Spontaneously occurring neutralizing antibodies against granulocyte–macrophage colony-stimulating factor in patients with autoimmune disease

There is increasing evidence that spontaneous anticytokine autoantibodies are associated with chronic infections and autoimmune diseases. We report the sporadic occurrence in autoimmune diseases of such autoantibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine involved in inflammation and the regulation of proliferation, differentiation and function of granulocytic and monocytic cell lineages. In 41 of 425 patients tested, we found low to moderate levels of autoantibodies binding to GM-CSF in serum or plasma. These were most prevalent in patients with myasthenia gravis (MG). However, neutralizing autoantibodies against GM-CSF were very rare, being found in only three patients. Two had autoimmune MG, one with thymoma (Patient A) and the other (Patient B) with 'seronegative' MG, i.e. without the antiacetylcholine receptor autoantibodies characteristic of most MG patients, and a third (Patient D) had multiple sclerosis. Only very limited amounts of Patient A and Patient D serum/plasma were available for analysis and therefore further studies were carried out on the more plentiful samples from Patient B. The anti-GM-CSF autoantibodies of Patient B were predominantly polyclonal immunoglobulin G and strongly neutralized recombinant human (rh) GM-CSF derived from different expression systems. They had similar immunological and immunochemical characteristics to anti-GM-CSF antibodies that developed in immunocompetent colorectal carcinoma patients following (rh)GM-CSF therapy. In serial samples from Patient B, the anti-GM-CSF autoantibodies were undetectable from diagnosis at age 8 years until at least age 13, but then developed spontaneously during (temporary) withdrawal of immunosuppressive treatment. Their neutralizing activity has persisted since their first detection at age 15 years 1 month, and was at its highest level recently at age 17 years 7 months. There was no obvious association with other autoimmune phenomena, nor were any haematological deficiencies overtly manifested, suggesting that any loss of GM-CSF function may have been compensated for by other cytokines.     

Human monocyte-derived dendritic cells produce macrophage colony-stimulating factor: enhancement of c-fms expression by interleukin-10

Human monocyte-derived dendritic cells (DC) generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 express c-fms (CD115), the receptor for macrophage-CSF (M-CSF). Expression of c-fms on monocyte-derived DC has been interpreted as the susceptibility of these cells to M-CSF-induced macrophage development. We show here that homogenous cultures of CD14⁻ DC constitutively produced large amounts of M-CSF. However, presence of M-CSF neither induced macrophage development nor did it prevent terminal maturation into CD83⁺ DC. M-CSF production by DC was driven by GM-CSF and inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin. M-CSF synthesis was rapidly induced during the first 24 h of DC culture and then declined during the 5-day culture period. Replating of the cells, which was associated by a transient adherence, always induced a strong up-regulation of M-CSF synthesis. Addition of recombinant IL-10 to DC cultures enhanced c-fms expression and induced macrophage development as measured by the strong up-regulation of CD14 expression as well as by enhanced expression of the Fcγ receptors I, II, and III (CD64, CD32, CD16). Our data demonstrate that immature monocyte-derived DC produce M-CSF which does not induce macrophage development, despite the surface expression of c-fms on DC. IL-10 appears to induce macrophage development by up-regulating c-fms and, thereby, enhancing the sensitivity of the cells to endogenously produced M-CSF.     

Regulation of the cellular subpopulation ratios of normal human endometrial stromal cells by macrophage colony-stimulating factor

Normal human endometrial stromal cells (ESCs) stimulated with 8-Br-cAMP secrete significantly large amounts of PRL and granulocyte colony-stimulating factor (G-CSF), whereas unstimulated stromal cells secreted little. However, there is no relation between PRL and G-CSF levels secreted from the stimulated cells, which suggests that PRL-secreting stromal cells may not completely coincide with the G-CSF-producing stromal cells. Recently we found that macrophage colony-stimulating factor (M-CSF) inhibits 8-Br-cAMP-induced decidualization, differentiation to prolactin (PRL)-secreting cells, by suppressing viable decidualizing cells. Therefore, we investigated the effects of M-CSF on G-CSF secretion from normal human endometrial stromal cells using an in vitro decidualization activity assay. M-CSF did not show any significant effects on viable cell numbers of unstimulated ESCs, while M-CSF dose-dependently enhanced G-CSF release from the non-decidualized stromal cells. A high concentration of M-CSF strongly suppressed the viable cell numbers and PRL secretion of stromal cells co-stimulated with 8-Br-cAMP and M-CSF, although G-CSF release from the co-stimulated stromal cells was not affected by M-CSF. Moreover, M-CSF did not affect viable cell numbers, PRL secretion or G-CSF secretion of 8-Br-cAMP-stimulated cells. These results indicate that M-CSF enhances G-CSF secretion from 8-Br-cAMP-unstimulated human endometrial stromal cells but not from 8-Br-cAMP-stimulated stromal cells, thus suggesting that there exists a functional subpopulation of G-CSF-secreting stromal cells that are different from the predecidualized ESCs that differentiate into PRL-secreting ESCs under stimuli with 8-Br-cAMP. Hence, M-CSF may autoregulate functional cellular subpopulations of human endometrial stromal cells in an autocrine or a paracrine manner.     

Characterization of a macrophage lineage cell colony-stimulating factor produced by thymic myoid cells.

Thymic myoid cells produced macrophage lineage cell stimulatory factors. Activities were separated into two factors on DEAE-Sepharose CL-6B chromatography: one eluted at lower concentrations of NaCl and the other at higher concentrations of NaCl. The latter fraction was purified to homogeneity with an apparent molecular weight of 100,000. This factor stimulated the growth of macrophage-lineage cells from the bone marrow, but not that of granulocytes, megakaryocytes or erythroblasts. The 100,000 MW factor was able to induce Ia antigens on proliferating bone marrow cells. These results suggest that myoid cell-derived 100,000 MW factor plays significant roles in the generation of Ia-positive macrophage lineage cells which are important for T-cell development in the thymus.     

Evaluation of a genetically modified reassortant H5N1 influenza A virus vaccine candidate generated by plasmid-based reverse genetics

Avian influenza A H5N1 viruses similar to those that infected humans in Hong Kong in 1997 continue to circulate in waterfowl and have reemerged in poultry in the region, raising concerns that these viruses could reappear in humans. The currently licensed trivalent inactivated influenza vaccines contain hemagglutinin (HA) and neuraminidase genes from epidemic strains in a background of internal genes derived from the vaccine donor strain, A/Puerto Rico/8/34 (PR8). Such reassortant candidate vaccine viruses are currently not licensed for the prevention of human infections by H5N1 influenza viruses. A transfectant H5N1/PR8 virus was generated by plasmid-based reverse genetics. The removal of the multibasic amino acid motif in the HA gene associated with high pathogenicity in chickens, and the new genotype of the H5N1/PR8 transfectant virus, attenuated the virus for chickens and mice without altering the antigenicity of the HA. A Formalin-inactivated vaccine prepared from this virus was immunogenic and protected mice from subsequent wild-type H5N1 virus challenge. This is the first successful attempt to develop an H5N1 vaccine seed virus resembling those used in currently licensed influenza A vaccines with properties that make it a promising candidate for further evaluation in humans.     

Production of macrophage colony-stimulating factor by adult murine parenchymal liver cells (hepatocytes).

The activity of macrophage colony-stimulating factor (M-CSF) was found in the culture supernatant of mouse parenchymal liver cell fractions in a bone marrow colony-forming assay. The activity of an M-CSF-like substance purified by a four-step procedure was neutralized by goat anti-mouse M-CSF antiserum. M-CSF mRNA was detected in cellular RNA prepared from cultured parenchymal liver cell fractions by Northern blot analysis and also in cultured parenchymal liver cells by in situ hybridization. These results indicate that parenchymal liver cells have the capacity to produce M-CSF. We discuss the role of M-CSF in hematopoiesis, the immune response, and other biological phenomena.     

Dendritic cell vaccine therapy by immunization with fusion cells of interleukin-2 gene-transduced, spleen-derived dendritic cells and tumour cells

We examined the preventive and therapeutic effects of fusion cells prepared from spleen-derived dendritic cells (DCs) transduced with the interleukin-2 (IL-2) gene and QRsP fibrosarcoma cells in a mouse lung metastasis model. The IL-2 or LacZ gene was introduced into spleen-derived DCs using an adenoviral vector. Irradiated QRsP tumour cells were fused with IL-2 gene-transduced DCs (fusion/IL-2) or LacZ gene-transduced DCs (fusion/LacZ) by polyethylene glycol. These fusion cells expressed major histocompatibility complexes (MHC) class I and II, CD86, CD11c and CD8alpha. Splenocytes from mice vaccinated with fusion cells showed increased production of interferon-gamma (IFN-gamma) and cytotoxic T-lymphocyte (CTL) activity as compared with those vaccinated with DCs or tumour cells alone, and CTL levels were higher in fusion/IL-2-vaccinated mice than in fusion/LacZ-vaccinated mice. In our experiments on the protective and therapeutic effects on lung metastasis, mice vaccinated with fusion/IL-2 fusion/LacZ or fusion showed a significant reduction in pulmonary metastasis compared with those given DCs, tumour or phosphate-buffered saline. The introduction of the IL-2 gene into fusion cells produced more potent preventive and therapeutic effects. These results suggest that immunization with fusion cells prepared from spleen-derived DCs and tumour cells is capable of inducing preventive and therapeutic anti-tumour immunity against lung metastasis, and modification by the IL-2 gene may increase anti-tumour efficacy.     

Granulocyte-macrophage colony-stimulating factor induces a unique set of STAT factors in murine dendritic cells

Cytokine-mediated signaling pathways were studied in mouse dendritic cells (DC) by analysis of the activation pattern of STAT factors. Electrophoretic mobility shift assays were performed to detect STAT isoform-specific complexes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) simultaneously induced complexes containing STAT1, STAT3, STAT5A, STAT5B and STAT6. In non-DC, a similar broad activation pattern of STAT factors by GM-CSF or other cytokines has not been observed so far. By comparison, in peritoneal macrophages, GM-CSF induced a complex with the properties of a truncated form of STAT5. Other cytokines tested on DC either failed to induce STAT factors [interleukin (IL)-1β, IL-2, IL-15], or activated the same STAT factors as observed in peritoneal macrophages (IL-4, IFN-γ). Our results implicate a specific effect of GM-CSF on STAT signaling in DC which might account for the cell type-specific effect of this cytokine on development and function.     

Colonization of in vitro-formed cervical human papillomavirus- associated (pre)neoplastic lesions with dendritic cells: role of granulocyte/macrophage colony-stimulating factor

The purpose of this study was to investigate the ability of CD1a+ Langerhans/dendritic cells (LCs/DCs) to infiltrate human papillomavirus (HPV)-associated (pre)neoplastic lesions of the uterine cervix. Migration of LCs/DCs in the presence of keratinocytes derived from normal cervix and HPV-transformed cell lines was evaluated in Boyden chambers and in organotypic cultures and correlated with granulocyte/macrophage colony-stimulating factor (GM-CSF) production by the cells, as determined by ELISA. Conditioned media of HPV-transformed keratinocytes contained lower amounts of GM-CSF and induced a decreased motile response of LCs/DCs in the Boyden chamber assay compared with those of normal cervical keratinocytes. The migration of LCs/DCs in the presence of conditioned media from normal keratinocytes could be blocked by an anti-GM-CSF antibody, and the migration of LCs/DCs in the presence of conditioned media from HPV-transformed keratinocytes could be increased by supplementing the media with recombinant GM-CSF. GM-CSF was also a potent factor in enhancing the colonization of LCs/DCs into organotypic cultures of HPV-transformed keratinocytes, as the infiltration of LCs/DCs in the in vitro-formed (pre)neoplastic epithelium was minimal under basal conditions and dramatically increased after the addition of GM-CSF to the cultures. These results suggest that GM-CSF could play an important role in the recruitment of LCs/DCs into the HPV-transformed (pre)neoplastic cervical epithelium and be useful as a new immunotherapeutic approach for cervical (pre)cancers.     

Production of specific antibody and T helper 1-dominant cytokine elicited by dendritic cells genetically modified with an adenovirus vector

In this study we examined how dendritic cells (DCs) transduced with an adenovirus vector encoding a model tumor antigen (beta-galactosidase; beta-gal) would influence the humoral immune response to this antigen. Mice immunized with LacZ transduced DCs by an adenovirus vector could produce more anti-beta-gal antibody, especially of IgG2a subclass, than mice immunized with DCs alone, although the amount of serum IgG antibody did not increase. Compared with mice immunized with DCs alone, splenocytes of mice immunized with LacZ transduced DCs could produce more interferon-gamma (IFN-gamma) against the beta-gal derived, H-2Ld-restricted nonapeptide (TPHPARIGL) in a dose-dependent manner. These data suggest that DCs transduced with an adenovirus vector encoding the model tumor antigen could induce the T helper 1 (Th1) dominant response against the model tumor antigen.     

Feasibility to generate monocyte-derived dendritic cell from coculture with melanoma tumor cells in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4

OBJECTIVE: We investigated how melanoma cells and membrane-bound granulocyte/macrophage colony stimulating factor (mbGM-CSF) melanoma cell lines affect the differentiation of dendritic cells (DC) from CD14+ monocytes. METHOD OF STUDY: The malignant melanoma cell lines (Conley and Jorp) and mbGM-CSF-positive cell lines (Conley B-F8 and Jorp C-E6) were cultured and cell-free supernatants were collected from each cell line cultures to assess the GM-CSF level. Adherent monocytes were cocultured for 6-7 days with irradiated mbGM-CSF and wild type melanoma cells (50 Gy) at each 1:1 and 0.1:1 ratio in six-well culture plates in ex vivo culture medium containing interleukin (IL)-4. Immunophenotyping was performed by triple color immunofluorescence staining with flow cytometry analysis. RESULTS: GM-CSF was detected at low levels in the culture supernatants of Conley B-F8 (0.48 ng/10(6) cells/24 hr), whereas there was no detectable GM-CSF in that of Conley melanoma cell line. Monocytes cultured with GM-CSF/IL-4 generated the expression of high levels of HLA DR, CD1a and CD86, while the expression of CD14 and CD83 were in low amounts. However, the addition of GM-CSF to these cultures resulted in an increased expression of these markers and decreased that of CD14. Monocytes cocultured with Jorp C-E6 illustrated similar expression pattern of CD1a, HLA DR and CD14 in the presence or absence of GM-CSF as Conley B-F8 melanoma cell line. Monocytes cocultured with Conley B-F8 melanoma cells at 1:1 and 0.1:1 ratio showed no significant difference in expression of CD1a, CD14 and CD83 between the two ratios. CONCLUSION: Our results illustrate the feasibility to generate monocyte-derived DC from coculture with melanoma tumor cells in the presence of GM-CSF and IL-4. However, mbGM-CSF tumor cells did not significantly enhance the DC differentiation through juxtacrine stimulation unless soluble GM-CSF was added in the culture media.     

肝癌细胞增殖受M-CSF胞内和胞外自分泌的双重调控

目的：研究胞内M-CSF及其受体在肝癌SMMC 7721细胞的表达与性质,探讨胞内M-CSF对SMMC 7721细胞增殖的影响及其机制。 方法： 以高表达M-CSF的人肝癌细胞系（SMMC 7721细胞）为模型，以免疫组化、流式细胞计数、反义技术与蛋白印迹等方法观测胞内M-CSF对SMMC 7721细胞增殖的影响及其机制。 结果： M-CSF 及其受体主要在SMMC 7721细胞的胞质、胞核中表达，胞内的M-CSF的相对分子量为20 000，M-CSFR的相对分子量为120 000；免疫共沉淀分析证明M-CSF在细胞内与M-CSFR以复合物的形式存在；M-CSF的单克隆抗体及其反义寡聚核苷酸能抑制SMMC 7721细胞的增殖、下调cyclinD1/E的表达和上调p16的表达，且M-CSF的单克隆抗体及其反义寡聚核苷酸的联合使用能进一步加强对SMMC 7721细胞抑制作用和增加下调cyclinD1/E和上调p16的表达幅度。 结论： SMMC 7721细胞受M-CSF胞外自分泌和胞内自分泌的双重调控。     

Successful granulocyte‐colony stimulating factor treatment of Crohn's disease is associated with the appearance of circulating interleukin‐10‐producing T cells and increased lamina propria plasmacytoid dendritic cells

Granulocyte‐colony stimulating factor (G‐CSF) has proved to be a successful therapy for some patients with Crohn's disease. Given the known ability of G‐CSF to exert anti‐T helper 1 effects and to induce interleukin (IL)‐10‐secreting regulatory T cells, we studied whether clinical benefit from G‐CSF therapy in active Crohn's disease was associated with decreased inflammatory cytokine production and/or increased regulatory responses. Crohn's patients were treated with G‐CSF (5 µg/kg/day subcutaneously) for 4 weeks and changes in cell phenotype, cytokine production and dendritic cell subsets were measured in the peripheral blood and colonic mucosal biopsies using flow cytometry, enzyme‐linked immunosorbent assay and immunocytochemistry. Crohn's patients who achieved a clinical response or remission based on the decrease in the Crohn's disease activity index differed from non‐responding patients in several important ways: at the end of treatment, responding patients had significantly more CD4⁺ memory T cells producing IL‐10 in the peripheral blood; they also had a greatly enhanced CD123⁺ plasmacytoid dendritic cell infiltration of the lamina propria. Interferon‐γ production capacity was not changed significantly except in non‐responders, where it increased. These data show that clinical benefit from G‐CSF treatment in Crohn's disease is accompanied by significant induction of IL‐10 secreting T cells as well as increases in plasmacytoid dendritic cells in the lamina propria of the inflamed gut mucosa.     

p40/LAIR-1 regulates the differentiation of peripheral blood precursors to dendritic cells induced by granulocyte-monocyte colony-stimulating factor

p40/LAIR-1, a member of the immunoglobulin superfamily, is a surface molecule broadly distributed among leukocytes which has been shown to down-regulate T and NK cell activation. In this study, we show that p40/LAIR-1 is highly expressed in CD14⁺ peripheral blood mononuclear cells (PBMC). When cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) for 10 – 14 days, CD14⁺ cells acquired morphologic and phenotypic features ( i.e. loss of CD14 and expression of CD80^bright and CD86^bright ) typical of dendritic cells (DC) and lost the expression of p40/LAIR-1. Engagement of p40/LAIR-1 (but not of CD58) by specific monoclonal antibodies prevented CD14⁺ PBMC differentiation into DC; when cultured in the presence of GM- CSF upon p40/LAIR-1 cross-linking, the resulting cells were CD14⁺ CD80^dull CD86^dull and displayed a macrophage-like morphology. We have recently demonstrated that peripheral blood CD14⁺ cells co-expressing the CD34 progenitor marker represent the circulating precursors of CD83⁺ DC. Herein we show that cross-linking of p40/LAIR-1 prevented the maturation of CD14⁺ CD34⁺ cells into CD83⁺ DC. This effect appears to be consequent to the impairment of GM-CSF receptor-mediated activation signaling. Indeed, triggering of GM-CSF receptors in both CD14⁺ and CD14⁺ CD34⁺ cells led to increases in the intracellular free calcium concentrations which were inhibited by p40/LAIR-1 engagement. Taken together, these data suggest a possible regulating role played by p40/LAIR-1 in the process of differentiation from peripheral blood precursors into DC induced by GM-CSF.     

Size fractions of ambient particulate matter induce granulocyte macrophage colony-stimulating factor in human bronchial epithelial cells by mitogen-activated protein kinase pathways

Environmental pollutants, including ambient particulate matter (PM), increase respiratory morbidity. Studies of model PM particles, including residual oil fly ash and freshly generated diesel exhaust particles, have demonstrated that PM affects inflammatory airway responses. Neither of these particles completely represents ambient PM, and therefore questions remain about ambient particulates. We hypothesized that ambient PM of different size fractions collected from an urban environment (New York City air), would activate primary culture human bronchial epithelial cells (HBECs). Because of the importance of granulocyte-macrophage colony-stimulating factor (GM-CSF) on inflammatory and immunomodulatory processes, we focused our studies on this cytokine. We demonstrated that the smallest size fraction (ultrafine/fine; < 0.18 micro m) of ambient PM (11 micro g/cm(2)), upregulated GM-CSF production (2-fold increase). The absence of effect of carbon particles of similar size, and the day-to-day variation in response, suggested that the chemical composition, but not the particle itself, was necessary for GM-CSF induction. Activation of the extracellular signal-regulated kinase and the p38 mitogen-activated protein kinase was associated with, and necessary for, GM-CSF release. These studies serve to corroborate and extend those on model particles. Moreover, they emphasize the role of the smallest size ambient particles in airway epithelial cell responses.     

Evaluation of the effectiveness and safety of a genetically modified live vaccine in broilers challenged with Salmonella Heidelberg

Salmonellosis ranks among the major diseases of commercial poultry, and its presence in poultry flocks is responsible for economic losses and risks related to public health. Vaccines are an important tool within integrated programmes to control salmonellosis. The purpose of this study was to assess cross-protection provided by the Poulvac® ST vaccine in the control of Salmonella Heidelberg in experimentally challenged 3- and 21-day-old birds. Eighty birds were identified and separated into four treatments (T1: vaccinated and challenged at 3 days of age, T2: unvaccinated and challenged at 3 days of age, T3: vaccinated and challenged at 21 days of age, and T4: unvaccinated and challenged at 21 days of age). The inoculum was produced from a Brazilian field strain of SH. At the end of the experiment, caecum and liver/spleen samples were collected for quantitative and qualitative analysis of SH, respectively. Analysis of the liver/spleen showed that Poulvac® ST significantly (P?≤?0.05) reduced the percentage of SH positivity in the group challenged at 3 days of age, while in the group challenged at 21 days this difference was almost considered significant (P?=?0.1818). On the other hand, there was no statistically significant difference in SH count in the caecum (CFU/g) in the group challenged at 3 days, but for the group challenged at 21 days the SH counts were significantly (P?≤?0.05) lower in the vaccinated group when compared to the positive control.