RESUMO
Asthma is a highly prevalent chronic allergic inflammatory disease of the airways affecting people worldwide. House dust mite (HDM) is the most common allergen implicated in human allergic asthma. HDM-induced allergic responses are thought to depend upon activation of pathways involving Toll-like receptors and their adaptor protein myeloid differentiation factor 88 (MyD88). We sought here to determine the role of MyD88 in myeloid and type II lung epithelial cells in the development of asthma-like allergic disease using a mouse model. Repeated exposure to HDM caused allergic responses in control mice characterized by influx of eosinophils into the bronchoalveolar space and lung tissue, lung pathology and mucus production and protein leak into bronchoalveolar lavage fluid. All these responses were abrogated in mice with a general deficiency of MyD88 but unaltered in mice with MyD88 deficiency, specifically in myeloid or type II lung epithelial cells. We conclude that cells other than myeloid or type II lung epithelial cells are responsible for MyD88-dependent HDM-induced allergic airway inflammation.
Assuntos
Asma/imunologia , Células Epiteliais/imunologia , Hipersensibilidade/imunologia , Células Mieloides/fisiologia , Fator 88 de Diferenciação Mieloide/metabolismo , Pneumonia/imunologia , Pyroglyphidae/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Movimento Celular , Células Epiteliais/patologia , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genéticaRESUMO
Human bronchial epithelial (HBE) cells play an essential role during bacterial infections of the airways by sensing pathogens and orchestrating protective immune responses. We here sought to determine which metabolic pathways are utilized by HBE cells to mount innate immune responses upon exposure to a relevant bacterial agonist. Stimulation of HBE cells by the bacterial component flagellin triggered activation of the mTOR pathway resulting in an increased glycolytic flux that sustained the secretory activity of immune mediators by HBE cells. The mTOR inhibitor rapamycin impeded glycolysis and limited flagellin-induced secretion of immune mediators. The role of the mTOR pathway was recapitulated in vivo in a mouse model of flagellin-triggered lung innate immune responses. These data demonstrate that metabolic reprogramming via the mTOR pathway modulates activation of the respiratory epithelium, identifying mTOR as a potential therapeutic target to modulate mucosal immunity in the context of bacterial infections.
Assuntos
Brônquios/patologia , Células Epiteliais/imunologia , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae/fisiologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Células Cultivadas , Reprogramação Celular , Modelos Animais de Doenças , Feminino , Flagelina/metabolismo , Glicólise , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Klebsiella pneumoniae is a common cause of nosocomial pneumonia. Osteopontin (OPN) is a phosphorylated glycoprotein involved in inflammatory processes, some of which is mediated by CD44. The aim of this study was to determine the role of OPN during K. pneumoniae-induced pneumonia. Wild-type (WT) and OPN knockout (KO) mice were intranasally infected with 104 colony forming units of K. pneumoniae, or administered Klebsiella lipopolysaccharides (LPS). In addition, recombinant OPN (rOPN) was intranasally administered to WT and CD44 KO mice. During Klebsiella pneumonia, WT mice displayed elevated pulmonary and plasma OPN levels. OPN KO and WT mice showed similar pulmonary bacterial loads 6 h after infection; thereafter, Klebsiella loads were higher in lungs of OPN KO mice and the mortality rate in this group was higher than in WT mice. Early neutrophil recruitment into the bronchoalveolar space was impaired in the absence of OPN after intrapulmonary delivery of either Klebsiella bacteria or Klebsiella LPS. Moreover, rOPN induced neutrophil migration into the bronchoalveolar space, independent from CD44. In vitro, OPN did not affect K. pneumoniae growth or neutrophil function. In conclusion, OPN levels were rapidly increased in the bronchoalveolar space during K. pneumoniae pneumonia, where OPN serves a chemotactic function towards neutrophils, thereby facilitating an effective innate immune response.
Assuntos
Infecções por Klebsiella/imunologia , Klebsiella pneumoniae/imunologia , Osteopontina/imunologia , Pneumonia Bacteriana/imunologia , Animais , Carga Bacteriana , Citocinas/sangue , Citocinas/imunologia , Receptores de Hialuronatos/imunologia , Klebsiella pneumoniae/isolamento & purificação , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/imunologia , Osteopontina/sangueRESUMO
AIMS: Type 2 diabetes is frequently associated with infectious complications. Swift activation of leucocytes is important for an adequate immune response. We determined the selective effects of hyperglycaemia and hyperinsulinaemia on lipopolysaccharide (LPS)-induced proinflammatory gene expression and cytokine production in leucocytes and on neutrophil functions. METHODS: Six healthy humans were studied on four occasions for 6 h during: (i) lower insulinaemic euglycaemic clamp, (ii) lower insulinaemic hyperglycaemic clamp, (iii) hyperinsulinaemic euglycaemic clamp, and (iv) hyperinsulinaemic hyperglycaemic clamp. Target levels of plasma glucose were 12.0 mmol/l (hyperglycaemic clamps) or 5.0 mmol/l (euglycaemic clamps). Target plasma insulin levels were 400 pmol/l (hyperinsulinaemic clamps) or 100 pmol/l (lower insulinaemic clamps). RESULTS: Hyperglycaemia reduced LPS-induced mRNA expression of nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (NFKBIA), interleukin-1 alpha (IL1A) and chemokine (C-C motif) ligand 3 (CCL3), whereas during hyperinsulinaemia enhanced mRNA levels occurred in six out of eight measured inflammation-related genes, irrespective of plasma glucose levels. Combined hyperglycaemia and hyperinsulinaemia led to enhanced IL1A, interleukin-1 beta (IL1B) and CCL3 mRNA levels upon LPS stimulation. Neither hyperglycaemia nor hyperinsulinaemia altered cytokine protein production, neutrophil migration, phagocytic capacity or oxidative burst activity. CONCLUSIONS: These results suggest that short-term hyperglycaemia and hyperinsulinaemia influence the expression of several inflammatory genes in an opposite direction, that the acute effects of hyperinsulinaemia on inflammatory mRNA levels may be stronger than those of hyperglycaemia, and that the effects of insulin, in particular, may be relevant in the concurrent presence of hyperglycaemia.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Hiperglicemia/metabolismo , Hiperinsulinismo/metabolismo , Neutrófilos/metabolismo , RNA Mensageiro/metabolismo , Adulto , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/genética , Técnica Clamp de Glucose , Humanos , Proteínas I-kappa B , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Masculino , Inibidor de NF-kappaB alfa , RNA Mensageiro/genéticaRESUMO
BACKGROUND: Sepsis and endotoxemia are associated with concurrent activation of inflammation and the hemostatic mechanism, which both contribute to organ dysfunction and death. Electrical vagus nerve stimulation (VNS) has been found to inhibit tumor necrosis factor (TNF)-alpha release during endotoxemia in rodents. OBJECTIVE: To determine the effect of VNS on activation of coagulation and fibrinolysis. METHODS: Rats received a sublethal i.v. dose of lipopolysaccharide (LPS) after electrical VNS or sham stimulation. Activation of coagulation and fibrinolysis, as well as cytokine release, was measured before LPS injection and 2, 4 and 6 h thereafter. RESULTS: LPS induced activation of the coagulation system (increases in the plasma concentrations of thrombin-antithrombin complexes and D-dimer, and a decrease in antithrombin) and biphasic changes in the fibrinolytic system [early rises of plasminogen activator activity and tissue-type plasminogen activator, followed by a delayed increase in plasminogen activator inhibitor type 1 (PAI-1)]. VNS strongly inhibited all LPS-induced procoagulant responses and more modestly attenuated the fibrinolytic response. In addition, VNS attenuated the LPS-induced increases in plasma and splenic concentrations of the proinflammatory cytokines TNF-alpha and interleukin-6 (IL-6), while not influencing the release of the anti-inflammatory cytokine IL-10. CONCLUSION: These data illustrate a thus far unrecognized effect of VNS and suggest that the cholinergic anti-inflammatory pathway not only impacts on inflammation but also on the coagulant-anticoagulant balance.
Assuntos
Coagulação Sanguínea , Terapia por Estimulação Elétrica , Endotoxemia/terapia , Fibrinólise , Nervo Vago , Animais , Estimulação Elétrica , Endotoxemia/patologia , Inflamação/prevenção & controle , Interleucina-10/análise , Interleucina-6/análise , Cinética , Lipopolissacarídeos/administração & dosagem , Masculino , Ratos , Ratos Endogâmicos Lew , Fator de Necrose Tumoral alfa/análiseRESUMO
PURPOSE: To investigate the density, distribution, and morphology of macrophages (bone marrow-derived microglia) and major histocompatibility complex (MHC) class II-positive cells in the retina of Lewis rats and the dynamics of these cells after systemic lipopolysaccharide (LPS) injection. METHODS: Immunohistochemistry was carried out using monoclonal antibodies specific to monocytes and macrophages (ED1, ED2) and MHC class II-positive cells (OX-6) on whole-mounts of the retina obtained from Lewis rats before and at different time points after footpad injection of 200 micrograms of LPS. RESULTS: The inner layers of the normal retina contained a network of macrophages, whereby ED1 and ED2 staining revealed similar results. Macrophages were either dendritiform or pleiomorphic in morphology, with the former predominant. The density of positive cells was higher at the peripheral part and the periequatorial part (271 +/- 10 cells/mm2 and 267 +/- 9 cells/mm2, respectively) than at the posterior part (196 +/- 11 cells/mm2; P < 0.0001 in both cases). Lipopolysaccharide injection induced an early adherence of monocytes to retinal blood vessels, followed by a massive influx of the macrophages into the retina. The ED1-ED2 positive cells showed a variety of morphologic appearances: large round cells, pleiomorphic cells, and dendritiform cells. Pleiomorphic cells were striking at 48 hours, whereas dendritiform cells were predominant in the whole retina at 72 hours and thereafter. On day 14, the dendritiform cell numbers returned to approximately preinjection levels. Major histocompatibility class II-positive cells could not be found in the normal retina, nor after LPS injection. CONCLUSIONS: The network of MHC class II-negative microglia in the retina were studied. These cells may play an important role in immunoregulation and stability of the immunologic microenvironment within the retina. Systemic LPS injection was followed by a massive influx of macrophages into the retina. The absence of MHC class II-positive cells in the retina after LPS challenge may be an important protective mechanism against possible autoimmune damage.
Assuntos
Macrófagos/imunologia , Retina/imunologia , Uveíte/imunologia , Animais , Anticorpos Monoclonais , Quimiotaxia de Leucócito/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Técnicas Imunoenzimáticas , Imunofenotipagem , Contagem de Leucócitos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Ratos , Ratos Endogâmicos Lew , Retina/citologia , Retina/efeitos dos fármacos , Salmonella typhimurium , Uveíte/induzido quimicamente , Uveíte/patologiaRESUMO
PURPOSE: Ample evidence is available demonstrating that cytokines play a pivotal role in the pathogenesis of uveitis. Because little is known concerning the site of cytokine synthesis in the eye, cytokine mRNA expression was analyzed in the uvea, retina, and cornea during endotoxin-induced uveitis (EIU) in the rat. METHODS: RNA was isolated from the iris, ciliary body, choroid-sclera, retina, and cornea at different points in time after foot-pad injection of 200 micrograms lipopolysaccharide (LPS) in Lewis rats. Reverse-transcription polymerase chain reaction analysis was used to determine tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), IL-6, IL-10, interferon gamma (IFN-gamma), monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), and IL-1 receptor antagonist (IL-1RA) mRNA expression. RESULTS: Maximal mRNA expression of all cytokines examined already was observed in the uvea 4 hours after systemic LPS injection, before the onset of clinical uveitis. Elevated expression of TNF-alpha, IL-1 beta, IL-6, IFN-gamma, MCP-1, and MIP-2 was also observed concomitant with maximal uveitis, at 22 to 24 hours. Except for IL-10 and IFN-gamma, all cytokines investigated were induced in the retina, with maximal expression at 22 to 24 hours. Expression of IL-1RA was detected in the uvea and retina at 4 hours and remained elevated up to 48 hours, when the clinical uveitis started to decline. LPS did not induce cytokine expression in the cornea. Strikingly, a considerable expression of IL-1RA was found in normal corneas, suggesting an inherent control mechanism for IL-1-mediated responses. CONCLUSIONS: Systemic LPS injection induces elevated mRNA expression of multiple cytokines and IL-1RA in the uvea and retina during various stages of EIU. This suggests that these mediators may contribute to the development and recovery of this intraocular inflammation.
Assuntos
Citocinas/biossíntese , Retina/imunologia , Sialoglicoproteínas/biossíntese , Úvea/imunologia , Uveíte/imunologia , Animais , Toxinas Bacterianas , Sequência de Bases , Córnea/imunologia , Citocinas/genética , Primers do DNA , Modelos Animais de Doenças , Endotoxinas , Expressão Gênica , Proteína Antagonista do Receptor de Interleucina 1 , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos Lew , Salmonella , Sialoglicoproteínas/genéticaRESUMO
PURPOSE: Previous studies have shown that experimental autoimmune encephalomyelitis (EAE) and anterior uveitis (AU) develop in Lewis rats immunized with myelin basic protein (MBP). The purpose of this study was to characterize the dynamics, distribution, and phenotype of infiltrating cells in the iris during EAE-associated AU. METHODS: Lewis rats were immunized with MBP emulsified in complete Freund's adjuvant (CFA) or with CFA alone. Cellular infiltration of the iris was analyzed at various time points by immunohistochemistry of wholemounts, flow cytometry, and immunoelectron microscopy, by using monoclonal antibodies specific for monocytes/macrophages (ED1), T lymphocytes (R73, W3.25, OX8), T-cell activation markers (OX39, OX40), granulocytes (HIS48), major histocompatibility complex (MHC) class II (OX6), and neurofilament (2H3). RESULTS: MBP-immunized rats showed development of characteristic monophasic EAE, followed, after resolution of paralysis, by mild self-limited AU. Initially, focal infiltrates of round MHC class II(+) and ED1(+) cells were found in the iris. During the course of AU, the midiris became massively infiltrated with ED1(+) monocytes-macrophages, R73(+) T cells, granulocytes (HIS48(+)), and MHC class II(+) cells. The influx of T cells consisted of CD4(+) and CD8(+) cells, of which only a small fraction (<14 and 11%, respectively) expressed activation markers. The infiltrating cells accumulated in proximity to myelinated and nonmyelinated nerve bundles and in the vicinity of blood vessels in the iris. No evidence was found for demyelination or nerve degradation. Neither EAE nor AU developed in CFA-treated control rats. CONCLUSIONS: These data show that EAE-associated AU is characterized by a transient mixed cellular infiltrate consisting of monocytes-macrophages, granulocytes, and CD4 and CD8 T cells. The preferential accumulation of inflammatory cells in the vicinity of nerve fibers suggests that AU in this model may result from autoreactivity to nerve antigens.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Iris/imunologia , Leucócitos/imunologia , Uveíte Anterior/imunologia , Animais , Anticorpos Monoclonais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/ultraestrutura , Encefalomielite Autoimune Experimental/patologia , Feminino , Citometria de Fluxo , Imunidade Celular , Técnicas Imunoenzimáticas , Iris/ultraestrutura , Leucócitos/ultraestrutura , Ativação Linfocitária/imunologia , Macrófagos/imunologia , Macrófagos/ultraestrutura , Microscopia Imunoeletrônica , Monócitos/imunologia , Monócitos/ultraestrutura , Proteína Básica da Mielina , Ratos , Ratos Endogâmicos Lew , Organismos Livres de Patógenos Específicos , Uveíte Anterior/patologiaRESUMO
PURPOSE: To determine the kinetics of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in serum and aqueous humor of rats with different susceptibilities to endotoxin-induced uveitis (EIU), after footpad injection of lipopolysaccharide (LPS). METHODS: Samples were collected from EIU-susceptible Lewis rats and EIU-resistant Brown Norway (BN) rats for up to 72 hours after LPS injection. Specific bioassays were used to measure TNF and IL-6 activity. Northern blot analysis was used to assess intraocular IL-6 mRNA expression. RESULTS: High levels of TNF and IL-6 were detected in serum of both rat strains early after LPS injection. A second rise in serum TNF was observed at 18 to 20 hours in Lewis rats only. In aqueous humor of Lewis rats, high levels of TNF and IL-6 were observed early after LPS injection (2 to 8 hours) and concomitant with maximal uveitis (18 to 24 hours). Low levels of TNF and IL-6 were found in aqueous humor of BN rats. Ocular IL-6 mRNA was detected at the same time as IL-6 activity was measured in aqueous humor. CONCLUSIONS: The results of this study indicate that both TNF and IL-6 may play a role in the pathogenesis of EIU. The early release of TNF in aqueous humor during EIU suggests that this cytokine may serve as an initial mediator of intraocular inflammation. Furthermore, Northern blot analysis indicates that IL-6 is produced locally during EIU.
Assuntos
Interleucina-6/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Uveíte Anterior/metabolismo , Animais , Humor Aquoso/metabolismo , Toxinas Bacterianas , Northern Blotting , Linhagem Celular , Células Cultivadas , Endotoxinas , Enterotoxinas , Interleucina-6/genética , Cinética , Masculino , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos Lew , Salmonella , Uveíte Anterior/induzido quimicamenteRESUMO
PURPOSE: Corneal allograft rejection in rats can be prevented by subconjunctival injections of liposomes containing dichloromethylene diphosphonate (clodronate-LIP), which selectively eliminate macrophages. In this study, the effect of clodronate-LIP treatment on cytokine mRNA levels in corneal allografts was examined. METHODS: AO rats received corneal grafts of PVG rats. Rats were either not treated or injected subconjunctivally with clodronate-LIP on the day of transplantation and on postoperative days (PODs) 2, 4, 6, and 8. RNA was isolated from the graft and rim of corneas at different times after transplantation and from normal controls. Interleukin (IL)-1beta, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, IL-10, IL-12p40, tumor necrosis factor (TNF)-alpha, TNF-beta/lymphotoxin (LT), interferon (IFN)-gamma, monocyte chemotactic protein 1 (MCP-1), and macrophage inflammatory protein 2 (MIP-2) mRNA levels were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Corneal rejection, observed in all untreated rats by POD 12, was associated with increased mRNA levels of all cytokines investigated in grafts and rims. Clodronate-LIP treatment prevented allograft rejection and strongly decreased the levels of IL-1beta, IL-1RA, IL-2, IL-4, IL-6, IL-10, IFN-gamma, TNF-beta/LT, MCP-1, and MIP-2 mRNA in grafts and IL-1 beta, IL-2, IL-4, IL-6, and IFN-gamma mRNA in rims. Interleukin-12p40 mRNA levels were unaltered in clodronate-treated rats, except for a transient increase in grafts at POD 3. TNF-alpha mRNA levels were increased by clodronate-LIP in grafts and rims early after transplantation (PODs 3 and 7). Despite a normal appearance, long-term accepted corneal grafts (POD 100) contained mRNA for IL-10, IL-12p40, TNF-alpha, MCP-1, and MIP-2. CONCLUSIONS: Clodronate-liposome treatment markedly altered the mRNA levels of all cytokines investigated in corneal allografts. These results may explain in part the mechanism by which clodronate-LIP treatment prevents corneal allograft rejection.
Assuntos
Ácido Clodrônico/administração & dosagem , Córnea/efeitos dos fármacos , Transplante de Córnea , Citocinas/genética , Rejeição de Enxerto/prevenção & controle , RNA Mensageiro/metabolismo , Animais , Southern Blotting , Quimiocina CCL2/genética , Quimiocina CXCL2 , Córnea/metabolismo , Citocinas/metabolismo , Portadores de Fármacos , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto , Lipossomos , Monocinas/genética , Ratos , Ratos Endogâmicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HomólogoRESUMO
PURPOSE: Idiopathic inflammation of the cornea, keratitis, has been proposed to result from an autoimmune process, but thus far no convenient animal model of keratitis exists. An attempt was made to establish an animal model for keratitis, to investigate possible autoimmune mechanisms. METHODS: T-cell lines were established from lymph node cells removed from rats immunized with bovine corneal epithelium (BCE) extract. After restimulation in vitro with BCE or a specific corneal antigen, the cells were transferred by intraperitoneal injection into naive rats, rats subjected to total body irradiation, or rats in which only one eye was irradiated. RESULTS: Neither direct immunization with corneal antigens nor transfer of activated anti-corneal T-cells into naive rats gave any signs of keratitis. Irradiation alone did not induce corneal inflammation. Transfer of corneal-specific activated T cells into irradiated rats produced keratitis starting around day 4 and culminating around day 8. The disease was self-limiting and the severity dependent on the dose and site of radiation. Keratitis was characterized by corneal haze, conjunctival and episcleral hyperemia, episcleral hemorrhages, chemosis, corneal infiltrates, and vascularization. Immunohistochemistry showed T-cell and macrophage infiltration of epithelium and stroma in the affected corneas. CONCLUSIONS: Thus, keratitis may be produced by T cells reactive to corneal antigens, provided that the target tissue has been made susceptible by irradiation. The effectiveness of T-cell vaccination in preventing adoptive keratitis suggests that systemic as well as local tissue factors may regulate the disease process.
Assuntos
Transferência Adotiva/efeitos adversos , Doenças Autoimunes/etiologia , Córnea/imunologia , Modelos Animais de Doenças , Ceratite/etiologia , Linfócitos T/imunologia , Animais , Antígenos/imunologia , Doenças Autoimunes/patologia , Linhagem Celular , Córnea/patologia , Proteínas do Olho/imunologia , Feminino , Técnicas Imunoenzimáticas , Ceratite/patologia , Ativação Linfocitária , Doses de Radiação , Ratos , Ratos Endogâmicos Lew , Linfócitos T/patologia , Irradiação Corporal TotalRESUMO
AIMS/BACKGROUND: Endotoxin induced uveitis has been regarded as a model for acute anterior uveitis and until now little was known about choroidal involvement. The aim of this study was to investigate changes in macrophages and MHC class II positive cells in the choroid of Lewis rats during endotoxin induced uveitis. METHODS: Choroid-sclera wholemounts were isolated from normal Lewis rats and at different time points--4, 8, 16, 24, 48, 72, and 96 hours, and 7, 10, and 14 days after a footpad injection of 200 micrograms of lipopolysaccharide (LPS). Immunohistochemistry was performed using the monoclonal antibodies ED1 (monocytes, macrophages, dendritic cells), and OX6 (MHC class II antigen). RESULTS: In normal rats, two layers of macrophages were identified in the choroid; a layer located immediately beneath the retinal pigment epithelium (RPE) and a layer bordering the sclera. The density of ED1 positive cells in the layer bordering the RPE cells was 902 (SD 132) cells/mm2 whereas the scleral layer had a cell density of 389 (73) cells/mm2. Based on morphology, positive cells could be divided into two main categories; pleomorphic/round cells and dendritiform cells with varying appearances, with the latter being predominant in normal eyes. A network of MHC class II positive dendritic cells was found in the choroid, beneath the RPE, with a density of 659 (96) cells/mm2. No MHC class II positive cells were found in the macrophage layer bordering the sclera. LPS injection caused a massive influx of ED1 positive macrophages in the area below the RPE cells but did not result in an influx of macrophages at the scleral side of the choroid. The infiltrate reached a maximum at 16 hours following LPS injection and decreased at 96 hours. The morphology of the infiltrating cells was pleomorphic/round at early stages of inflammation and changed into a dendritiform cell population later. The number of MHC class II positive cells on the anterior side of the choroid increased 8 hours after injection and reached a peak at 72-96 hours. MHC class II positive cells were not observed in the vicinity of the sclera at any time after LPS injection. Both resident and MHC class II positive dendritic cell numbers returned to normal values at day 14 following LPS injection. CONCLUSIONS: These results indicate that the choroid is severely inflamed after systemic LPS administration to Lewis rats and suggests that endotoxin induced uveitis may serve as a model for generalised uveitis in humans.
Assuntos
Corioide/imunologia , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/análise , Macrófagos/imunologia , Uveíte/imunologia , Animais , Corioide/citologia , Células Dendríticas/metabolismo , Endotoxinas , Imuno-Histoquímica , Masculino , Monócitos/imunologia , Ratos , Ratos Endogâmicos Lew , Uveíte/etiologia , Uveíte/patologiaRESUMO
AIMS: Previous studies have implied that interferon gamma (IFN-gamma) is involved in the pathogenesis of endotoxin induced uveitis (EIU) in the rat. This study investigated the source of IFN-gamma in the iris during EIU. METHODS: Whole mounts of iris were isolated from Lewis rats before and at different times (from 4 hours to 14 days) after foot pad injection of 200 micrograms Salmonella typhimurium lipopolysaccharide (LPS). Immunohistological analysis was performed using monoclonal antibodies (mAbs) specific to rat IFN-gamma (DB12 and DB13). mAbs specific to monocytes, macrophages, and dendritic cells and MHC class II were used to asses the inflammatory response in the eye (ED-1, ED-2, and OX-6). An antibody specific to neurofilaments (2H3) was used to stain nerve fibres in the normal iris. RESULTS: LPS administration induced acute intraocular inflammation, characterised by a massive infiltration of monocytes/macrophages and increased numbers of MHC class II positive cells in the iris. IFN-gamma immunoreactive cells were not detected in iris whole mounts of control rats. Strikingly, IFN-gamma immunoreactivity was found in fibres from 4 hours until 10 days after LPS injection, with the most intense staining at 48-72 hours. Other DB12 or DB13 positive cells were not detected in the iris. The pattern of DB12 and DB13 staining in the inflamed iris was similar to the 2H3 staining of neurons in the iris of control rats. CONCLUSION: These results show that systemic LPS administration induces IFN-gamma immunoreactivity in iris fibres and suggest that iris nerve fibres may be a source of IFN-gamma during EIU. The IFN-gamma immunoreactive material in the iris nerve fibres may be identical to neuronal IFN-gamma.
Assuntos
Interferon gama/análise , Iris/inervação , Uveíte/imunologia , Animais , Endotoxinas , Iris/imunologia , Lipopolissacarídeos , Masculino , Ratos , Ratos Endogâmicos Lew , Salmonella typhimurium , Uveíte/etiologiaRESUMO
Although the exact pathogenic mechanisms underlying uveitis are unknown, cytokines appear to be involved in this inflammatory disorder. This review describes the studies in which the uveitogenic properties of several cytokines, including tumor necrosis factor (TNF), interleukin 1 (IL-1), IL-6, IL-8 and interferon gamma (IFN-gamma), were investigated and the reports on intraocular expression of cytokines, such as TNF, IL-2, IL-6 and IFN-gamma, during uveitis. The exact contribution of these mediators to uveitis remains to be determined. This may provide new clues in the treatment of uveitis.
Assuntos
Citocinas/imunologia , Uveíte/imunologia , Animais , Modelos Animais de Doenças , Imunidade , Neutrófilos/imunologiaRESUMO
The human cornea has been shown to express a number of inflammatory cytokines including IL-1, IL-6 and IL-8. In view of the potent proinflammatory activities of interleukin-1 (IL-1), regulatory mechanisms should be present in the human cornea to control IL-1 mediated inflammatory and immune responses. This is important for the maintenance of the integrity and transparency of the cornea. To test this hypothesis, the authors determined the presence of IL-1 receptor antagonist (IL-tra) in the normal human cornea using an enzyme-linked immunosorbent assay (ELISA). IL-tra is a natural antagonist of IL-1 and competes with IL-1 for the binding to its receptors thereby blocking the inflammatory response. Corneas were either tested immediately or after a 24-hour culture period. Furthermore, the authors separately analyzed the three layers of the cornea. Their results present evidence for the constitutive expression of the IL-tra protein in the normal human cornea and show that both epithelial and stromal cells produce IL-1ra. The epithelial cells are the major source of corneal IL-1ra immunoreactivity, and secrete IL-1ra during culture. Stromal cells contain detectable, albeit low amounts of cell associated IL-1ra. No IL-1ra was detected in the endothelial cell layer. A more accurate understanding of the balance between IL-1 and IL-1ra in ocular tissues and the role of the IL-1ra under physiologie and pathophysiologic conditions will be necessary for an eventual use of IL-1 receptor antagonist as a therapeutical tool.
RESUMO
OBJECTIVE: To investigate the longitudinal changes of macrophages and major histocompatibility complex (MHC) class II-positive cells in the iris and ciliary body of Lewis rats after lipopolysaccharide (LPS) injection. METHODS: Immunohistochemistry was performed using monoclonal antibodies to monocytes and macrophages (ED1) and MHC class II-positive cells (OX6) on wholemounts of the iris and ciliary body in endotoxin-induced uveitis (EIU). RESULTS: A network of macrophages (ED1+ cell) and MHC class II-positive cells, was present in the iris and ciliary body of normal Lewis rats. Most cells in the iris and ciliary body displayed dendritiform appearance. A severe involvement of the iris and ciliary body, as evidenced by a rapid influx of monocytes and macrophages and remarkable increase of MHC class II-positive cells, was observed after LPS injection. CONCLUSIONS: A network of macrophages and MHC class II-positive cells in the iris and ciliary body may play an important role in immune surveillance. LPS injection induces a severe inflammation in the anterior segment of the eye, which may serve as a model for acute anterior uveitis in human.
Assuntos
Modelos Animais de Doenças , Uveíte Anterior/imunologia , Animais , Corpo Ciliar/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Iris/imunologia , Lipopolissacarídeos , Macrófagos/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Ratos , Ratos Endogâmicos Lew , Salmonella typhimurium , Uveíte Anterior/induzido quimicamenteRESUMO
Sepsis is a very heterogeneous clinical syndrome broadly defined as the systemic host response to an infection. Until very recently, the prevailing concept of the pathogenesis of sepsis was that mortality is the consequence of an uncontrolled hyperinf lammatory response of the host. The disappointing results of nearly 40 years of anti-inflammatory strategies and the development of animal models that more closely mimic clinical sepsis have led to the reconsideration of the pathophysiology of sepsis. Sepsis is now considered a misbalance between proinflammatory reactions (designed to kill invading pathogens but at the same time responsible for tissue damage) and anti-inflammatory responses (designed to limit excessive inflammation, but at the same time making the host more vulnerable for secondary infections). This review discusses key components of the pro- and anti-inflammatory response to sepsis, listing potential novel interventional strategies along the way.
Assuntos
Bacteriemia , Animais , Modelos Animais de Doenças , HumanosRESUMO
Toll-like receptors (TLRs) are pattern-recognition receptors that have been implicated in the initiation of innate immune responses upon the first encounter with invading pathogens. The airways are frequently exposed to various types of lipopolysaccharide (LPS) from the environment or from pathogens. The current study was designed to determine the effect of LPS on TLR gene expression in human alveolar macrophages in vivo. In total, 16 healthy subjects were enrolled in a single-blinded, placebo-controlled study. Subjects inhaled 100 microg LPS or normal saline (n = 8 per group). Measurements were performed in alveolar macrophages purified from bronchoalveolar lavage fluid obtained 6 h post-challenge. Inhalation of LPS by healthy human volunteers resulted in enhanced alveolar macrophage expression of mRNAs encoding TLRs 1, 2, 7, 8 and CD14, and reduced expression of mRNAs encoding TLR4 and lymphocyte antigen 96. In conclusion, lipopolysaccharide differentially influences the toll-like receptor mRNA expression profile in human alveolar macrophages in vivo.
Assuntos
Endotoxinas/toxicidade , Expressão Gênica , Lipopolissacarídeos/toxicidade , Macrófagos Alveolares/imunologia , Receptores Toll-Like/genética , Administração por Inalação , Adulto , Endotoxinas/administração & dosagem , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Humanos , Lipopolissacarídeos/administração & dosagem , Macrófagos Alveolares/efeitos dos fármacos , Masculino , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Receptores Toll-Like/efeitos dos fármacosRESUMO
PURPOSE: To investigate changes of the cornea induced by lipopolysaccharide (LPS). METHODS: Immunohistochemical study using monoclonal antibodies to monocytes, macrophages (ED1, ED2) and MHC class II antigen (OX6) was performed on the corneal wholemounts obtained from normal lewis rats and those after LPS injection. RESULTS: Macrophages were noted to be present in whole cornea with a gradually decreased cell density from limbus to central part in normal lewis rats. However, major histocompatibility complex class II antigen (MHC class II)-positive cells were only distributed at limbus. Footpad injection of LPS induced an increase of monocytes and macrophages in whole cornea and a dramatical changes of these cells morphologically. MHC class II-positive cells were only and shortly noted on the surface of the cornea endothelium at early stage after LPS injection. CONCLUSION: LPS-induced increase of macrophages in the cornea may be an important defense mechanism in response to LPS challenge. On the other hand, the absence of MHC class II-positive cells both in the normal and in the inflamed cornea may be contributed to the stability of immunological microenvironment within this tissue.