Enhanced CD16 expression associated with high TGF-ß1 and GM-CSF levels suggests polymorphonuclear cells activation in chronic rheumatic fever
Martha C. RiveroViviane Maria Montenegro TorresVilma dos Santos Trindade VianaCleide BellezaErnesto DallaverdeCristiane Y. FukudaMária KissRenato C. Monteiro
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Multiple sclerosis (MS) is an autoimmune disease of the CNS. Studies in animal models of MS have shown that GM-CSF produced by T cells is necessary for the development of autoimmune CNS inflammation. This suggests that GM-CSF may have a pathogenic role in MS as well, and a clinical trial testing its blockade is ongoing. However, there have been few reports on GM-CSF production by T cells in MS. The objective of this study was to characterize GM-CSF production by T cells of MS patients and to determine the effect of IFN-β therapy on its production. GM-CSF production by peripheral blood (PB) T cells and the effects of IFN-β were characterized in samples of untreated and IFN-β-treated MS patients versus healthy subjects. GM-CSF production by T cells in MS brain lesions was analyzed by immunofluorescence. Untreated MS patients had significantly greater numbers of GM-CSF(+)CD4(+) and CD8(+) T cells in PB compared with healthy controls and IFN-β-treated MS patients. IFN-β significantly suppressed GM-CSF production by T cells in vitro. A number of CD4(+) and CD8(+) T cells in MS brain lesions expressed GM-CSF. Elevated GM-CSF production by PB T cells in MS is indicative of aberrant hyperactivation of the immune system. Given its essential role in animal models, abundant GM-CSF production at the sites of CNS inflammation suggests that GM-CSF contributes to MS pathogenesis. Our findings also reveal a potential mechanism of IFN-β therapy, namely suppression of GM-CSF production.
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Abstract Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Studies in animal models of MS have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T cells is necessary for development of autoimmune CNS inflammation. This suggests that GM-CSF may have a pathogenic role in MS as well. The objective of this study was to characterize GM-CSF production by T cells of MS patients, and to determine the effect of interferon-beta (IFN-β) therapy on its production. GM-CSF production by peripheral blood (PB) T cells and the effects of IFN-β were characterized in samples of untreated and IFN-β-treated MS patients vs. healthy subjects. GM-CSF production by T cells in MS brain lesions was analyzed by immunofluorescence. Untreated MS patients had significantly greater numbers of GM-CSF+ CD4+ and CD8+ T cells in PB compared to healthy controls and IFN-β-treated MS patients. IFN-β strongly suppressed GM-CSF production by T cells in vitro. A number of CD4+ and CD8+ T cells in MS brain lesions expressed GM-CSF. Elevated GM-CSF production by PB T cells in MS is indicative of aberrant hyperactivity of the immune system. Given its essential role in animal models, abundant GM-CSF production at the sites of CNS inflammation suggests that GM-CSF contributes to MS pathogenesis. Our findings also reveal a potential mechanism of IFN-β therapy, namely suppression of GM-CSF production.
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Objective Granulocyte–macrophage colony stimulating factor (GM‐CSF) is a potent inflammatory mediator that is responsible for recruitment and activation of innate immune cells. Recent data from murine studies have identified Th17 cells as a key source of GM‐CSF and suggest that T cell–derived GM‐CSF is instrumental in the induction of autoimmune disease. The present study was undertaken to analyze the expression of T cell–derived GM‐CSF in the joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the differentiation of Th17 cells and how this relates to GM‐CSF+ T helper cells. Methods Synovial fluid (SF) and peripheral blood (PB) samples from 24 patients with JIA were analyzed, by flow cytometry and reverse transcription–polymerase chain reaction, for expression of GM‐CSF and the Th17 marker CD161. A cytokine capture assay was used to purify Th17 cells and test the plasticity of cytokine production in response to interleukin‐12 (IL‐12) and IL‐23. Results The frequency of GM‐CSF–producing T helper cells was significantly enriched in SF mononuclear cells compared to PB mononuclear cells from the patients with JIA (24.1% of CD4+ T cells versus 2.9%) and closely correlated with the erythrocyte sedimentation rate (r 2 = 0.91, P < 0.001). Synovial GM‐CSF+ T cells were predominantly CD161+ and coexpressed interferon‐γ (IFNγ), but not IL‐17. Culture of Th17 cells in the presence of IL‐12 led to rapid up‐regulation of GM‐CSF and IFNγ, recapitulating the phenotype of GM‐CSF–expressing cells within the joint. Conclusion Our results identify a novel outcome of Th17 plasticity in humans that may account for the enrichment of GM‐CSF–expressing T cells in the joints of patients with JIA. The association of GM‐CSF expression with systemic inflammation highlights the potential role of Th17‐related cytokines in the pathology of JIA.
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Background and Objectives
Th17 cells represent a subset of T helper lymphocytes that produces several inflammatory cytokines, including interleukin-17A, -17F, -21, -22, and tumor necrosis factor. Increased Th17 cell differentiation and IL-17 production have been observed in rheumatoid arthritis (RA) and in several other autoimmune diseases. IL-17 contributes to development of inflammation and promotes osteoclast differentiation in RA. We have studied the differentiation of Th17 cells.Materials and Methods
CD4 positive T cells were negatively separated by magnetic method from peripheral blood mononuclear cells (PBMC) of healthy volunteers. The cells were treated for 5-10 days with anti-CD3 and anti-CD28 antibodies and with TGFβ (2.5ng/ml), IL-6 (25ng/ml) and IL-1 (10ng/ml) cytokines, furthermore with anti-IL-4 (10μg/ml) and anti-IFNγ (10μg/ml) blocking antibodies. The IL-17 and IL-22 production was measured by ELISPOT and ELISA, the RORγt expression was measured by real-time PCR and by Western blot methods, cell viability was monitored by Trypan blue staining and by Annexin V binding.Results
Anti-CD3/CD28 treatment increased the IL-17 production, but did not alter the RORγt expression. The anti-CD3/CD28, TGFβ, IL-6, and IL-1 induced RORγt expression was further increased by the anti-IL-4 and anti-IFNγ antibody treatment. Anti-CD3/CD28-induced IL-22 production was inhibited by TGFβ, IL-6, IL-1, anti-IL-4 and anti-IFNγ antibody treatment. The applied treatments did not change the viability of the cells.Conclusion
Our present results suggest that IL-17 and IL-22 production are differentially regulated during CD4 T cell activation and Th17 differentiation.ELISPOT
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Canine Steroid-responsive
Meningitis-Arteritis (SRMA) is a systemic
inflammatory disease with predominant manifestation within the cervical meninges. Laboratory characteristics include simultaneous
elevation of immunoglobulin A (IgA) concentrations
in serum and cerebrospinal fluid (CSF) and marked neutrophilic
pleocytosis. The first part of the presented study
addressed the hypothesis that increased IgA levels
are due to a Th2-dominated immune response. In a second and third part
factors potentially facilitating neutrophil
migration into the CSF were investigated. These included leukocytic
matrix metalloproteinase (MMP) expression and b2 integrin
expression on peripheral blood polymorphonuclear
cells (PBPMNs). Whereas MMPs
have the potential to induce blood-CSF-barrier permeabilization,
b2 integrins are
responsible for neutrophil-endothelium-interaction,
which represents a crucial event for subsequent extravasation.
Throughout the study
values deriving from dogs in the acute phase of SRMA were compared to those
of dogs with SRMA under glucocorticosteroid
treatment and individuals suffering from various neurological disorders. To
detect Th2-dominance, pellets containing peripheral blood mononuclear cells (PBMNCs) and CSF white blood cells (CSF WBCs) were examined for their cytokine mRNA expression.
Cytokines that were investigated included Th1-associated interleukin (IL)-2
and interferon (IFN)-g as well as Th2 signature cytokines IL-4, -5, and -10. All results
were normalized to the expression of 3 reference genes. A similar approach
allowed quantitation of MMP-2 and -9 expression in PBMNCs, PBPMNs and CSF WBCs. In
addition, mRNA expression for tissue inhibitors of metalloproteinases
(TIMP)-1 and -2 was investigated. Beta2integrin
expression (CD11a, CD11b, CD11c) on PBPMNs was quantitated by means
of indirect immunophenotyping and subsequent flow cytomentry.
The immune response occurring
in SRMA is characterized by high levels of IL-4 and low IL-2 and IFN-g expression, whereas IL-5 and -10
levels are similar among all disease categories. These results are consistent
with a Th2 skewed immune reaction in the acute phase of SRMA, which may well
explain previous findings of high IgA and B cell
levels. In addition, increased IL-10 levels are found in CSF WBCs of affected dogs. This may reflect a
counter-regulatory reaction that prevents involvement and damage of the
central nervous system (CNS) parenchyma. Cerebrospinal fluid WBCs produce both, MMP-2 and -9, suggesting their
contribution to leukocyte migration into the subarachnoidal
space by disruption of the blood-CSF-barrier. These cells additionally
produce TIMP-1 and -2, which may represent another mechanism that prevents
destruction of the adjacent CNS parenchyma. Investigation of the b2 integrin
expression showed that selective CD11a expression occurs in SRMA, whereas
CD11b and CD11c expression remains unchanged. The surface molecule CD11a is
necessary for leukocyte adhesion to endothelial cells and up-regulation is
likely to display another mechanism that contributes to high CSF neutrophil counts in the acute phase of SRMA.
In conclusion, our study
reveals that SRMA is associated with a Th2-dominated immune response and that
facilitation of leukocyte migration into the subarachnoidal
space is mediated by MMP-2 and -9 production as well as CD11a up-regulation.
Pleocytosis
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Behçet's disease (BD) is an inflammatory disease mainly affecting men along the ancient Silk Route. In the present study we describe a Dutch family suffering from BD-like disease with extreme pathergic responses, but without systemic inflammation. Genetic assessment revealed a combination of the human leukocyte antigen (HLA)-B*51 risk-allele together with a rare heterozygous variant in the CSF2 gene (c.130A>C, p.N44H) encoding for granulocyte-macrophage colony-stimulating factor (GM-CSF) found by whole exome sequencing. We utilized an over-expression vector system in a human hepatocyte cell line to produce the aberrant variant of GM-CSF. Biological activity of the protein was measured by signal transducer and activator of transcription 5 (STAT-5) phosphorylation, a downstream molecule of the GM-CSF receptor, in wild-type peripheral mononuclear cells (PBMCs) using flow cytometry. Increased STAT-5 phosphorylation was observed in response to mutated GM-CSF when compared to the wild-type or recombinant protein. CSF2 p.N44H results in disruption of one of the protein's two N-glycosylation sites. Enzymatically deglycosylated wild-type GM-CSF also enhanced STAT-5 phosphorylation. The patient responded well to anti-tumor necrosis factor (TNF)-α treatment, which may be linked to the capacity of TNF-α to induce GM-CSF in phorbol 12-myristate 13-acetate (PMA)-treated PBMCs, while GM-CSF itself only induced dose-dependent interleukin (IL)-1Ra production. The identified CSF2 pathway could provide novel insights into the pathergic response of BD-like disease and offer new opportunities for personalized treatment.
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Objective Toinvestigate apoptosis and expression of mRNA encoding IL－ 5 and GM－ CSF of peripheral lymphatic cell of asthmatic guinea pig.Method 30 guinea pigs were equally aasigned to asthmatic or control group. tumel was used to determine the rate of apoptosis and in situ hybridization(ISH)was used to examine the expression of mRNA encoking IL－ 5 and GM－ CSF of perpheral lymphatic cell. Result the rate of apoptosis in asthmatic group(3.50± 1.00％ )was much lower than that in control group (6.80± 1.50％ )(p0.01).the positive rates of expression of mena encoding IL－ 5 and GM－ CSF in asthmatic group(58.00± 6.00％ ,49.00± 4.00％ )were much higher than that in control group(30.00± 2.00％ ,24.00± 2.00％ ).Conclusion The hyper－ expression of mRNA encoding IL－ 5 and GM－ CSF in perpheral lymphatic cell may be related with the activation as well as the decrease of apoptosis of lymphatic cell,which induced more secretion of cytokine.
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Abstract Introduction:Two different subsets of monocytes, classical(CD14+CD16-) and inflammatory(CD14+CD16+) monocytes have been identified. Recently, increased intermediate(CD14++CD16+) monocyte(Int-mo) and its decrease after treatment with anti-M-CSF antibody was reported in rheumatoid arthritis(RA), although the mechanism and involvement in RA remains unknown. The aim of this study is to elucidate the role of classical/inflammatory/intermediate monocytes in RA pathogenesis. Methods:Serum cytokines were measured by ELISA, CD14/CD16 expression was measured by flow cytometer and classical monocyte was sorted for in vitro culture. Results:Proportion of Int-mo, serum IL-6, IL-8, IL-10 but not M-CSF was increased in RA patients compared to healthy control subjects. Disease activity of RA positively correlated with serum IL-6, IL-10 and proportion of Int-mo but not with IL-8 and M-CSF. When sorted classical monocytes were stimulated with each cytokine, IL-10 and M-CSF but not other cytokines induced CD16 expression. Interestingly, CD16 expression was strongly induced by IL-10 at 14 hours whereas 72 hours was necessary for M-CSF. Discussion:Int-mo positively correlated with RA disease activity and was induced by IL-10 and M-CSF but not with other cytokines. Since, M-CSF is reported to induce IL-10 production and both cytokines are up-regulated in RA patients, our results suggest that IL-10 is the key cytokine that regulate Int-mo and presumably contribute to the anti-inflammatory process.
CD16
Monocyte
Pathogenesis
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Multiple sclerosis (MS) is thought to be a Th17-mediated dysimmune disease of the central nervous system. However, recent publications have questioned the pathogenicity of IL-17 per se and rather suggest the implication of other Th17-related inflammatory mediators. Therefore, we studied the expression of GM-CSF, IL-22, IL-24, IL-26 and CD39 in peripheral blood mononuclear cells (PBMCs) from MS patients during relapses, remission and following corticosteroid treatment. We performed qPCR to measure mRNA levels from ex vivo or in vitro-stimulated PBMCs. Cytokine levels were determined by ELISA. We used flow cytometry to assess GM-CSF+, IL-22+ and CD39+ cells in relationship to IL-17+ CD4+ T cells. Our results showed that IL-22 mRNA and IL-22+CD4+ lymphocytes are increased in circulating cells of relapsing MS patients compared to remitting patients while GM-CSF was unchanged. We have further shown that 12.9, 39 and 12.4% of Th17 cells from MS patients during relapses expressed IL-22, GM-CSF and CD39 respectively. No changes in these proportions were found in stable MS patients. However, the majority of GM-CSF+ or IL-22+ T cells did not co-express IL-17. GM-CSF mRNA, but not IL-22 mRNA, was dramatically decreased ex vivo by ivMP. Our results contribute to a better characterisation of Th17, Th22 and ThGM-CSF cells in the setting of MS and according to disease activity.
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