RESUMO
Dry eye is an allegedly autoimmune disorder for which the initiating mechanisms and the targeted antigens in the ocular surface are not known, yet there is extensive evidence that a localized T helper type 1 (Th1)/Th17 effector T cell response is responsible for its pathogenesis. In this work, we explore the reconciling hypothesis that desiccating stress, which is usually considered an exacerbating factor, could actually be sufficient to skew the ocular surface's mucosal response to any antigen and therefore drive the disease. Using a mouse model of dry eye, we found that desiccating stress causes a nuclear factor kappa B (NF-κB)- and time-dependent disruption of the ocular surface's immune tolerance to exogenous ovalbumin. This pathogenic event is mediated by increased Th1 and Th17 T cells and reduced regulatory T cells in the draining lymph nodes. Conversely, topical NF-κB inhibitors reduced corneal epithelial damage and interleukin (IL)-1ß and IL-6 levels in the ocular surface of mice under desiccating stress. The observed effect was mediated by an augmented regulatory T cell response, a finding that highlights the role of mucosal tolerance disruption in dry eye pathogenesis. Remarkably, the NF-κB pathway is also involved in mucosal tolerance disruption in other ocular surface disorders. Together, these results suggest that targeting of mucosal NF-κB activation could have therapeutic potential in dry eye.
Assuntos
Síndromes do Olho Seco/imunologia , Epitélio Corneano/fisiopatologia , Tolerância Imunológica/imunologia , NF-kappa B/metabolismo , Estresse Fisiológico/imunologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Epitélio Corneano/imunologia , Epitélio Corneano/lesões , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Linfonodos/citologia , Linfonodos/imunologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , NF-kappa B/antagonistas & inibidores , Ovalbumina/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologiaRESUMO
The impact of topical eye drops with benzalkonium chloride (BAK) as a preservative could involve more than the reported toxic effects on the ocular surface epithelium and ultimately affect the immune balance of the conjunctiva. We found that BAK not only impairs tolerance induction in a murine model, but leads to mild systemic immunization. Contrasting with antigen only-treated mice, there was no induction of interleukin 10-producing antigen-specific CD4(+) cells in BAK-treated animals. Moreover, the tolerogenic capacity of migrating dendritic cells (DCs) was reduced, apparently involving differential conditioning by soluble epithelial factors. Accordingly, epithelial cells exposed in vitro to BAK were less suppressive and failed to induce tolerogenic DCs in culture. As this effect of BAK was dependent on epithelial nuclear factor κB pathway activation, our findings may provide new therapeutic targets. Thus, tolerance breakdown by BAK should be considered an important factor in the management of glaucoma and immune-mediated ocular surface disorders.
Assuntos
Compostos de Benzalcônio/farmacologia , Túnica Conjuntiva/efeitos dos fármacos , Túnica Conjuntiva/imunologia , Tolerância Imunológica/efeitos dos fármacos , Animais , Compostos de Benzalcônio/toxicidade , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Camundongos , NF-kappa B/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologiaRESUMO
We examined the ability of TNF-alpha to modulate human neutrophil apoptosis. Neutrophils cultured with TNF-alpha alone undergo a low but significant increase in the number of apoptotic cells. More interestingly, when neutrophils were pretreated with TNF-alpha for 1-2 min at 37 degrees C and then were exposed to a variety of agents such as immobilized IgG, IgG-coated erythrocytes, complement-treated erythrocytes, zymosan, PMA, zymosan-activated serum, fMLP, Escherichia coli, and GM-CSF for 3 h at 37 degrees C, a marked stimulation of apoptosis was observed. Similar results were obtained in neutrophils pretreated with TNF-alpha for 30 min, 1 h, 3 h, and 18 h. Dose-dependent studies showed that TNF-alpha enhances neutrophil apoptosis at concentrations ranging from 1 to 100 ng/ml. In contrast to the observations made in neutrophils pretreated with TNF-alpha, there was no stimulation of apoptosis when TNF-alpha was added to neutrophils previously activated by conventional agonists. Experiments performed to establish the mechanism through which TNF-alpha promotes neutrophil apoptosis showed that neither reactive oxygen intermediates nor the Fas/Fas ligand system appear to be involved. Our results suggest that TNF-alpha plays a critical role in the control of neutrophil survival by virtue of its ability to induce an apoptotic death program which could be triggered by a variety of conventional agonists.
Assuntos
Apoptose/imunologia , Neutrófilos/citologia , Neutrófilos/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Anexina A5/metabolismo , Sangue/imunologia , Membrana Celular/imunologia , Membrana Celular/metabolismo , Sobrevivência Celular/imunologia , Células Cultivadas , Meios de Cultivo Condicionados , Citocinas/fisiologia , Relação Dose-Resposta Imunológica , Proteína Ligante Fas , Humanos , Interfase/imunologia , Ligantes , Glicoproteínas de Membrana/fisiologia , Neutrófilos/metabolismo , Fosfatidilserinas/metabolismo , Ligação Proteica/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptor fas/fisiologiaRESUMO
The interaction of immunoglobulin G (IgG) antibodies with FcgammaR constitutes a critical mechanism through which IgG antibody effector functions are mediated. In the current work we have examined whether human neutrophil FcgammaR exhibit pH dependence in their association with IgG. Binding assays were performed in culture medium adjusted to different pH values. It was found that the binding of either heat-aggregated human IgG (AIgG), soluble immune complexes (sIC) or IgG-coated erythrocytes (IgG-E) was markedly higher at pH 6.5 than at pH 7.3. This effect was not observed when saturation of FcgammaR was achieved, suggesting that acidic pH increases the avidity of FcgammaR for IC without modifying the total binding capacity. Similar results were observed for the binding of AIgG to either monocytes, natural killer (NK) or K562 cells, suggesting that acidic pH increases the avidity of both, FcgammaRII and FcgammaRIII. Additional experiments were performed to analyse whether the binding of IgG to FcgammaRI also showed pH dependence. To this aim, we employed interferon-gamma-treated human neutrophils and mouse inflammatory macrophages, previously incubated with blocking antibodies directed to FcgammaRII and FcgammaRIII. Acidic pH did not enhance the binding of AIgG nor monomeric IgG under these experimental conditions. Further studies are required to determine whether the enhancement of FcgammaR avidity for IC could be attributed to titration of histidine(s) residues on the Fc fragment of IgG.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Neutrófilos/imunologia , Receptores de IgG/imunologia , Células Cultivadas , Citometria de Fluxo , Humanos , Concentração de Íons de Hidrogênio , Leucócitos/imunologia , Ligação ProteicaRESUMO
In the current work, we evaluated the effect of extracellular acidification on neutrophil physiology. Neutrophils suspended in bicarbonate-buffered RPMI 1640 medium adjusted to acidic pH values (pH 6.5-7.0) underwent: 1) a rapid transient increase in intracellular free calcium concentration levels; 2) an increase in the forward light scattering properties; and 3) the up-regulation of surface expression of CD18. By contrast, extracellular acidosis was unable to induce neither the production of H2O2 nor the release of myeloperoxidase. Acidic extracellular pH also modulated the functional profile of neutrophils in response to conventional agonists such as FMLP, precipiting immune complexes, and opsonized zymosan. It was found that not only calcium mobilization, shape change response, and up-regulation of CD18 expression but also production of H2O2 and release of myeloperoxidase were markedly enhanced in neutrophils stimulated in acidic pH medium. Moreover, extracellular acidosis significantly delayed neutrophil apoptosis and concomitantly extended neutrophil functional lifespan. Extracellular acidification induced an immediate and abrupt fall in the intracellular pH, which persisted over the 240-s analyzed. A similar abrupt drop in the intracellular pH was detected in cells suspended in bicarbonate-supplemented PBS but not in those suspended in bicarbonate-free PBS. A role for intracellular acidification in neutrophil activation is suggested by the fact that only neutrophils suspended in bicarbonate-buffered media (i.e., RPMI 1640 and bicarbonate-supplemented PBS) underwent significant shape changes in response to extracellular acidification. Together, our results support the notion that extracellular acidosis may intensify acute inflammatory responses by inducing neutrophil activation as well as by delaying spontaneous apoptosis and extending neutrophil functional lifespan.
Assuntos
Espaço Extracelular/metabolismo , Ativação de Neutrófilo , Complexo Antígeno-Anticorpo/farmacologia , Antígenos CD18/biossíntese , Antígenos CD18/sangue , Sinalização do Cálcio/imunologia , Tamanho Celular/imunologia , Sobrevivência Celular/imunologia , Citoplasma/metabolismo , Espaço Extracelular/imunologia , Espaço Extracelular/fisiologia , Humanos , Ácido Clorídrico , Peróxido de Hidrogênio/sangue , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Soluções Isotônicas , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Peroxidase/sangue , Peroxidase/metabolismo , Zimosan/farmacologiaRESUMO
We analyzed the effect of nitric oxide (NO) on oxygen-dependent cytotoxic responses mediated by neutrophils against unopsonized erythrocytes using three NO donors: S-nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine (SNAP), and sodium nitroprusside (SNP). Neutrophils were treated with these compounds for 1-2 min at 37 degrees C and cytotoxicity was then triggered in the presence of NO donors by precipitating immune complexes, aggregated IgG, the chemotactic peptide FMLP, or opsonized zymosan. GSNO induced, in all cases, a marked increase in cytotoxic responses, while SNAP moderately increased cytotoxicity triggered by immune complexes, aggregated IgG, or Z, opsonized zymosen, without modifying those responses induced by FMLP. By contrast, SNP dramatically suppressed cytotoxicity triggered by all of the stimuli assessed. The enhancing effects mediated by GSNO and SNAP did not depend on the stimulation of guanylyl cyclase and were prevented by the NO scavengers hemoglobin and PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl 3-oxide). The inhibitory activity of SNP, on the other hand, was not prevented by NO scavengers, suggesting that it cannot be ascribed to the release of NO. In another set of experiments, neutrophils were pretreated with GSNO or SNAP for different times. Then cells were washed to remove NO donors from the culture medium, and cytotoxicity was triggered by different stimuli. It was found that neutrophils must be pretreated with NO donors for at least 4 h to increase cytotoxic responses, and pretreatment for longer periods (i.e., 8 or 18 h) further increased cytotoxicity. Not only cytotoxic responses, but also the production of O2- and H2O2, and the release of myeloperoxidase were increased under these conditions.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Neutrófilos/imunologia , Doadores de Óxido Nítrico/farmacologia , Oxigênio/farmacologia , Guanilato Ciclase/fisiologia , Humanos , Peróxido de Hidrogênio/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Zimosan/farmacologiaRESUMO
In the present study we examined whether immune complexes (IC) are able to modulate human neutrophil apoptosis. We observed different effects depending on the type of IC employed. Precipitating IC (pIC) and Ab-coated erythrocytes (E-IgG) triggered a marked stimulation of apoptosis, while heat-aggregated IgG and soluble IC, significantly delayed spontaneous apoptosis. Blocking Abs directed to Fcgamma receptor type II (FcgammaRII), but not to FcgammaRIII, markedly diminished the acceleration of apoptosis triggered by either pIC or E-IgG, supporting a critical role for FcgammaRII in apoptosis stimulation. This phenomenon, on the other hand, does not appear to involve IC phagocytosis or the participation of CR3. Acceleration of neutrophil apoptosis triggered by either pIC or E-IgG seems to require the activation of the respiratory burst, as suggested by 1) the ability of catalase to prevent apoptosis stimulation; 2) the effect of azide, an heme enzyme inhibitor, which dramatically enhanced apoptosis induced by pIC or E-IgG; and 3) the inability of pIC or E-IgG to accelerate apoptosis of neutrophils isolated from CGD patients. It is well established that IC affect the course of inflammation by inducing the release of inflammatory cytokines, proteolytic enzymes, oxidative agents, and other toxic molecules. Our results suggest that IC may also affect the course of inflammation by virtue of their ability to modulate neutrophil apoptosis.
Assuntos
Complexo Antígeno-Anticorpo/fisiologia , Apoptose/imunologia , Neutrófilos/imunologia , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Cálcio/metabolismo , Precipitação Química , Citosol/metabolismo , Eritrócitos/imunologia , Proteína Ligante Fas , Doença Granulomatosa Crônica/imunologia , Doença Granulomatosa Crônica/patologia , Temperatura Alta , Humanos , Imunoglobulina G/fisiologia , Ligantes , Antígeno de Macrófago 1/fisiologia , Glicoproteínas de Membrana/fisiologia , Ativação de Neutrófilo/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Fagocitose , Espécies Reativas de Oxigênio/fisiologia , Receptores de IgG/fisiologia , Explosão Respiratória/imunologia , Solubilidade , Receptor fas/fisiologiaRESUMO
Losartan, a selective antagonist of AT1 receptors for angiotensin II, is widely used clinically to manage hypertension. We report here that losartan markedly inhibits neutrophil shape change, adherence and chemiluminescence responses triggered by N-formylmethionyl-leucyl-phenylalanine (fMLP), without affecting responses induced by immune complexes, zymosan or concanavalin A. Neither saralasin, another antagonist of angiotensin II receptors, nor captopril, an angiotensin-converting enzyme inhibitor, reproduced the effects of losartan. It was also observed that neutrophil responses triggered by fMLP were not affected by exogenously added angiotensin II. The effect of losartan on the binding of fMLP was measured using [3H]fMLP. It was found that losartan inhibits the binding of [3H]fMLP to neutrophil receptors. As observed for neutrophils, studies performed with monocytes showed that losartan inhibits chemiluminescence emission triggered by fMLP, without affecting chemiluminescence responses triggered by immune complexes, zymosan or concanavalin A.
Assuntos
Angiotensina I/metabolismo , Antagonistas de Receptores de Angiotensina , Compostos de Bifenilo/farmacologia , Imidazóis/farmacologia , N-Formilmetionina Leucil-Fenilalanina/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Tetrazóis/farmacologia , Anti-Hipertensivos/farmacologia , Humanos , Losartan , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/metabolismo , Receptores de Angiotensina/metabolismoRESUMO
In the absence of appropriate stimuli, polymorphonuclear neutrophils rapidly undergo characteristic changes indicative of programmed cell death or apoptosis. We report here that neutrophils cultured in the presence of platelets (neutrophil:platelet ratios of 1:50, 1:25, and 1:10) show a dramatic inhibition of apoptosis compared with neutrophils cultured alone. Similar degrees of apoptosis delay were induced by viable unstimulated platelets, fixed unstimulated platelets, or fixed activated (1 U/ml thrombin) platelets. Inhibition of apoptosis was associated with prolongation of the functional lifespan of the neutrophil, as indicated by the higher capacity of platelet-treated neutrophils to display chemiluminescence responses triggered by FMLP, immune complexes, and zymosan. The mechanism responsible for the inhibition of neutrophil apoptosis by platelets has not yet been defined. However, it seems that classical recognition systems such as those mediated by the interaction between platelet P-selectin (CD62) or glycoprotein IIb/IIIa complex and their counter-receptors expressed by neutrophils are not involved.
Assuntos
Apoptose/imunologia , Plaquetas/imunologia , Neutrófilos/imunologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/imunologia , Técnicas de Cocultura , Humanos , Neutrófilos/efeitos dos fármacos , Selectina-P/fisiologiaRESUMO
The role of nitric oxide in the regulation of adrenal steroidogenesis was examined in BALB/c mice by employing the nitric oxide synthase inhibitors NG-nitro L-arginine methyl ester (L-NAME) and NG-nitro L-arginine (L-NNA). The administration of a single dose of nitric oxide inhibitors (50 mg kg-1 body wt. i.p.) induced a fourfold increase in plasma corticosterone. Treatment with L-arginine (750 mg kg-1 body wt. s.c.), but not D-arginine, completely prevented corticosterone increases induced by L-NAME. To analyse whether the activation of adrenal steroidogenesis induced by nitric oxide synthase inhibitors involved the stimulation of the hypothalamo-pituitary-adrenal axis. ACTH levels were assessed. It was found that L-NAME significantly enhanced plasma ACTH concentrations. Genetic variations in this regulatory pathway are suggested by the fact that L-NAME increased corticosterone levels in BALB/c. C3H/He and DBA-2 mice, but not in C57BI/c mice, a strain characterized by a low steroid response to stress.