RESUMO
BACKGROUND: TNF-like ligand 1A (TL1A), a recently recognized member of the TNF superfamily, and its death domain receptor 3 (DR3), firstly identified for their relevant role in T lymphocyte homeostasis, are now well-known mediators of several immune-inflammatory diseases, ranging from rheumatoid arthritis to inflammatory bowel diseases to psoriasis, whereas no data are available on their involvement in sarcoidosis, a multisystemic granulomatous disease where a deregulated T helper (Th)1/Th17 response takes place. METHODS: In this study, by flow cytometry, real-time PCR, confocal microscopy and immunohistochemistry analyses, TL1A and DR3 were investigated in the pulmonary cells and the peripheral blood of 43 patients affected by sarcoidosis in different phases of the disease (29 patients with active sarcoidosis, 14 with the inactive form) and in 8 control subjects. RESULTS: Our results demonstrated a significant higher expression, both at protein and mRNA levels, of TL1A and DR3 in pulmonary T cells and alveolar macrophages of patients with active sarcoidosis as compared to patients with the inactive form of the disease and to controls. In patients with sarcoidosis TL1A was strongly more expressed in the lung than the blood, i.e., at the site of the involved organ. Additionally, zymography assays showed that TL1A is able to increase the production of matrix metalloproteinase 9 by sarcoid alveolar macrophages characterized, in patients with the active form of the disease, by reduced mRNA levels of the tissue inhibitor of metalloproteinase (TIMP)-1. CONCLUSIONS: These data suggest that TL1A/DR3 interactions are part of the extended and complex immune-inflammatory network that characterizes sarcoidosis during its active phase and may contribute to the pathogenesis and to the progression of the disease.
RESUMO
Polymorphonuclear leukocytes (PMN) have been identified as cells capable of producing a number of pro- and antiinflammatory cytokines in response to specific agonists. Previously, we showed that tumor necrosis factor (TNF), interleukin-1 beta (IL-1 beta), and IL-8, are produced by PMN after stimulation with agonists, such as lipopolysaccharide (LPS). In this study, we demonstrate that LPS is also a potent stimulus for the mRNA expression and release of the IL-1 receptor antagonist (IL-1ra). In addition, we show that the release of IL-1ra from LPS-stimulated PMN is markedly potentiated in the presence of IL-10 (from two to threefold after 18 h of stimulation). Moreover, we observed that this upregulation of IL-1ra production by IL-10 in LPS-stimulated PMN took place through IL-1ra mRNA stabilization. Indeed, the half-life of IL-1ra mRNA was prolonged in PMN stimulated in the presence of IL-10 and LPS, as compared with cells stimulated with LPS alone. That IL-10 selectively upregulates IL-1ra production in LPS-activated PMN, while it inhibits the production of IL-1 beta, TNF, and IL-8 under the same conditions, suggests that IL-10 may be an important physiologic regulator of cytokine production from PMN, and emphasizes the potential role of IL-10 in inflammatory responses.
Assuntos
Interleucina-10/metabolismo , Lipopolissacarídeos/farmacologia , Neutrófilos/metabolismo , Receptores de Interleucina-1/antagonistas & inibidores , Sialoglicoproteínas/biossíntese , Células Cultivadas , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
Innate lymphoid cells (ILCs) have a central role in innate defenses against pathogens, lymphoid organogenesis, and tissue remodeling. They have been detected in human decidua, however, their role in this tissue remains unclear. Successful pregnancy requires an early inflammatory phase favoring implantation and tissue remodeling as well as a subsequent regulatory phase to prevent fetal rejection and supporting neoangiogenesis. Here, we show that, during the first trimester of pregnancy, neutrophils infiltrate decidua basalis and are more abundant in normal pregnancy than in spontaneous miscarriages. Decidual neutrophils localize in proximity of NCR+ILC3, which may influence neutrophil migration and survival given their production of CXCL8 and granulocyte macrophage colony-stimulating factor (GM-CSF). Moreover, NCR+ILC3-derived GM-CSF was found to induce the expression of heparin-binding EGF-like growth factor and IL1ra in neutrophils, two proteins/cytokines involved in tissue remodeling and maintenance of pregnancy. Our data suggest that the simultaneous presence of NCR+ILC3 and neutrophils in decidual tissues and their possible cross talk, may have a role in the early phases of pregnancy.
Assuntos
Quimiotaxia de Leucócito/imunologia , Decídua/imunologia , Decídua/metabolismo , Imunidade Inata , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Antígenos CD/metabolismo , Biomarcadores , Sobrevivência Celular/imunologia , Citocinas/metabolismo , Feminino , Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/biossíntese , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Humanos , Ativação Linfocitária/imunologia , Infiltração de Neutrófilos/imunologia , Fenótipo , Gravidez , Primeiro Trimestre da GravidezRESUMO
In recent years, several investigators have addressed the question of whether mature polymorphonuclear neutrophils (PMN) are able to secrete cytokines. Their studies have brought forward new and exciting discoveries, by establishing that the release of inflammatory cytokines constitutes a novel and important aspect of the neutrophil biology, thereby emphasizing that PMN should no longer be regarded as cells that only release preformed mediators. Although it is still premature to assess the true biological significance of cytokine production by neutrophils, this new aspect of neutrophil biology opens novel perspectives as to the potential role of these cells in the inflammatory and immune responses. In this context, a correct methodological analysis and a detailed molecular investigation of the mechanisms regulating cytokine production by neutrophils in vitro is a critical and fundamental step to better understand how the release of cytokines by PMN may influence pathophysiological processes in vivo. We now describe and discuss the approach that we typically used throughout most of the last decade to characterize cytokine production by human neutrophils, as illustrated herein for a protein that is expressed and released by PMN, namely, vascular endothelial growth factor (VEGF).
Assuntos
Fatores de Crescimento Endotelial/análise , Linfocinas/análise , Neutrófilos/química , Células Cultivadas , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/metabolismo , Humanos , Interleucina-10/farmacologia , Lipopolissacarídeos/farmacologia , Linfocinas/biossíntese , Linfocinas/metabolismo , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , RNA Mensageiro/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
GRO alpha, a member of the chemokine superfamily, exerts potent stimulatory actions on granulocytes. In this report, we show that activated human polymorphonuclear leukocytes (PMN) are able to produce significant amounts of GRO alpha. Lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and yeast particles opsonized with IgG (Y-IgG) had the ability to induce GRO alpha release, with Y-IgG being the most potent stimulus. The extracellular production of GRO alpha was also modulated by both interferon-gamma (IFN gamma) and interleukin-10 (IL-10). IFN gamma significantly inhibited the production of GRO alpha by PMN stimulated for 2 hr with LPS, TNF alpha, or Y-IgG, but potentiated the production of GRO alpha in cells stimulated for 18 hr with LPS and TNF alpha. IL-10 moderately suppressed the Y-IgG-induced production of GRO alpha, but strongly inhibited the action of LPS and potentiated the effect of TNF alpha. As revealed by Northern blot analysis, the extracellular production of GRO alpha under the experimental conditions used did not always correlate with parallel changes at the level GRO alpha mRNA expression, suggesting that production of GRO alpha by PMN might be regulated at post-transcriptional, translational, or post-translational level. These findings identify a novel biological function of PMN, likely involved in the modulation of the acute inflammatory response.
Assuntos
Quimiocinas CXC , Fatores Quimiotáticos/biossíntese , Inibidores do Crescimento/biossíntese , Substâncias de Crescimento/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular , Neutrófilos/metabolismo , Quimiocina CXCL1 , Fatores Quimiotáticos/genética , Sinergismo Farmacológico , Expressão Gênica , Inibidores do Crescimento/genética , Substâncias de Crescimento/genética , Humanos , Imunoglobulina G/farmacologia , Interferon gama/farmacologia , Interleucina-10/farmacologia , Lipopolissacarídeos/farmacologia , Proteínas Opsonizantes , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
B-lymphocyte stimulator (BLyS) acts as survival factor for B lymphocytes. As Hodgkin and Reed-Sternberg (HRS) cells express receptors through which BLyS promotes their growth and chemotherapy resistance, we investgated whether this molecule was increased in sera from patients with classical Hodgkin lymphoma (cHL) and whether it correlates with clinical-pathological features and outcomes. Enzyme-linked immunosorbent assay was used to measure soluble BLyS (sBLyS) in sera from 87 patients and 33 donors; higher levels were detected in patients (mean +/- standard error 4493.9 +/- 264.9 pg/ml vs. 2687.0 +/- 200.9 pg/ml; P < 0.0001). Levels above the median value (4242.0 pg/ml) were associated with age > or = 45 years (P = 0.042), advanced stages of disease (P = 0.005), systemic symptoms (P = 0.014) and extranodal involvement (P = 0.009). Five-year failure-free survival (FFS) of patients with sBLyS below or equal to median levels was 88.6% as compared to 65.1% of those with levels above the median (P = 0.009). Statistical analyses confirmed the prognostic significance of sBLyS (P = 0.046). When patients were analysed according to variables associated with high levels, sBLyS showed an independent predictive power in terms of FFS. Our findings support the involvement of BLyS in cHL pathogenesis. The association between high serum levels and an inferior FFS indicates that sBLyS is a possible prognostic predictor with a potential significance as a therapeutic target.
Assuntos
Fator Ativador de Células B/sangue , Biomarcadores Tumorais/sangue , Doença de Hodgkin/sangue , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Doença de Hodgkin/mortalidade , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Resultado do TratamentoRESUMO
In this report, we show that interferon-gamma (IFN-gamma) modulates the production of IL-1ra in activated human neutrophils. In lipopolysaccharide-stimulated cells, IFN-gamma increased the release of IL-1ra without modulating IL-1ra mRNA accumulation; under these conditions, IFN-gamma only marginally enhanced IL-1ra de novo synthesis, while IL-1ra was more efficiently secreted. In response to the formylated peptide, fMLP, neutrophils released small but significant amounts of IL-1ra, yet without an increase in IL-1ra mRNA over constitutive levels. Following IFN-gamma treatment, however, the fMLP-elicited IL-1ra production was greatly potentiated, and this was accompanied by a transient increased accumulation of IL-1ra mRNA. Finally, opsonized yeast particles were found to induce IL-1ra formation at late incubation times, and prior treatment of neutrophils with IFN-gamma moderately enhanced this response. Collectively, our results demonstrate that in neutrophils activated by different classes of agonists, IFN-gamma modulates the release of IL-1ra by acting through distinct mechanisms.
Assuntos
Interferon gama/farmacologia , Neutrófilos/metabolismo , Sialoglicoproteínas/biossíntese , Células Cultivadas , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Lipopolissacarídeos/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , RNA Mensageiro/análise , Sialoglicoproteínas/genéticaRESUMO
It is widely accepted that the activation of the NADPH oxidase of phagocytes is linked to the stimulation of protein kinase C by diacylglycerol formed by hydrolysis of phospholipids. The main source would be choline containing phospholipid via phospholipase D and phosphatidate phosphohydrolase. This paper presents a condition where the activation of the respiratory burst by FMLP correlates with the formation of phosphatidic acid, via phospholipase D, and not with that of diacylglycerol. In fact: 1) in neutrophils treated with propranolol, an inhibitor of phosphatidate phosphohydrolase, FMLP plus cytochalasin B induces a respiratory burst associated with a stimulation of phospholipase D, formation of phosphatidic acid and complete inhibition of that of diacylglycerol. 2) The respiratory burst by FMLP plus cytochalasin B lasts a few minutes and may be restimulated by propranolol which induces an accumulation of phosphatidic acid. 3) In neutrophils stimulated by FMLP in the absence of cytochalasin B propranolol causes an accumulation of phosphatidic acid and a marked enhancement of the respiratory burst without formation of diacylglycerol. 4) The inhibition of the formation of phosphatidic acid via phospholipase D by butanol inhibits the respiratory burst by FMLP.
Assuntos
Diglicerídeos/fisiologia , Glicerídeos/fisiologia , NADH NADPH Oxirredutases/metabolismo , Neutrófilos/fisiologia , Ácidos Fosfatídicos/fisiologia , Fosfolipase D/fisiologia , Fosfolipases/fisiologia , Butanóis/farmacologia , Citocalasina B/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Técnicas In Vitro , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases , Consumo de Oxigênio/efeitos dos fármacos , Propranolol/farmacologiaRESUMO
We examined the mechanisms responsible for the regulation by interferon-gamma (IFN-gamma) of the expression of the genes encoding the high affinity IgG-Fc receptor (Fc gamma R-I, CD64) and the NADPH oxidase 47-kDa cytosolic factor (p47-phox) in human polymorphonuclear leukocytes (PMN). Nuclear run-on transcriptional assays demonstrated that the Fc gamma R-I gene transcription is undetectable in untreated PMN but is significantly induced by IFN-gamma. Unlike Fc gamma R-I, p47-phox gene transcription is constitutively active in resting PMN and is down-regulated by a 2-h treatment of these cells with IFN-gamma. The transcriptional modulation by IFN-gamma of Fc gamma R-I and p47-phox genes is not influenced by the protein synthesis inhibitor cycloheximide. Moreover, Northern blot analysis revealed that cycloheximide superinduces p47-phox mRNA expression by increasing its half-life and without affecting p47-phox gene transcription. These findings indicate that human PMN can regulate gene expression by transcriptional and posttranscriptional events.
Assuntos
Antígenos de Diferenciação/genética , Interferon gama/farmacologia , NADH NADPH Oxirredutases/genética , Neutrófilos/fisiologia , Receptores Fc/genética , Transcrição Gênica/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Células Cultivadas , Cicloeximida/farmacologia , Citosol/enzimologia , Dactinomicina/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulina G/metabolismo , Cinética , NADH NADPH Oxirredutases/sangue , NADPH Oxidases , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , RNA Mensageiro/sangue , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Receptores de IgG , Proteínas RecombinantesRESUMO
We recently showed that lipopolysaccharide (LPS) is a potent inducer of interleukin-8 (IL-8) expression in human polymorphonuclear leucocytes (PMN), at the level of both mRNA and protein, and that interferon-gamma (IFN gamma) inhibits IL-8 mRNA accumulation in stimulated PMN. To further define the molecular basis of the regulation of IL-8 gene expression in PMN, we investigated the effects of LPS and IFN gamma at both the transcriptional and post-transcriptional levels. As determined by Northern blot analysis, new protein synthesis was not required for the induction of IL-8 mRNA expression by LPS. Neither did the half-life of IL-8 mRNA in LPS-treated PMN differ from that observed in untreated cells. However, nuclear run-on analysis revealed that LPS increased the transcription of the IL-8 and IL-1 beta genes and that, in LPS-activated cells, IFN gamma markedly inhibited the rate of IL-8 gene transcription, but not that of IL-1 beta. IFN gamma did not affect IL-8 mRNA stability in LPS-treated PMN, indicating that the cytokine does not regulate LPS-induced IL-8 gene expression through post-transcriptional events. These results provide the first evidence that human granulocytes can actively transcribe the IL-8 gene, and that transcriptional inhibition is the mechanism by which IFN gamma inhibits IL-8 gene expression in PMN.
Assuntos
Expressão Gênica/efeitos dos fármacos , Granulócitos/imunologia , Interferon gama/farmacologia , Interleucina-8/biossíntese , Lipopolissacarídeos/farmacologia , Neutrófilos/imunologia , Transcrição Gênica/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Cicloeximida/farmacologia , Granulócitos/efeitos dos fármacos , Humanos , Interleucina-8/sangue , Lipopolissacarídeos/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Proteínas RecombinantesRESUMO
Accumulation of polymorphonuclear neutrophils (PMN) into the subarachnoidal space is one of the hallmarks of Neisseria meningitidis infection. In this study, we evaluated the ability of outer membrane vesicles (OMV) from N. meningitidis B to stimulate cytokine production by neutrophils. We found that PMN stimulated in vitro by OMV produce proinflammatory cytokines and chemokines including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-8, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-1beta. A considerable induction of gamma interferon (IFN-gamma)-inducible protein 10 (IP-10) mRNA transcripts, as well as extracellular IP-10 release, was also observed when neutrophils were stimulated by OMV in combination with IFN-gamma. Furthermore, PMN stimulated by OMV in the presence of IFN-gamma demonstrated an enhanced capacity to release TNF-alpha, IL-1beta, IL-8, and MIP-1beta compared to stimulation with OMV alone. In line with its downregulatory effects on neutrophil-derived proinflammatory cytokines, IL-10 potently inhibited TNF-alpha, IL-1beta, IL-8, and MIP-1beta production triggered by OMV. Finally, a neutralizing anti-TNF-alpha monoclonal antibody (MAb) did not influence the release of IL-8 and MIP-1beta induced by OMV, therefore excluding a role for endogenous TNF-alpha in mediating the induction of chemokine release by OMV. In contrast, the ability of lipopolysaccharide from N. meningitidis B to induce the production of IL-8 and MIP-1beta was significantly inhibited by anti-TNF-alpha MAb. Our results establish that, in response to OMV, neutrophils produce a proinflammatory profile of cytokines and chemokines which may not only play a role in the pathogenesis of meningitis but may also contribute to the development of protective immunity to serogroup B meningococci.
Assuntos
Quimiocinas CXC/biossíntese , Interleucina-1/biossíntese , Interleucina-8/biossíntese , Proteínas Inflamatórias de Macrófagos/biossíntese , Neisseria meningitidis/imunologia , Neutrófilos/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CXCL10 , Quimiocinas CXC/genética , Humanos , Interferon gama/farmacologia , Interleucina-1/genética , Interleucina-10/farmacologia , Interleucina-8/genética , Proteínas Inflamatórias de Macrófagos/genética , Meningite Meningocócica/etiologia , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/genéticaRESUMO
The neuropeptide substance P (SP), a main mediator of neurogenic inflammation, has been shown to have direct and modulatory effects on functional responses of polymorphonuclear leucocytes (PMNL). In this study, we further investigated the effects exerted by SP on human PMNL functions. Pretreatment of PMNL with SP resulted in an increase of superoxide anion (O2-) production in response to formyl-methionyl-leucyl-phenylalanine (FMLP), concanavalin A (Con A) and opsonized zymosan (STZ). In contrast, the O2- production induced by tumour necrosis factor (TNF) was strongly inhibited by pretreatment with SP. Both enhancement and inhibition of O2- response were exerted by SP in a dose-dependent manner and at concentrations which did not directly stimulate O2- production. These effects were rapid in onset, and occurred after 5 min of preincubation of cells with the neuropeptide. At concentrations that modulated O2- production by PMNL, SP also directly stimulated release of the chemotactic cytokine interleukin-8 (IL-8). Induction of IL-8 release required a longer incubation time (1 hr) with SP and was preceded by an increase of IL-8 mRNA steady-state levels. Furthermore, as well as directly stimulating IL-8 production, SP was also able to enhance the IL-8 release induced by other stimuli such as FMLP and TNF. The results of this study indicate that, in addition to the rapid and differential modulation of O2- production, SP also induces long-term changes such as IL-8 synthesis and release, and can thus amplify the process of PMNL recruitment to the inflammatory site.
Assuntos
Interleucina-8/sangue , Neutrófilos/efeitos dos fármacos , Substância P/farmacologia , Superóxidos/sangue , Concanavalina A/farmacologia , Interações Medicamentosas , Humanos , Interleucina-8/genética , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , RNA Mensageiro/análise , Fator de Necrose Tumoral alfa/farmacologia , Zimosan/farmacologiaRESUMO
Neutrophils are known to play an important role in inflammatory responses by virtue of their ability to perform a series of effector functions that collectively represent a major mechanism of innate immunity against injury and infection. In recent years, however, it has become obvious that the contribution of neutrophils to host defence and natural immunity extends well beyond their traditional role as professional phagocytes. Indeed, neutrophils can be induced to express a number of genes whose products lie at the core of inflammatory and immune responses. These include not only Fc receptors, complement components, cationic antimicrobial and NADPH oxidase proteins, but also a variety of cytokines (including tumour necrosis factor-alpha, interleukin (IL)-1beta, IL-1R alpha, IL-12 and vascular endothelial growth factor), and chemokines such as IL-8, growth-related gene product, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, interferon-gamma-inducible protein of 10 kDa and monokine induced by interferon-gamma. Because these chemokines are primarily chemotactic for neutrophils, monocytes, immature dendritic cells and T-lymphocyte subsets, a potential role for neutrophils in orchestrating the sequential recruitment of distinct leukocyte types to the inflamed tissue is likely to occur. The purpose of this review is to summarize the essential features of the production of chemokines by polymorphonuclear neutrophil leukocytes and the contribution that we have made to characterize some aspects of this newly discovered crucial function of neutrophils.
Assuntos
Quimiocinas/imunologia , Neutrófilos/imunologia , Animais , Humanos , Inflamação , Ativação de NeutrófiloRESUMO
Interferon-gamma (IFN-gamma)-inducible protein-10 (IP-10), a member of the C-X-C sub-family of chemokines, is known to be produced by monocytes, lymphocytes, keratinocytes and endothelial cells in response to IFN-gamma. Here, we show that human polymorphonuclear neutrophils (PMN) also have the ability to produce IP-10. IFN-gamma alone had a modest effect on IP-10 mRNA accumulation, whereas tumor necrosis factor-alpha (TNF-alpha), yeast particles opsonized with IgG (Y-IgG), lipopolysaccharide (LPS), and formyl-methionyl-leucyl-phenylalanine (fMLP) all failed to up-regulate IP-10 gene expression. However, stimulation of neutrophils with IFN-gamma in combination with either TNF-alpha or LPS (but not with Y-IgG or fMLP) resulted in a considerable induction of IP-10 mRNA transcripts, as well as in the extracellular release of the protein. In contrast, the best inducer of IP-10 release from peripheral blood mononuclear cells was IFN-gamma alone. Furthermore, mRNA stabilization analyses demonstrated that IP-10 mRNA isolated from PMN stimulated with IFN-gamma only, or with IFN-gamma plus either TNF-alpha or LPS, had similar half-lives. Finally, we found that interleukin-10, a known inhibitor of chemokine production in PMN, moderately suppressed the extracellular production of IP-10 in neutrophils stimulated with IFN-gamma plus either LPS or TNF-alpha. Since IP-10 is a potent chemoattractant for activated T lymphocytes, the ability of neutrophils to produce IP-10 might contribute to the evolution and progression of the inflammatory response.
Assuntos
Quimiocinas CXC , Citocinas/biossíntese , Neutrófilos/metabolismo , Células Cultivadas , Quimiocina CXCL10 , Dactinomicina/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , RNA Mensageiro/genética , Fatores de TempoRESUMO
To understand how expression of IL-8 mRNA is regulated, we studied the effects of Staurosporine, H7, phorbol-myristate-acetate and Dexamethasone in human neutrophils subsequently treated with IFN-gamma. Our results can be summarized as follows: a) IL-8 mRNA steady state levels were enhanced in a dose dependent fashion by both Staurosporine and phorbol-myristate-acetate, were not influenced by H7, but were decreased by Dexamethasone; b) the negative effect of IFN-gamma on IL-8 mRNA accumulation was prevented by Staurosporine and phorbol-myristate-acetate, was not influenced by H7, and was potentiated by Dexamethasone; c) IL-8 mRNA induction caused by Staurosporine was accompanied by a time and dose dependent release of IL-8 into the PMN supernatants.
Assuntos
Alcaloides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-8/genética , NADPH Oxidases , Neutrófilos/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Northern Blotting , Grupo dos Citocromos b/genética , Dexametasona/farmacologia , Sinergismo Farmacológico , Humanos , Isoquinolinas/farmacologia , Piperazinas/farmacologia , RNA Mensageiro/metabolismo , Estaurosporina , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
We have recently shown that IL-10 fails to trigger Stat3 and Stat1 tyrosine phosphorylation in freshly isolated human neutrophils. In this study, we report that IL-10 can nonetheless induce Stat3 tyrosine phosphorylation and the binding of Stat1 and Stat3 to the IFN-gamma response region or the high-affinity synthetic derivative of the c-sis-inducible element in neutrophils that have been cultured for at least 3 h with LPS. Similarly, the ability of IL-10 to up-regulate suppressor of cytokine signaling (SOCS)-3 mRNA was dramatically enhanced in cultured neutrophils and, as a result, translated into the SOCS-3 protein. Since neutrophils' acquisition of responsiveness to IL-10 required de novo protein synthesis, we assessed whether expression of IL-10R1 or IL-10R2 was modulated in cultured neutrophils. We detected constitutive IL-10R1 mRNA and protein expression in circulating neutrophils, at levels which were much lower than those observed in autologous monocytes or lymphocytes. In contrast, IL-10R2 expression was comparable in both cell types. However, IL-10R1 (but not IL-10R2) mRNA and protein expression was substantially increased in neutrophils stimulated by LPS. The ability of IL-10 to activate Stat3 tyrosine phosphorylation and SOCS-3 synthesis and to regulate IL-1 receptor antagonist and macrophage-inflammatory protein 1beta release in LPS-treated neutrophils correlated with this increased IL-10R1 expression, and was abolished by neutralizing anti-IL-10R1 and anti-IL-10R2 Abs. Our results demonstrate that the capacity of neutrophils to respond to IL-10, as assessed by Stat3 tyrosine phosphorylation, SOCS-3 expression, and modulation of cytokine production, is very dependent on the level of expression of IL-10R1.
Assuntos
Interleucina-10/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de Interleucina/biossíntese , Proteínas Repressoras , Fatores de Transcrição , Regulação para Cima/imunologia , Separação Celular , Células Cultivadas , Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Soros Imunes/farmacologia , Interleucina-10/farmacologia , Interleucina-10/fisiologia , Lipopolissacarídeos/farmacologia , Fosforilação , Ligação Proteica/imunologia , Biossíntese de Proteínas , Proteínas/genética , Proteínas/fisiologia , RNA Mensageiro/biossíntese , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia , Receptores de Interleucina-10 , Fator de Transcrição STAT3 , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Transativadores/metabolismo , Tirosina/metabolismoRESUMO
We have recently shown that, in human neutrophils, interleukin-10 (IL-10) fails to induce specific DNA-binding activities to the gamma-interferon response region (GRR), a regulatory element located in the FcgammaRI gene promoter, which is required for transcriptional activation by IL-10 and interferon gamma (IFNgamma) in monocytic cells. In this study, we report that IL-10 is also unable to induce the binding of STAT1 or STAT3 to the serum-inducible element (hSIE/m67), despite the fact that both proteins are expressed in neutrophils. Whereas IFNgamma and granulocyte colony-stimulating factor (G-CSF) are efficient inducers of STAT1 and STAT3 tyrosine phosphorylation in polymorphonuclear neutrophils (PMN), IL-10 fails to trigger STAT1 and STAT3 tyrosine and serine phosphorylation, therefore explaining its inability to induce the FcgammaRI expression in these cells. By contrast, we demonstrate that IL-10 alone represents an efficient stimulus of CIS3/SOCS3 mRNA expression in neutrophils. CIS3/SOCS3 belongs to the recently cloned cytokine-inducible SH2-containing protein (CIS) gene family (which also includes CIS1, CIS2, CIS4, CIS5, and JAB) that is believed to be, at least in part, under the control of STAT transcription factors and whose products are potential modulators of cytokine signaling. Moreover, IL-10 synergizes with lipopolysaccharide (LPS) in upregulating CIS3/SOCS3 mRNA expression in PMN through a mechanism that involves mRNA stabilization. In contrast to CIS3/SOCS3, mRNA transcripts encoding other family members are unaffected by IL-10 in neutrophils. Finally, transfection of CIS3/SOCS3 in murine M1 myeloid cells suppresses LPS-induced growth arrest, macrophage-like differentiation, and nitric oxide synthesis, but not IL-6 mRNA expression. Collectively, our data suggest that, in neutrophils, the activation of STAT1 and STAT3 phosphorylation is neither required for CIS3/SOCS3 induction by IL-10 nor involved in the regulatory effects of IL-10 on cytokine production.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-10/farmacologia , Neutrófilos/efeitos dos fármacos , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Proteínas Repressoras , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Humanos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Família Multigênica , Neutrófilos/metabolismo , Óxido Nítrico/biossíntese , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas/genética , RNA Mensageiro/genética , Fator de Transcrição STAT1 , Fator de Transcrição STAT3 , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/metabolismo , Transfecção , Domínios de Homologia de srcRESUMO
Monokine induced by IFN-gamma (MIG), IFN-inducible T cell alpha chemoattractant (I-TAC), and IFN-gamma-inducible protein of 10 kDa (IP-10) are related members of the CXC chemokine subfamily that bind to a common receptor, CXCR3, and that are produced by different cell types in response to IFN-gamma. We have recently reported that human polymorphonuclear neutrophils (PMN) have the capacity to release IP-10. Herein, we show that PMN also have the ability to produce MIG and to express I-TAC mRNA in response to IFN-gamma in combination with either TNF-alpha or LPS. While IFN-gamma, alone or in association with agonists such as fMLP, IL-8, granulocyte (G)-CSF and granulocyte-macrophage (GM)-CSF, failed to influence MIG, IP-10, and I-TAC gene expression, IFN-alpha, in combination with TNF-alpha, LPS, or IL-1beta, resulted in a considerable induction of IP-10 release by neutrophils. Furthermore, IL-10 and IL-4 significantly suppressed the expression of MIG, IP-10, and I-TAC mRNA and the extracellular production of MIG and IP-10 in neutrophils stimulated with IFN-gamma plus either LPS or TNF-alpha. Finally, supernatants harvested from stimulated PMN induced migration and rapid integrin-dependent adhesion of CXCR3-expressing lymphocytes; these activities were significantly reduced by neutralizing anti-MIG and anti-IP-10 Abs, suggesting that they were mediated by MIG and IP-10 present in the supernatants. Since MIG, IP-10, and I-TAC are potent chemoattractants for NK cells and Th1 lymphocytes, the ability of neutrophils to produce these chemokines might contribute not only to the progression and evolution of the inflammatory response, but also to the regulation of the immune response.