Blockade of collagen-induced arthritis post-onset by antibody to granulocyte-macrophage colony-stimulating factor (GM-CSF): requirement for GM-CSF in the effector phase of disease

There is mounting evidence for a role of the growth factor granulocyte-macrophage colony-stimulating factor (GM-CSF) in inflammatory disease, including arthritis. In the present study, we examined the effectiveness of treatment of collagen-induced arthritis (CIA) with a neutralizing mAb to GM-CSF. DBA/1 mice were immunized for the development of CIA and treated at different times, and with different doses, with neutralizing mAb to GM-CSF or isotype control mAb. Anti-GM-CSF mAb treatment prior to the onset of arthritis, at the time of antigen challenge, was effective at ameliorating the ensuing disease. Modulation of arthritis was seen predominantly as a reduction in overall disease severity, both in terms of the number of limbs affected per mouse and the clinical score of affected limbs. Importantly, anti-GM-CSF mAb treatment ameliorated existing disease, seen both as a reduction in the number of initially affected limbs progressing and lower numbers of additional limbs becoming affected. By histology, both inflammation and cartilage destruction were reduced in anti-GM-CSF-treated mice, and the levels of tumor necrosis factor-a and IL-1? were also reduced in joint tissue washouts of these mice. Neither humoral nor cellular immunity to type II collagen, however, was affected by anti-GM-CSF mAb treatment. These results suggest that the major effect of GM-CSF in CIA is on mediating the effector phase of the inflammatory reaction to type II collagen. The results also highlight the essential role of GM-CSF in the ongoing development of inflammation and arthritis in CIA, with possible therapeutic implications for rheumatoid arthritis.     

Molecular modeling of the GM-CSF and IL-3 receptor complexes.

A model for the structure of the cytokine interleukin-3 (IL-3) is presented based on the structural homology of the hematopoietic cytokines and utilizing the crystal structures of interleukin-5 and granulocyte macrophage colony stimulating factor (GM-CSF). In addition, models of the receptor complexes of GM-CSF and IL-3 are presented based on the structural homology of the hematopoietic receptors to growth hormone. Several key interactions between the ligands and their receptors are discovered, some in agreement with previous mutagenesis studies and others that have not yet been the subject of mutagenesis studies. The models provide insights into the binding of GM-CSF and IL-3 to their receptors.     

Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody

X-ray structure analysis of 4 antibody Fab fragments, each in complex with human granulocyte macrophage colony stimulating factor (GM-CSF), was performed to investigate the changes at the protein-protein binding interface during the course of in vitro affinity maturation by phage display selection. The parental antibody MOR03929 was compared to its derivatives MOR04252 (CDR-H2 optimized), MOR04302 (CDR-L3 optimized) and MOR04357 (CDR-H2 and CDR-L3 optimized). All antibodies bind to a conformational epitope that can be divided into 3 sub-epitopes. Specifically, MOR04357 binds to a region close to the GM-CSF N-terminus (residues 11–24), a short second sub-epitope (residues 83–89) and a third at the C-terminus (residues 112–123). Modifications introduced during affinity maturation in CDR-H2 and CDR-L3 led to the establishment of additional hydrogen bonds and van der Waals contacts, respectively, providing a rationale for the observed improvement in binding affinity and neutralization potency. Once GM-CSF is complexed to the antibodies, modeling predicts a sterical clash with GM-CSF binding to GM-CSF receptor α and β chain. This predicted mutually exclusive binding was confirmed by a GM-CSF receptor α chain ligand binding inhibition assay. Finally, high throughput sequencing of clones obtained after affinity maturation phage display pannings revealed highly selected consensus sequences for CDR-H2 as well for CDR-L3, which are in accordance with the sequence of the highest affinity antibody MOR04357. The resolved crystal structures highlight the criticality of these strongly selected residues for high affinity interaction with GM-CSF.     

Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis

Neutrophils occur in tissues of the female reproductive tract (FRT) under non-infected conditions. These cells generally enter tissues under the influence of chemoattractants called chemokines. Primary epithelial cells (EC) from FRT were a potent source of chemokines, IL-8 being the chief neutrophil chemoattractant secreted. Blocking with neutralizing anti-IL-8 showed that IL-8 did not account for all of the chemoattraction observed. A mixture of 25 ng/mL rIL-8 and 1 ng/mL rGM-CSF mediated 2.7-fold more chemotaxis than that expected if the two agents were additive. We then found that GM-CSF was produced by EC in amounts that synergised strongly with IL-8 to enhance chemotaxis. Treatment of uterine EC conditioned medium with saturating doses of anti-IL-8 plus anti-GM-CSF antibodies produced an 84% inhibition of chemotaxis. These findings demonstrate that the majority of neutrophil chemoattractant activity produced by FRT EC results from the synergistic effects of IL-8 and GM-CSF.     

Endothelium-derived GM-CSF influences expression of oncostatin M

During and after transendothelial migration, neutrophils undergo a number of phenotypic changes resulting from encounters with endothelium-derived factors. This report uses an in vitro model with human umbilical vein endothelial cells and isolated human neutrophils to examine the effects of two locally derived cytokines, granulocyte (G)-macrophage (M) colony-stimulating factor (GM-CSF) and G-CSF, on oncostatin M (OSM) expression. Neutrophils contacting activated HUVEC expressed and released increased amounts of oncostatin M (OSM), a proinflammatory cytokine known to induce polymorphonuclear neutrophil adhesion and chemotaxis. Neutrophil transendothelial migration resulted in threefold higher OSM expression and protein levels compared with nontransmigrated cells. Addition of anti-GM-CSF neutralizing antibody reduced OSM expression level but anti-G-CSF was without effect. GM-CSF but not G-CSF protein addition to cultures of isolated neutrophils resulted in a significant increase in OSM protein secretion. However, inhibition of β(2) integrins by neutralizing antibody significantly reduced GM-CSF-induced OSM production indicating this phenomenon is adhesion dependent. Thus cytokine-stimulated endothelial cells can produce sufficient quantities of GM-CSF to influence in an adhesion-dependent manner, the phenotypic characteristics of neutrophils resulting in the latter's transmigration. Both transmigration and adhesion phenomenon lead to increased production of OSM by neutrophils that then play a major role in inflammatory response.     

The granulocyte macrophage colony stimulating factor (GM-CSF) regulates amyloid beta (Abeta) production

One of the hallmarks of Alzheimer’s disease (AD) is the accumulation of amyloid beta (Aβ) plaques in the brain parenchyma. An inflammatory component to AD has been suggested in association with increased cytokine release. We have previously shown that CD40L stimulation of microglia induces increases in pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, IL-8 and GM-CSF. We have also shown that CD40L stimulation increases Aβ levels in HEK-293 cells over-expressing both the amyloid precursor protein (APP) and CD40 (HEK/APPsw/CD40). In this study, we show that GM-CSF neutralizing antibodies mitigate the CD40L-induced production of Aβ in HEK/APPsw/CD40 cells. In addition, we demonstrate that treatment of these cells with recombinant GM-CSF significantly increases Aβ levels. Furthermore, we show that shRNA silencing of the GM-CSF receptor gene significantly reduces Aβ levels to below base line in non-stimulated HEK/APPsw/CD40 cells. Analysis of cell surface proteins revealed that silencing of the GM-CSF receptor also decreases APP endocytosis (therefore reducing the availability of APP to be cleaved in the endosomes). Taken together, our results suggest that GM-CSF operates downstream of CD40/CD40L interaction and that GM-CSF modulates Aβ production by influencing APP trafficking. GM-CSF signaling may be a suitable therapeutic target against Aβ production in AD.     

Two neutralizing monoclonal antibodies against human granulocyte-macrophage colony-stimulating factor recognize the receptor binding domain of the molecule.

Using a series of mutant and chimeric human-mouse granulocyte-macrophage-CSF molecules the binding epitopes of two neutralizing mAb antibodies to human GM-CSF have been mapped. Both intact antibody and Fab fragments neutralize the biologic activity of human GM-CSF. The epitope of one of the antibodies contains residues widely separated in the primary structure of the growth factor that suggests that these two regions are adjacent in the tertiary structure of the molecule. In addition, evidence is presented that both mAb neutralize the activity of this cytokine by blocking the receptor binding domain of human GM-CSF.     

Innate immune responses to LPS in mouse lung are suppressed and reversed by neutralization of GM-CSF via repression of TLR-4

The innate immune inflammatory response to lipopolysaccharide (LPS, an endotoxin) is essential for lung host defense against infection by gram-negative bacteria but is also implicated in the pathogenesis of some lung diseases. Studies on genetically altered mice implicate granulocyte-macrophage colony-stimulating factor (GM-CSF) in lung responses to LPS; however, the physiological effects of GM-CSF neutralization are poorly characterized. We performed detailed kinetic and dose-response analyses of the lung inflammation response to LPS in the presence of the specific GM-CSF-neutralizing antibody 22E9. LPS instilled into the lungs of BALB/c mice induced a dose-dependent inflammation comprised of intense neutrophilia, macrophage infiltration and proliferation, TNF-alpha and matrix metalloproteinase release, and macrophage inflammatory protein-2 induction. The neutralization of anti-GM-CSF in a dose-dependent fashion suppressed these inflammatory indexes by 85% when given before or after LPS or after repeat LPS challenges. Here we report for the first time that the physiological expression of Toll-like receptor-4 in lung is reduced by anti-GM-CSF. We observed that lower Toll-like receptor-4 expression correlated with a similar decline in peak TNF- levels in response to endotoxin. Consequently, sustained expression of key inflammatory mediators over 24 h was reduced. These data expand the understanding of the contribution of GM-CSF to innate immune responses in lung and suggest that blocking GM-CSF might benefit some lung diseases where LPS has been implicated in etiology.     

A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor

BACKGROUNDS: Previously, we demonstrated that neutralizing capacity but not the concentration of GM-CSF autoantibody was correlated with the disease severity in patients with autoimmune pulmonary alveolar proteinosis (PAP)^1-3. As abrogation of GM-CSF bioactivity in the lung is the likely cause for autoimmune PAP^4,5, it is promising to measure the neutralizing capacity of GM-CSF autoantibodies for evaluating the disease severity in each patient with PAP.Until now, neutralizing capacity of GM-CSF autoantibodies has been assessed by evaluating the growth inhibition of human bone marrow cells or TF-1 cells stimulated with GM-CSF^6-8. In the bioassay system, however, it is often problematic to obtain reliable data as well as to compare the data from different laboratories, due to the technical difficulties in maintaining the cells in a constant condition.OBJECTIVE: To mimic GM-CSF binding to GM-CSF receptor on the cell surface using cell-free receptor-binding-assay.METHODS: Transgenic silkworm technology was applied for obtaining a large amount for recombinant soluble GM-CSF receptor alpha (sGMRα) with high purity^9-13. The recombinant sGMRα was contained in the hydrophilic sericin layers of silk threads without being fused to the silk proteins, and thus, we can easily extract from the cocoons in good purity with neutral aqueous solutions^14,15. Fortunately, the oligosaccharide structures, which are critical for binding with GM-CSF, are more similar to the structures of human sGMRα than those produced by other insects or yeasts.RESULTS: The cell-free assay system using sGMRα yielded the data with high plasticity and reliability. GM-CSF binding to sGMRα was dose-dependently inhibited by polyclonal GM-CSF autoantibody in a similar manner to the bioassay using TF-1 cells, indicating that our new cell-free assay system using sGMRα is more useful for the measurement of neutralizing activity of GM-CSF autoantibodies than the bioassay system using TF-1 cell or human bone marrow cells.CONCLUSIONS: We established a cell-free assay quantifying the neutralizing capacity of GM-CSF autoantibody.Download video file.^{(45M, mov)}     

鸡GM-CSF和IL-2增强表达新城疫病毒F蛋白的重组杆状病毒疫苗的免疫效果

为比较鸡粒细胞-巨噬细胞集落刺激因子(Granulocyte macrophage colony stimulating factor,GM-CSF)及鸡白细胞介素2(Interleukin 2,IL-2)对杆状病毒疫苗的免疫增强效果,通过基因工程手段构建重组杆状病毒疫苗(Recombinant Baculovirus,BV)rBV-LMI-F,并联合GM-CSF及IL-2进行鸡体免疫。对中和抗体水平及细胞因子含量比较GM-CSF和IL-2的免疫增强效果进行对比。结果显示,在第一次免疫28 d或42 d后,GM-CSF联合免疫组可诱导鸡体产生更高的抗体和细胞因子水平(P0.01)。表明鸡GM-CSF能更有效地刺激机体产生较强的抗体和细胞因子反应,提高重组杆状病毒疫苗的免疫效果。     

Induction of IL-4 release and upregulated expression of protease activated receptors by GM-CSF in P815 cells

GM-CSF has been showed to be able to induce up-regulated receptor and cytokine expression in mast cells in inflammatory conditions. However, little is known of its effects on protease activated receptor (PAR) expression and Th2 cytokine secretion from mast cells. In the present study, we examined potential influence of GM-CSF on mast cell PAR expression and IL-4 and IL-10 release by using flow cytometry analysis, quantitative real time PCR, ELISA and cellular activation of signaling ELISA (CASE) techniques. The results showed that GM-CSF induced up to 3.0-fold increase in IL-4 release from P815 cells, and FSLLRY-NH₂ and trans-cinnamoyl (tc)-YPGKF-NH₂ did not affect GM-CSF induced IL-4 release. GM-CSF reduced tryptase and trypsin induced IL-4 release by up to approximately 55.8% and 70.3%, respectively. GM-CSF elicited the upregulated expression of PAR-1, PAR-2, PAR-3 and PAR-4 mRNAs, but enhanced only PAR-4 protein expression in P815 cells. U0126, PD98059 and LY204002 almost completely abolished GM-CSF induced IL-4 release when they were preincubated with P815 cells for 30 min, indicating ERK and Akt cell signaling pathways may be involved in the event. In conclusion, GM-CSF can stimulate IL-4 release from mast cells through an ERK and Akt cell signaling pathway dependent, but PAR independent mechanism. GM-CSF may serve as a regulator for IL-4 production in mast cells and through which participates in the mast cell related inflammation.     

MLN51 and GM-CSF involvement in the proliferation of fibroblast-like synoviocytes in the pathogenesis of rheumatoid arthritis

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease of unclear etiology. This study was conducted to identify critical factors involved in the synovial hyperplasia in RA pathology. We applied cDNA microarray analysis to profile the gene expressions of RA fibroblast-like synoviocytes (FLSs) from patients with RA. We found that the MLN51 (metastatic lymph node 51) gene, identified in breast cancer, is remarkably upregulated in the hyperactive RA FLSs. However, growth-retarded RA FLSs passaged in vitro expressed small quantities of MLN51. MLN51 expression was significantly enhanced in the FLSs when the growth-retarded FLSs were treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or synovial fluid (SF). Anti-GM-CSF neutralizing antibody blocked the MLN51 expression even though the FLSs were cultured in the presence of SF. In contrast, GM-CSF in SFs existed at a significant level in the patients with RA (n = 6), in comparison with the other inflammatory cytokines, IL-1beta and TNF-alpha. Most RA FLSs at passage 10 or more recovered from their growth retardation when cultured in the presence of SF. The SF-mediated growth recovery was markedly impaired by anti-GM-CSF antibody. Growth-retarded RA FLSs recovered their proliferative capacity after treatment with GM-CSF in a dose-dependent manner. However, MLN51 knock-down by siRNA completely blocked the GM-CSF/SF-mediated proliferation of RA FLSs. Taken together, our results imply that MLN51, induced by GM-CSF, is important in the proliferation of RA FLSs in the pathogenesis of RA.     

A chimeric HIV-1 envelope glycoprotein trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain induces enhanced antibody and T cell responses

An effective HIV-1 vaccine should ideally induce strong humoral and cellular immune responses that provide sterilizing immunity over a prolonged period. Current HIV-1 vaccines have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the ability to induce an antibody response of sufficient quantity and quality. We hypothesized that Env immunogenicity could be improved by embedding an immunostimulatory protein domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of gp120. Probing with neutralizing antibodies showed that both the Env and GM-CSF components of the chimeric protein were folded correctly. Furthermore, the embedded GM-CSF domain was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric Env(GM-CSF) enhanced Env-specific antibody and T cell responses compared with wild-type Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within the protein can improve the immunogenicity of Env subunit vaccines.     

Natural and therapeutically-induced antibodies to cytokines

Serum samples obtained from non-immunocompromised patients treated therapeutically with recombinant cytokines (e.g. Il-1α; Il-1β; Il-2 to Il-18; IFNα; GM-CSF; G-CSF; etc.) often reveal the presence of high affinity anti-cytokine antibodies. Antibody Fab binding in a saturable manner by ELISA and RIA or western immunoblotting prove their specificity. Antibody level often increases in these patients with repeated cytokine administration, suggesting boosts of antigen stimulation. However, the appearance in circulation of auto-antibodies to exogenous cytokine is not always associated with a decreased clinical response to therapy. The demonstration that non-neutralizing auto-antibodies to several natural cytokines can be found even in sera of normal healthy individuals never treated before with cytokines and particularly during the last trimester of pregnancy and in cord-blood, suggests that these naturally- occurring and therapeutically-induced auto-antibodies may exert different functions, not only as inhibitors or antagonists but also as benificial physiological cytokine carriers or regulators of their activity.     

Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis

Evidence derived from human and animal studies strongly supports the notion that dysfunctional alveolar epithelial cells (AECs) play a central role in determining the progression of inflammatory injury to pulmonary fibrosis. We formed the hypothesis that impaired production of the regulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) by injured AECs plays a role in the development of pulmonary fibrosis. To test this hypothesis, we used the well-characterized model of bleomycin-induced pulmonary fibrosis in rats. GM-CSF mRNA is expressed at a constant high level in the lungs of untreated or saline-challenged animals. In contrast, there is a consistent reduction in expression of GM-CSF mRNA in the lung during the first week after bleomycin injury. Bleomycin-treated rats given neutralizing rabbit anti-rat GM-CSF IgG develop increased fibrosis. Type II AECs isolated from rats after bleomycin injury demonstrate diminished expression of GM-CSF mRNA immediately after isolation and in response to stimulation in vitro with endotoxin compared with that in normal type II cells. These data demonstrate a defect in the ability of type II epithelial cells from bleomycin-treated rats to express GM-CSF mRNA and a protective role for GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.     

Intracerebral administration of recombinant rabies virus expressing GM-CSF prevents the development of rabies after infection with street virus

Recently it was found that prior immunization with recombinant rabies virus (RABV) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) (LBNSE-GM-CSF) resulted in high innate/adaptive immune responses and protection against challenge with virulent RABV (Wen et al., JVI, 2011). In this study, the ability of LBNSE-GM-CSF to prevent animals from developing rabies was investigated in mice after infection with lethal doses of street RABV. It was found that intracerebral administration of LBNSE-GM-CSF protected more mice from developing rabies than sham-treated mice as late as day 5 after infection with street RABV. Intracerebral administration of LBNSE-GM-CSF resulted in significantly higher levels of chemokine/cytokine expression and more infiltration of inflammatory and immune cells into the central nervous system (CNS) than sham-administration or administration with UV-inactivated LBNSE-GM-CSF. Enhancement of blood-brain barrier (BBB) permeability and increases in virus neutralizing antibodies (VNA) were also observed in mice treated with LBNSE-GM-CSF. On the other hand, intracerebral administration with UV-inactivated LBNSE-GM-CSF did not increase protection despite the fact that VNA were induced in the periphery. However, intracerebral administration with chemoattractant protein-1 (MCP-1, also termed CCL2) increased significantly the protective efficacy of UV-inactivated LBNSE-GM-CSF. Together these studies confirm that direct administration of LBNSE-GM-CSF can enhance the innate and adaptive immunity as well as the BBB permeability, thus allowing infiltration of inflammatory cells and other immune effectors enter into the CNS to clear the virus and prevent the development of rabies.     

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

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