5 receptors (beta(c)) immunoprecipitated both GMR alpha and beta(c) from the surface of primary myeloid cells, myeloid cell lines, and transfected cells in the absence of GM-CSF. Further association of the two chains could be induced by the addition of GM-CSF. The preformed complex required only the extracellular regions of GMR alpha and beta(c), as shown by the ability of soluble beta(c) to associate with membrane-anchored GMR alpha or soluble GMR alpha. Kinetic experiments on eosinophils and monocytes with radiolabeled GM-CSF, IL-3, and IL-5 showed association characteristics unique to GM-CSF. Significantly, receptor phosphorylation experiments showed that not only GM-CSF but also IL-3 and IL-5 stimulated the phosphorylation of GMR alpha-associated beta(c). These results indicate a pattern of assembly of the heterodimeric GM-CSF receptor that is unique among receptors of the cytokine receptor superfamily. These results also suggest that the preformed GM-CSF receptor complex mediates the instantaneous binding of GM-CSF and is a target of phosphorylation by IL-3 and IL-5, raising the possibility that some of the biologic activities of IL-3 and IL-5 are mediated through the GM-CSF receptor complex.