Activity Of Granulocyte-macrophage Colony-stimulating Factor Research Articles

CMRF-56 Immunoselected Blood Dendritic Cell Preparations Activated With GM-CSF Induce Potent Antimyeloma Cytotoxic T-cell Responses

The efficient antigen-presenting function of dendritic cells (DC) makes them an attractive cellular adjuvant for clinical immunotherapeutic protocols aimed at eradicating minimal residual disease after conventional treatment of multiple myeloma (MM) and other malignancies. We used single-step positive immunoselection with biotinylated CMRF-56 monoclonal antibody to generate a CD11c blood DC (BDC) enriched antigen-presenting cell population, which, after exposure to activation stimuli for as little as 2 hours, displayed a mature costimulatory BDC phenotype and secreted inflammatory cytokines. Of the activation stimuli tested, granulocyte macrophage colony-stimulating factor (GM-CSF) provided optimal activation of the CMRF-56 immunoselected preparations and primed efficient cytotoxic T cell (CTL) responses using MART-1 peptide as a model tumor-associated antigen. In addition, GM-CSF activated CMRF-56 immunoselected cells cross-presented MM cell lysate and improved the MM-specific polyclonal CTL response (no activation 18.8%+/-4.3% vs. GM-CSF activation 40.9%+/-7.3%, P=0.051). CMRF-56 immunoselected BDC migrated in vitro both spontaneously and specifically toward the secondary lymphoid chemokine CCL21. Their migration was also significantly improved by GM-CSF and prostaglandin E2 activation and a greater percentage of activated BDC migrated specifically compared with monocyte-derived DC. These results indicate that the CMRF-56 immunoselected BDC preparations can cross-present antigen for effective anti-MM CTL responses and that limited exposure to maturation stimuli can produce phenotypically and functionally mature migrating DC. CMRF-56 immunoselected cells are suitable for use as part of an immunotherapeutic anti-MM vaccine.

MFG-E8–mediated uptake of apoptotic cells by APCs links the pro- and antiinflammatory activities of GM-CSF

Granulocyte-macrophage colony-stimulating factor (GM-CSF) enhances protection against tumors and infections, but GM-CSF-deficient mice develop inflammatory disease. Here we show that GM-CSF is required for the expression of milk fat globule EGF 8 (MFG-E8) in antigen-presenting cells, and that MFG-E8-mediated uptake of apoptotic cells is a key determinant of GM-CSF-triggered tolerance and immunity. Upon exposure to apoptotic cells, GM-CSF-deficient antigen-presenting cells (APCs) produce an altered cytokine profile that results in decreased Tregs and increased Th1 cells, whereas concurrent ablation of IFN-gamma promotes Th17 cells. In wild-type mice, MFG-E8 attenuates the vaccination activity of GM-CSF-secreting tumor cells through Treg induction, whereas a dominant-negative MFG-E8 mutant potentiates GM-CSF-stimulated tumor destruction through Treg inhibition. These findings clarify the immunoregulatory effects of apoptotic cells and suggest new therapeutic strategies to modulate CD4(+) T cell subsets in cancer and autoimmunity.

The effects of fluconazole and cytokines on human mononuclear cells

Candida infections are common infections and fluconazole is one of the most frequently administered antifungal agents in their treatment. The resistance developed against antifungal agents has necessitated the improvement of new treatments. This study focuses on the investigation of the effect of fluconazole and cytokines such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF) on chemokine production and anticandidal activity of human monocytes. In the study it was observed that GM-CSF caused an increase in candidacidal activity of monocytes. Anticandidal activity of GM-CSF + IFN-gamma combination was not found to be more effective than GM-CSF or IFN-gamma alone. The presence of cytokine and fluconazole caused an increase in the levels of CCL3 and CCL4 chemokines. Accordingly, it was considered that chemokines could contribute to the efficacy of fluconazole in C. albicans infections. Besides, in order to strengthen the immune system some cytokines might be used in addition to antifungal agents for the treatment.

Granulocyte–Macrophage Colony-Stimulating Factor Is Essential for Normal Wound Healing

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent growth factor, which plays an important role during the process of wound healing. In clinical settings it has occasionally been employed in the treatment of cutaneous wounds of diverse etiologies. In a previous study, we have shown the positive influence of GM-CSF on full thickness excisional wounds in transgenic mice overexpressing GM-CSF in the basal layer of the epidermis. Direct GM-CSF action as well as indirect processes through the induction of secondary cytokines were proposed to contribute towards the beneficial effects. In this study, we analyzed the process of wound healing in transgenic mice overexpressing a GM-CSF antagonist in the epidermis. These mice not only exhibited a delayed scab rejection and reepithelialization but also neovascularization was reduced. The newly formed tissue was of poor quality as exhibited by the presence of extensive fibrosis. We suggest that the presence of GM-CSF in the repair process is of basic importance and its absence leads not only to delayed wound healing but it is also detrimental for the quality of the newly formed tissue.

GM-CSF and the impaired pulmonary innate immune response following hyperoxic stress

We have previously demonstrated that mice exposed to sublethal hyperoxia (an atmosphere of >95% oxygen for 4 days, followed by return to room air) have significantly impaired pulmonary innate immune response. Alveolar macrophages (AM) from hyperoxia-exposed mice exhibit significantly diminished antimicrobial activity and markedly reduced production of inflammatory cytokines in response to stimulation with LPS compared with AM from control mice in normoxia. As a consequence of these defects, mice exposed to sublethal hyperoxia are more susceptible to lethal pneumonia with Klebsiella pneumoniae than control mice. Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a growth factor produced by normal pulmonary alveolar epithelial cells that is critically involved in maintenance of normal AM function. We now report that sublethal hyperoxia in vivo leads to greatly reduced alveolar epithelial cell GM-CSF expression. Systemic treatment of mice with recombinant murine GM-CSF during hyperoxia exposure preserved AM function, as indicated by cell surface Toll-like receptor 4 expression and by inflammatory cytokine secretion following stimulation with LPS ex vivo. Treatment of hyperoxic mice with GM-CSF significantly reduced lung bacterial burden following intratracheal inoculation with K. pneumoniae, returning lung bacterial colony-forming units to the level of normoxic controls. These data point to a critical role for continuous GM-CSF activity in the lung in maintenance of normal AM function and demonstrate that lung injury due to hyperoxic stress results in significant impairment in pulmonary innate immunity through suppression of alveolar epithelial cell GM-CSF expression.

Gangliosides of the stroma layer participate in the interferon-gamma receptor-dependent controls of myelopoiesis

Stroma-mediated myelopoiesis depends upon growth-factors and an appropriate intercellular microenvironment, whose polarity is relevant for granulocyte-macrophage colony stimulating factor (GM-CSF) mediated myeloid cell proliferation. Here we have studied qualitative and quantitative aspects of ganglioside participation in controls of the microenvironment required to sustain myelopoiesis. We analysed ganglioside synthesis, expression and shedding by two primary liver stromal cell cultures isolated from wild type and interferon-gamma (IFNγ) receptor knockout mice. The latter one has a higher capacity to sustain myelopoiesis. FDC-P1 myeloid growth factor-dependent cell line was used as the reporter system, monitoring the cell survival and proliferation that reflect the bio-availability and the activity of GM-CSF. Although the two stromal cells synthesised the same gangliosides their relative content was quite different. FDC-P1 proliferation decreased in cultures in which ganglioside synthesis was inhibited in the stroma, as well as in presence of stroma cell supernatants in which GM3 was neutralised by the anti-GM3 monoclonal antibody. Addition of exogenous GM3 reverted the inhibition and sustained proliferation of FDC-P1 cells. FDC-P1 cells do not accumulate GM3, but they are able to take up the stroma-produced sphingolipids. Thus, stroma has a double role in sustaining myelopoiesis, providing both growth factor(s) and ganglioside(s) required for the optimal stimulation of the myeloid cell proliferation, and the IFNγ mediated stroma-dependent controls of myelopoiesis are determinant for this cell interaction.

Gangliosides of myelosupportive stroma cells are transferred to myeloid progenitors and are required for their survival and proliferation

In previous studies, we have shown that the myelopoiesis dependent upon myelosupportive stroma required production of growth factors and heparan-sulphate proteoglycans, as well as generation of a negatively charged sialidase-sensitive intercellular environment between the stroma and the myeloid progenitors. In the present study, we have investigated the production, distribution and role of gangliosides in an experimental model of in vitro myelopoiesis dependent upon AFT-024 murine liver-derived stroma. We used the FDC-P1 cell line, which is dependent upon GM-CSF (granulocyte/macrophage colony-stimulating factor) for both survival and proliferation, as a reporter system to monitor bioavailability and local activity of GM-CSF. G(M3) was the major ganglioside produced by stroma, but not by myeloid cells, and it was required for optimal stroma myelosupportive function. It was released into the supernatant and selectively incorporated into the myeloid progenitor cells, where it segregated into rafts in which it co-localized with the GM-CSF-receptor alpha chain. This ganglioside was also metabolized further by myeloid cells into gangliosides of the a and b series, similar to endogenous G(M3). In these cells, G(M1) was the major ganglioside and it was segregated at the interface by stroma and myeloid cells, partially co-localizing with the GM-CSF-receptor alpha chain. We conclude that myelosupportive stroma cells produce and secrete the required growth factors, the cofactors such as heparan sulphate proteoglycans, and also supply gangliosides that are transferred from stroma to target cells, generating on the latter ones specific membrane domains with molecular complexes that include growth factor receptors.

Heparin-binding Sites in Granulocyte-Macrophage Colony-stimulating Factor: LOCALIZATION AND REGULATION BY HISTIDINE IONIZATION

The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four alpha-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.

Kinetics of development and characteristics of antibodies induced in cancer patients against yeast expressed rDNA derived granulocyte macrophage colony stimulating factor (GM-CSF)

We have determined the presence and kinetics of granulocyte macrophage colony stimulating factor (GM-CSF) antibodies induced after repeated administration of a yeast expressed GM-CSF product in prostate cancer patients with minimal recurrent disease using a panel of assays for detection and characterization of antibodies. Results showed that all 15 prostate cancer patients treated with GM-CSF developed GM-CSF reactive antibodies during the course of therapy. Most patients (87%) developed GM-CSF reactive antibodies within 3 months while in other patients (13%), these antibodies were induced after additional cycles of GM-CSF treatment. For most patients, the timing of occurrence of these antibodies was the same regardless of whether the ELISA or surface plasmon resonance (SPR) assays were used for detection. However, in two patients, the recognition of GM-CSF reactive antibodies by SPR assays preceded their detection by ELISA. A significant number of patients ( n = 9 , 60%) developed GM-CSF antibodies which neutralized the biological activity of GM-CSF in vitro in a cell-line based bioassay. These antibodies also recognized GM-CSF protein from different expression systems including the non-glycosylated protein from E. coli indicating that the antibody response is directed towards the amino acid backbone of the protein. A significant effect of GM-CSF antibodies on PSA modulation was not observed in this small cohort of patients despite an alteration in PSA levels in some treated patients. The study design used here did not allow conclusions regarding the relationship between neutralizing antibodies and the PSA levels which were used as a marker for clinical outcome. Implementation of a clinical strategy which permits monitoring for antibody development and for levels of a relevant pre-determined clinical marker at appropriate time-points is necessary for assessing the impact of antibody development on the therapeutic efficacy of the protein.

Delivery of Granulocyte-Macrophage Colony-Stimulating Factor in Bioadhesive Hydrogel Stimulates Migration of Dendritic Cells in Models of Human Papillomavirus-Associated (Pre)Neoplastic Epithelial Lesions

Because of the central role of dendritic cells and/or Langerhans cells(DC/LC) in the induction of cellular immune responses, pharmacological agents that modulate the recruitment of these cells might have a clinical interest. The present study was designed to evaluate the capacity of several pharmaceutical formulations to topically deliver granulocyte-macrophage colony-stimulating factor (GM-CSF) on human papillomavirus (HPV)-associated genital (pre)neoplastic lesions. The formulations were evaluated for their bioactivity and for their potential to recruit DC in organotypic cultures of HPV-transformed keratinocytes. We found that a bioadhesive polycarbophil gel (Noveon) at pH 5.5 is able to maintain the bioactivity of GM-CSF at 4 or 37 degrees C for at least 7 days, whereas a decreased activity of GM-CSF was observed when the molecule is included in other polymer gels. GM-CSF incorporated in the polycarbophil gel was also a potent factor in enhancing the colonization of DC into organotypic cultures of HPV-transformed keratinocytes since the infiltration of DC in the in vitro-formed (pre)neoplastic epithelium was very low under basal conditions and dramatically increased in the presence of GM-CSF gel. We next demonstrated that GM-CSF incorporated in polycarbophil gel induces the recruitment of human DC in a human (pre)neoplastic epithelium grafted into NOD/SCID mice. The efficacy of GM-CSF in this formulation was equivalent to that observed with liquid GM-CSF. These results suggest that GM-CSF incorporated in polycarbophil gel could play an important role in the recruitment of DC/LC in mucosal surfaces and be useful as a new immunotherapeutic approach for genital HPV-associated (pre)neoplastic lesions.

Induction of neutralising antibodies restricts the use of human granulocyte/macrophage colony stimulating factor for vaccine studies in rhesus macaques

Granulocyte/macrophage-colony stimulating factor (GM-CSF) is a valuable adjuvant to enhance induction of cellular immune responses in rodents. Less information is available regarding its use as an adjuvant in primates or humans. We explored recombinant human GM-CSF for potential vaccine studies in rhesus macaques and focused on its effect on peripheral monocytes as progenitors of dendritic cells and its potential immunogenicity. Application of human GM-CSF to nine animals led to an average 32-fold increase in monocyte numbers. This was not observed upon re-treatment, which coincided with GM-CSF-specific neutralising antibodies. These also neutralised the activity of rhesus macaque GM-CSF. The data underscore the need to use species-specific GM-CSF for immunomodulation in primates.

Human immunodeficiency virus type 1 infection inhibits granulocyte-macrophage colony-stimulating factor-induced activation of STAT5A in human monocyte-derived macrophages.

Human immunodeficiency virus type 1 (HIV-1) infects cells of the monocyte/macrophage lineage. While infection of macrophages by HIV-1 is generally not cytopathic, it does impair macrophage function. In this study, we examined the effect of HIV-1 infection on intracellular signaling in human monocyte-derived macrophages (MDM) stimulated with the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF is an important growth factor for cells of both the macrophage and granulocyte lineages and enhances effector functions of these cells via the heterodimeric GM-CSF receptor (GM-CSFR). A major pathway which mediates the effects of GM-CSF on macrophages involves activation of the latent transcription factor STAT5A via a Janus kinase 2 (JAK2)-dependent pathway. We demonstrate that GM-CSF-induced activation of STAT5A is inhibited in MDM after infection in vitro with the laboratory-adapted R5 strain of HIV-1, HIV-1(Ba-L), but not after infection with adenovirus. HIV-1 infection of MDM did not decrease the STAT5A or JAK2 mRNA level or STAT5A protein level or result in increased constitutive activation of STAT5A. Surface expression of either the alpha-chain or common beta(c)-chain of GM-CSFR was also unaffected. We conclude that HIV-1 inhibits GM-CSF activation of STAT5A without affecting expression of the known components of the signaling pathway. These data provide further evidence of disruption of cellular signaling pathways after HIV-1 infection, which may contribute to immune dysfunction and HIV-1 pathogenesis.

Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy.

In this study, the development of neutralizing and non-neutralizing GM-CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli-derived GM-CSF and a colon carcinoma-reactive MoAb in the absence of any concomitant chemotherapy. The recombinant human GM-CSF was administered subcutaneously for 10 days every month for 4 months. Following the first cycle of treatment, no GM-CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM-CSF binding antibodies. However, only a proportion (40%) of the 19 antibody-positive patients developed antibodies that neutralized the biological activity of GM-CSF in an in vitro bioassay. The presence of GM-CSF neutralizing antibodies was associated with a significant reduction in GM-CSF-induced expansion of leucocytes, neutrophils and eosinophils. Such clinical effects were not apparent in patients with non-neutralizing antibodies. Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM-CSF preparations derived using different expression systems (Chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross-react with endogenously produced GM-CSF. These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response. Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy.

Stroma-mediated granulocyte-macrophage colony-stimulating factor (GM-CSF) control of myelopoiesis: spatial organisation of intercellular interactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is one of the major cytokines involved in control of haemopoiesis both in bone marrow and in extramedullar sites. Its biological activity depends upon the composition and physicochemical properties of the microenvironment provided by the supporting stroma. GM-CSF activity is modulated and controlled by the stromal heparan-sulphate proteoglycans, but their optimal interaction occurs only at low pH. We questioned whether the microenvironment organisation of the interface between stroma and haemopoietic cells provides such conditions. We studied myeloid progenitor proliferation in contact with bone marrow-derived and extramedullar stromas using electron microscopy and selective labelling of pericellular components. We present evidence that, upon interaction, the two cell types reorganise their interface both in shape and molecular composition. Haemopoietic cells extend projections that considerably increase the area of intercellular contact, and stromal cells form lamellipodia and carry out a redistribution of membrane-associated sialylated glycoconjugates and proteoglycans. Such rearrangements lead to extensive capping of negatively charged molecules at the interface between the supporting stroma and the haemopoietic cells, leading potentially to a local decrease in pH. Our results indicate that the distribution of negative charges at the cellular interface may be responsible for the selectivity of cell response to GM-CSF.

Granulocyte macrophage colony stimulating factor (GM-CSF) activity is regulated by a GM-CSF binding molecule in Wallerian degeneration following injury to peripheral nerve axons

The hematopoietic factor and inflammatory cytokine GM-CSF is involved in PNS and CNS injury and disease, and in macrophage and microglia function regulation. We presently document that injury to PNS axons induces in vivo production of GM-CSF-inhibitor and GM-CSF-augmenter activities. GM-CSF-inhibitor activity was detected in extract and conditioned medium (CM) of injured PNS but not in extract of intact PNS, and was removed from CM by GM-CSF affinity chromatography, suggesting it is carried by a secreted GM-CSF binding molecule. CM further displayed GM-CSF-augmenter activity along with GM-CSF-inhibitor activity but at contrasting concentrations; augmentation at lowest and inhibition at highest. GM-CSF activity is thus regulated during Wallerian degeneration (WD); augmenter activity characterizes the onset and inhibitor activity the later stages of WD.

Molecular assembly of the ternary granulocyte-macrophage colony-stimulating factor receptor complex

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine that stimulates the production and functional activity of granulocytes and macrophages, properties that have encouraged its clinical use in bone marrow transplantation and in certain infectious diseases. Despite the importance of GM-CSF in regulating myeloid cell numbers and function, little is known about the exact composition and mechanism of assembly of the GM-CSF receptor complex. We have now produced soluble forms of the GM-CSF receptor alpha chain (sGMRalpha) and beta chain (sbetac) and utilized GM-CSF, the GM-CSF antagonist E21R (Glu21Arg), and the betac-blocking monoclonal antibody BION-1 to define the molecular assembly of the GM-CSF receptor complex. We found that GM-CSF and E21R were able to form low-affinity, binary complexes with sGMRalpha, each having a stoichiometry of 1:1. Importantly, GM-CSF but not E21R formed a ternary complex with sGMRalpha and sbetac, and this complex could be disrupted by E21R. Significantly, size-exclusion chromatography, analytical ultracentrifugation, and radioactive tracer experiments indicated that the ternary complex is composed of one sbetac dimer with a single molecule each of sGMRalpha and of GM-CSF. In addition, a hitherto unrecognized direct interaction between betac and GM-CSF was detected that was absent with E21R and was abolished by BION-1. These results demonstrate a novel mechanism of cytokine receptor assembly likely to apply also to interleukin-3 (IL-3) and IL-5 and have implications for our molecular understanding and potential manipulation of GM-CSF activation of its receptor.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) acts independently of the beta common subunit of the GM-CSF receptor to prevent inner cell mass apoptosis in human embryos.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in the female reproductive tract during early pregnancy and can promote the growth and development of preimplantation embryos in several species. We have demonstrated with in vitro experiments that the incidence of blastulation in human embryos is increased approximately twofold when GM-CSF is present in the culture medium. In the present study, we investigated the mechanisms underlying the embryotrophic actions of GM-CSF. Using reverse transcription-polymerase chain reaction and immunocytochemistry, expression of mRNA and protein of the GM-CSF-receptor alpha subunit (GM-Ralpha) was detected in embryos from the first-cleavage through blastocyst stages of development, but the GM-CSF-receptor beta common subunit (betac) could not be detected at any stage. When neutralizing antibodies reactive with GM-Ralpha were added to embryo culture experiments, the development-promoting effect of GM-CSF was ablated. In contrast, GM-CSF activity in embryos was not inhibited either by antibodies to betac or by E21R, a synthetic GM-CSF analogue that acts to antagonize betac-mediated GM-CSF signaling. Unexpectedly, E21R was found to mimic native GM-CSF in promoting blastulation. When embryos were assessed for apoptosis and cell number by confocal microscopy after TUNEL and propidium iodine staining, it was found that blastocysts cultured in GM-CSF contained 50% fewer apoptotic nuclei and 30% more viable inner cell mass cells. Together, these data indicate that GM-CSF regulates cell viability in human embryos and that this potentially occurs through a novel receptor mechanism that is independent of betac.

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.

Interleukin-15 inhibits spontaneous apoptosis in human eosinophils via autocrine production of granulocyte macrophage-colony stimulating factor and nuclear factor-kappaB activation.

Prolonged eosinophil survival, i.e., reduced apoptosis, is implicated in the pathogenesis of chronic allergic inflammation. Here we demonstrate that interleukin (IL)-15, in the presence or absence of tumor necrosis factor (TNF)-alpha, reduces spontaneous apoptosis in freshly isolated human eosinophils. The prosurvival effect of IL-15 was abrogated by neutralizing antibody to granulocyte macrophage-colony stimulating factor (GM-CSF), although GM-CSF was not detected in conditioned media by ELISA. Additionally, the effect of IL-15 on spontaneous eosinophil apoptosis appeared to require nuclear factor-kappaB (NF-kappaB) activation based on evidence for NF-kappaB nuclear translocation and abrogation of the effect by the NF-kappaB inhibitor, Bay 11- 7082. Finally, the data demonstrate that IL-15 expression is higher in the submucosa of endobronchial tissues from subjects with moderate to severe asthma when compared with control subjects. Thus, our results suggest that IL-15, either alone or in combination with TNF-alpha, may perpetuate allergic inflammation by reduction of spontaneous eosinophil apoptosis through autocrine production of GM-CSF and NF-kappaB activation.

GPI-anchoring of GM-CSF results in active membrane-bound and partially shed cytokine

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the generation and activation of dendritic cells (DCs), the most potent of antigen presenting cells (APCs). Tumors secreting GM-CSF have been shown to induce strong anti-tumor immune responses. In this report, we have constructed a glycosylphosphatidyl-inositol (GPI) anchored form of GM-CSF (GPI-GM-CSF). This protein subsequently was found expressed on the cell membrane and sensitive to phosphatidyl-inositol-specific phospholipase C (PIPLC), confirming that it is GPI-anchored. However, GM-CSF was also found in the culture supernatant of cells expressing GPI-GM-CSF. Inhibition studies using brefeldin A and para-formaldehyde fixation revealed that GM-CSF found in the supernatant was not secreted, but due to shedding or proteolytic cleavage. Accumulation of GM-CSF in the media from isolated membranes was time and temperature-dependent. The released portion represented 10–15% of all membrane-bound GM-CSF after 72 h under culture conditions. GPI-GM-CSF retained functional activity to induce bone marrow cell proliferation and administration of GPI-GM-CSF expressing membranes induced the generation of DCs in vivo. These results demonstrate that GPI-anchored GM-CSF retains all functional activity of native GM-CSF while gaining the ability to attach to cell membranes. The ability of GPI-GM-CSF to be expressed on membranes and be partially released, can possibly lead to formation of a cytokine gradient, while retaining ability to target associated membrane antigens to DCs. This novel form of GM-CSF may have wide range of clinical applicability.