RESUMEN
PURPOSE: It is known that both human conjunctival fibroblasts (HCF) and corneal epithelial (HCE) cells contribute to the inflammatory process in the ocular surface by releasing inflammatory cytokines. In addition, nitric oxide (NO) has an important role in inflammatory responses in the ocular surface. In the present study, we aimed to characterize the capacity of these cells to release nitric oxide in response to cytokines and Lipopolysaccharide (LPS), and show that Alpha-linoleic acid (ALA) inhibits these responses. METHODS: HCF, HCE cells, peripheral blood mononuclear cells (PBMCs) and co-culture of HCF and PBMC were treated with different combinations of inflammatory inducers, including interleukin)IL- (6, tumor necrosis factors (TNF)-α, interferon (IFN)- γ and IL-1ß and LPS. Nitrite levels were measured in cell supernatants with and without ALA by the Griess reaction test at 24, 48 and 72 h respectively. Expression of nitric oxide synthase 2 (NOS-2) was evaluated by real-time PCR. RESULTS: All cytokine combinations had an inducible effect on nitrite secretion in HCF, PBMC and co-cultured PBMC and HCF, but not in HCE cells. Treatment with a combination of IL-6, LPS, TNF-α, IFN- γ and IL-1ß induced the highest nitrite secretion (2.91 fold, P < 0.01) as compared to cells incubated in medium alone. nitrite secretion was reduced by 38.9 % (P < 0.05) after treatment with ALA alone. Co-culturing PBMC with HCF with and without ALA treatment demonstrated similar results in nitrite level as,compared to PBMC alone. In addition, ALA significantly decreased NOS-2 expression in HCF by 48.9 % (P < 0. 001) after 72 h. CONCLUSIONS: The decrease in nitrite release and inhibition of NOS-2 expression indicate that ALA may have an anti-inflammatory effect both on HCF and on peripheral immune cells. This indicates that ALA may serve as a potent anti-inflammatory agent in ocular surface inflammation.
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We have previously shown that naturally occurring as well as acquired Abs against the Mycobacterium tuberculosis heat shock protein (HSP)65 protect against the induction of murine autoimmune inflammatory arthritis. In the present work, we have studied the anti-inflammatory effect of prozumab, a humanized anti-HSP mAb in murine inflammatory arthritis and colitis, and its effects on cytokine secretion. Prozumab was shown to bind to HSP60, the highly conserved mammalian homolog of the bacterial protein, and it was found to be effective in protecting and suppressing autoimmune arthritis in the models of adjuvant arthritis and collagen-induced arthritis in rats and mice, respectively, as well as in acute hapten-mediated colitis and chronic, spontaneous colitis models. Mechanistically, prozumab induces IL-10 secretion from naive human PBMCs and suppresses the secretion of IFN-γ and IL-6 from anti-CD3-activated human PBMCs. These findings make prozumab a promising potential drug for treating human rheumatoid arthritis and inflammatory bowel disease, as well as a wide range of autoimmune inflammatory diseases.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Chaperonina 60/antagonistas & inhibidores , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Animales , Proteínas Bacterianas/inmunología , Línea Celular Tumoral , Chaperonina 60/inmunología , Femenino , Humanos , Interferón gamma/metabolismo , Interleucina-10/biosíntesis , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Células Jurkat , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Unión Proteica/inmunología , Ratas , Ratas Endogámicas LewRESUMEN
BACKGROUND: Toll-like receptors (TLRs) are recognized as important contributors to the initiation and modulation of the inflammatory response in the eye. This study investigated the precise expression patterns and functionality of TLRs in human corneal epithelial cells (HCE) and in conjunctival fibroblasts (HCF). METHODS: The cell surface expression of TLRs 2-4, TLR7 and TLR9 in HCE and HCF was examined by flow cytometry with or without stimulation with lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C). The mRNA expression of the TLRs was determined by real-time PCR. The protein content levels of interleukin (IL)-6, IL-8, IL-1ß and tumor necrosis factor-α (TNF-α) were measured in HCE and HCF using multiplex fluorescent bead immunoassay (FBI). RESULTS: The surface expression of TLR3 and TLR4 was detected on both HCE and HCF. Following incubation with LPS, the percentage of HCE cells staining for TLR4 decreased from 10.18% to 0.62% (P < 0.001). Incubation with poly I:C lowered the percentage of HCE cells positive for TLR3 from 10.44% to 2.84% (P < 0.001). The mRNA expression of TLRs2, 4, 7 and 9 was detected in HCE only. Activation of HCE with LPS complex elicited protein secretion up to 4.51 ± 0.85-fold higher levels of IL-6 (P < 0.05), 2.5 ± 0.36-fold IL-8 (P > 0.05), 4.35 ± 1.12-fold IL-1ß (P > 0.05) and 29.35 ± 2.3-fold TNFα (P < 0.05) compared to cells incubated in medium. CONCLUSIONS: HCF and HCE both express TLRs that respond to specific ligands by increasing cytokine expression. Following activation, the surface expression of TLR3 and TLR4 on HCE is decreased, thus creating a negative feedback loop, mitigating the effect of TLR activation.
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Autoimmunity is the result of an abnormal immune response against constituents of body tissues. For many years, the study of animal models of human diseases was aimed at defining the factors participating in the autoimmune process. During the past two decades, much of the attention was diverted to another intriguing aspect of animal models: the mechanisms rendering some animal strains autoimmune-susceptible and others resistant. In this report, we focus on one experimental model, adjuvant arthritis (AA) which is inducible in the Lewis rat following stimulation of the immune system by heat-killed mycobacterium and its 65kDa heat shock protein. We describe genetic loci regulating the severity of this disease as well as the contribution of microbial flora and endocrine activity to susceptibility and resistance. In our opinion, a better understanding of the processes underlying susceptibility and resistance to AA is an important step towards the development of new therapeutic approaches to autoimmunity.
Asunto(s)
Artritis Experimental/genética , Predisposición Genética a la Enfermedad , Adyuvantes Inmunológicos/farmacología , Animales , Autoinmunidad , Linfocitos B/metabolismo , Susceptibilidad a Enfermedades , Sistema Endocrino/patología , Proteínas de Choque Térmico/metabolismo , Humanos , Sistema Inmunológico , Interleucina-10/metabolismo , Ratones , Ratas , Ratas Endogámicas Lew , Linfocitos T/metabolismoRESUMEN
Adjuvant arthritis (AA) is an experimental model of autoimmune arthritis that can be induced in susceptible strains of rats such as inbred Lewis upon immunization with CFA. AA cannot be induced in resistant strains like Brown-Norway or in Lewis rats after recovery from arthritis. We have previously shown that resistance to AA is due to the presence of natural as well as acquired anti-heat shock protein (HSP) Abs. In this work we have studied the fine specificity of the protective anti-HSP Abs by analysis of their interaction with a panel of overlapping peptides covering the whole HSP molecule. We found that arthritis-susceptible rats lack Abs to a small number of defined epitopes of the mycobacterial HSP65. These Abs are found naturally in resistant strains and are acquired by Lewis rats after recovery from the disease. Active vaccination of Lewis rats with the protective epitopes as well as passive vaccination with these Abs induced suppression of arthritis. Incubation of murine and human mononuclear cells with the protective Abs induced secretion of IL-10. Analysis of the primary and tertiary structure of the whole Mycobacterium tuberculosis HSP65 molecule indicated that the protective epitopes are B cell epitopes with nonconserved amino acid sequences found on the outer surface of the molecule. We conclude that HSP, the Ag that contains the pathogenic T cell epitopes in AA, also contains protective B cell epitopes exposed on its surface, and that natural and acquired resistance to AA is associated with the ability to respond to these epitopes.