RESUMO
PURPOSE: Previous studies have shown that in Pseudomonas aeruginosa ocular infection, IL-12 drives a Th1 T-cell response and IFN-gamma production in susceptible (cornea perforates) C57BL/6 (B6) mice, and that after similar infection of resistant (cornea heals) BALB/c mice, no IL-12 is detectable in cornea at either the mRNA or protein levels. Therefore, the purpose of this study was to test whether BALB/c mice are capable of responding to exogenous IL-12 administration, and whether disease responsiveness following P. aeruginosa challenge is modified. METHODS: Immunostaining, RT/PCR, recombinant cytokine injection, and histopathology were used. Statistical analysis was performed using an unpaired, two-tailed Student's t-test. RESULTS: Injection of BALB/c mice with recombinant (r) IL-12 converted these normally resistant animals to the susceptible phenotype as evidenced by corneal perforation within 5-7 days after infection. RT-PCR analysis of the corneas of rIL-12 vs PBS/BSA-treated mice showed a significant increase in IFN-gamma and TNF-alpha mRNA levels in the rIL-12 vs PBS/BSA (vehicle)-treated mice at 3 and 5 days p.i. In addition, similar analysis of IL-4 mRNA levels showed decreased amounts of the cytokine in rIL-12 vs vehicle-treated mice. Injection of rIL-4 into susceptible B6 mice, however, failed to rescue these animals from corneal perforation following P. aeruginosa challenge. CONCLUSIONS: These data provide evidence that BALB/c mice can respond to exogenous IL-12, that the cytokine promotes susceptibility by increasing IFN-gamma and TNF-alpha production, with a concomitant reduction in IL-4 levels; and that injected rIL-4 fails to rescue susceptible B6 mice from corneal perforation after bacterial challenge.
Assuntos
Úlcera da Córnea/imunologia , Infecções Oculares Bacterianas/imunologia , Interleucina-12/imunologia , Infecções por Pseudomonas/imunologia , Animais , Úlcera da Córnea/patologia , Progressão da Doença , Infecções Oculares Bacterianas/patologia , Feminino , Técnicas Imunoenzimáticas , Mediadores da Inflamação/metabolismo , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-12/toxicidade , Interleucina-4/biossíntese , Interleucina-4/genética , Interleucina-4/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Infecções por Pseudomonas/patologia , RNA Mensageiro/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/toxicidade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de Doença , Células Th1/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genéticaRESUMO
The role of macrophage inflammatory protein-1alpha (MIP-1alpha) in cell infiltration into Pseudomonas aeruginosa-infected cornea and subsequent disease was examined. Greater amounts of the chemokine (protein and mRNA) were found in the infected cornea of susceptible B6 ("cornea perforates") versus resistant BALB/c ("cornea heals") mice from 1 to 5 days postinfection. Treatment of BALB/c mice with recombinant (r) MIP-1alpha exacerbated disease and was associated with an increased number of neutrophils (PMNs) in the cornea. Treatment of BALB/c mice with rMIP-1alpha also induced recruitment of activated CD4+ T cells into the affected cornea, converting resistant to susceptible mice. Depleting CD4+ T cells in r-treated BALB/c mice significantly decreased PMNs in cornea tissue, suggesting that T cells regulate persistence of PMNs at this site. In B6 mice, administration of neutralizing MIP-1alpha polyclonal antibody also significantly reduced PMN numbers and pathology. Collectively, evidence is provided that MIP-1alpha directly contributed to CD4+ T cell recruitment and indirectly to PMN persistence in the infected cornea.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Doenças da Córnea/imunologia , Infecções Oculares Bacterianas/imunologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Neutrófilos/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Quimiocina CCL3 , Quimiocina CCL4 , Imunidade Celular/efeitos dos fármacos , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Proteínas Recombinantes/farmacologiaRESUMO
The anterior surface of the eye is composed of the cornea, conjunctiva, and the zone between the two called the limbus. The cornea must maintain optical clarity to retain good vision. However, the ocular surface is vulnerable to trauma, microbial infection, and exposure to environmental toxins. This places the cornea, especially, at risk for disruptions of the epithelial barrier and subsequent immunopathological events. Cell-cell and cell-matrix attachment junctions incorporating adhesion molecules ensure that the epithelial barrier remains intact. Protein components of the basement membrane, including laminins, are vital to the adhesion of corneal epithelial cells to the underlying stroma and function to enhance the strength of the bond between epithelium and connective tissue. Epithelial cells also play an early and crucial role in the initiation of ocular surface responses should a potentially antigenic molecule enter into deeper corneal tissues. For example, epithelial cells may produce and release cytokines such as interleukin-1 (IL-1). The delicate balance between the matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are central to mechanisms regulating dissolution of the extracellular matrix that may be a consequence of infection or wound healing. Adhesion molecules, cytokines and chemokines, and MMPs and TIMPs thus participate in the corneal response to immunologic challenge or wounding. They may also be involved in corneal pathologies associated with genetic diseases, diabetes, and vitamin A deficiency. In addition these molecules are components of cellular pathways underlying the clinical complications often observed with contact lens wear and refractive surgeries used to improve visual acuity.
Assuntos
Córnea/anatomia & histologia , Córnea/fisiologia , Animais , Córnea/metabolismo , Córnea/ultraestrutura , Doenças da Córnea/etiologia , Doenças da Córnea/patologia , Doenças da Córnea/fisiopatologia , Doenças da Córnea/terapia , Lesões da Córnea , HumanosRESUMO
PURPOSE: Previous data has shown that basement membrane associated perlecan serves as a binding site for Pseudomonas aeruginosa in the wounded mouse cornea. The current study determined whether it also provides a binding site for Pseudomonas aeruginosa in transformed human corneal epithelium. METHODS: Bacterial adherence to transformed human corneal epithelial cells grown in normal or in media containing various inhibitors of glycosaminoglycan synthesis was tested. Bacterial binding was similarly tested in wild-type and in mutant Chinese hamster ovary cell lines naturally deficient in glycosaminoglycan synthesis. Transformed human corneal epithelial extracellular matrix also was tested before and after treatment with anti-proteoglycan monoclonal antibodies or heparinase III before bacterial inoculation. Scanning electron microscopy was used to quantitate adherent bacteria. Intact transformed human corneal epithelial cells or extracellular matrix, the latter either treated or not treated with heparinase III or chondroitin ABC lyase were stained to localize perlecan. RESULTS: Examination of the binding of bacteria to transformed human corneal epithelial cells (normal media vs with inhibitors) and Chinese hamster ovary cell lines suggested that bacterial binding was not associated with the surface of either cell type. In contrast, anti-perlecan antibody, as well as heparinase III decreased the binding of bacteria to corneal extracellular matrix. Fluorescence staining localized perlecan to the extracellular matrix beneath the corneal epithelial cells. CONCLUSIONS: Perlecan localized to the extracellular matrix but not the apical surface of transformed human corneal epithelial cells, provides a binding site for Pseudomonas aeruginosa.
Assuntos
Aderência Bacteriana , Epitélio Corneano/metabolismo , Epitélio Corneano/microbiologia , Matriz Extracelular/metabolismo , Matriz Extracelular/microbiologia , Proteoglicanas de Heparan Sulfato/metabolismo , Pseudomonas aeruginosa/fisiologia , Animais , Anticorpos Monoclonais , Células CHO/microbiologia , Células Cultivadas , Cricetinae , Epitélio Corneano/ultraestrutura , Matriz Extracelular/ultraestrutura , Proteínas do Olho/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Microscopia Eletrônica de Varredura , Microscopia de FluorescênciaRESUMO
Evidence suggests that Pseudomonas aeruginosa stromal keratitis and corneal perforation (susceptibility) is a CD4(+) T cell-regulated inflammatory response following experimental P. aeruginosa infection. This study examined the role of Langerhans cells (LC) and the B7/CD28 costimulatory pathway in P. aeruginosa-infected cornea and the contribution of costimulatory signaling by this pathway to disease pathology. After bacterial challenge, the number of LC infiltrating the central cornea was compared in susceptible C57BL/6 (B6) vs resistant (cornea heals) BALB/c mice. LC were more numerous at 1 and 6 days postinfection (p.i.), but were similar at 4 days p.i., in susceptible vs resistant mice. Mature, B7 positive-stained LC in the cornea and pseudomonas Ag-associated LC in draining cervical lymph nodes also were increased significantly p.i. in susceptible mice. To test the relevance of these data, B6 mice were treated systemically and subconjunctivally with neutralizing B7 (B7-1/B7-2) mAbs. Treatment decreased corneal disease severity and reduced significantly the number of B7-positive cells as well as the recruitment and activation of CD4(+) T cells in the cornea. IFN-gamma mRNA levels also were decreased significantly in the cornea and in draining cervical lymph nodes of mAb-treated mice. When CD28(-/-) animals were tested, they exhibited a less severe disease response (no corneal perforation) than wild-type B6 mice and had a significantly lower delayed-type hypersensitivity response to heat-killed pseudomonas Ag. These results support a critical role for B7/CD28 costimulation in susceptibility to P. aeruginosa ocular infection.
Assuntos
Anticorpos Bloqueadores/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antígeno B7-1/fisiologia , Antígenos CD28/fisiologia , Doenças da Córnea/imunologia , Infecções por Pseudomonas/imunologia , Animais , Anticorpos Bloqueadores/análise , Anticorpos Monoclonais/análise , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/imunologia , Apirase/análise , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Antígeno B7-2 , Antígenos CD28/genética , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Movimento Celular/imunologia , Doenças da Córnea/enzimologia , Doenças da Córnea/patologia , Feminino , Predisposição Genética para Doença , Injeções Intraperitoneais , Células de Langerhans/enzimologia , Células de Langerhans/imunologia , Células de Langerhans/patologia , Linfonodos/imunologia , Linfonodos/patologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Pseudomonas/enzimologia , Infecções por Pseudomonas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Coloração e RotulagemRESUMO
PURPOSE: Alkaline protease has been associated with virulence in Pseudomonas aeruginosa corneal infections. To define the role of this enzyme in such infections, isogenic mutants of P. aeruginosa deficient in alkaline protease production were constructed. This study examines the ability of these mutants to adhere to scarified corneal tissue in vitro and to establish corneal infections in vivo. METHODS: Mutants were constructed by allelic exchange in two phenotypically different wild type strains, PAO1 (invasive) and ATCC 19660 (cytotoxic). Alkaline protease-deficient mutants were characterized by zymography and western blot analysis of bacterial culture supernatants. Allelic exchange was confirmed by PCR analysis of the disrupted aprA gene of the mutants. Adherence of wild type and mutant strains to scarified corneal epithelium was assessed by an in vitro organ culture assay, while ocular virulence of the strains was determined in vivo using a mouse scarification model of bacterial keratitis. RESULTS: Being isogenic, phenotypes of mutants were identical to their respective parents with the exception of the loss of alkaline protease production. The absence of alkaline protease did not alter corneal adherence or ocular virulence of the organisms when compared to similar wild type strains. CONCLUSIONS: These data provide evidence that alkaline protease produced by P. aeruginosa is not essential in the pathogenesis of P. aeruginosa keratitis.
Assuntos
Aderência Bacteriana , Córnea/microbiologia , Úlcera da Córnea/microbiologia , Infecções Oculares Bacterianas/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Serina Endopeptidases/fisiologia , Animais , Proteínas de Bactérias , Western Blotting , Úlcera da Córnea/patologia , Primers do DNA/química , DNA Bacteriano/análise , Endopeptidases/genética , Infecções Oculares Bacterianas/patologia , Feminino , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Reação em Cadeia da Polimerase , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/enzimologia , Serina Endopeptidases/deficiência , VirulênciaRESUMO
PURPOSE: To test the hypothesis that a membrane-spanning mucin, Muc1, facilitates the spread of tear film and protects against bacterial adherence. METHODS: Age-matched, Muc1 null mice and wild-type mice of C57BL/6 genetic background were used for comparison. Eyes were examined by slit lamp biomicroscopy with fluorescein solution to assess epithelial damage and tear film stability. Structure of the ocular surface epithelia was examined by light microscopy, scanning and transmission electron microscopy, and wholemount confocal microscopy. Bacterial adherence assay was performed on in vivo corneas with Pseudomonas aeruginosa containing a plasmid encoding green fluorescent protein, followed by wholemount confocal microscopy. Real-time reverse transcription-polymerase chain reaction was performed using Muc4-specific primers to quantitate Muc4 mRNA expression in ocular surface tissues. RESULTS: No differences were found between Muc1 null and control mice in any parameter tested. Ocular surface epithelia of Muc1 null mice of the C57BL/6 strain had a normal appearance of surface microplicae, a well-developed glycocalyx on the apical cell membrane, and a normal appearance of goblet cell mucin packets. There was no convincing evidence that bacterial adherence on the cornea was increased in Muc1 null mice. Muc4 mRNA expression was not upregulated in Muc1 null mice compared with control. No ocular surface infections were observed in Muc1 null mice of the C57BL/6 strain (n = 204), which were housed in the animal facility over a period of 26 months. CONCLUSIONS: Muc1 null mice of C57BL/6 background appeared normal in all respects tested. These data differ from the reported phenotype in the mice of the C57BL/6 x SVJ129 background, which show development of blepharitis and conjunctivitis.
Assuntos
Epitélio Corneano/ultraestrutura , Células Caliciformes/ultraestrutura , Mucina-1/fisiologia , Animais , Aderência Bacteriana/fisiologia , Primers do DNA/química , Epitélio Corneano/metabolismo , Epitélio Corneano/microbiologia , Células Caliciformes/metabolismo , Células Caliciformes/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Mucina-4 , Mucinas/genética , Mutação , Fenótipo , Pseudomonas aeruginosa/metabolismo , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vesículas Secretórias/metabolismo , Vesículas Secretórias/microbiologia , Vesículas Secretórias/ultraestruturaRESUMO
PURPOSE: Alterations in immune system function associated with aging may contribute to increased morbidity in this population of individuals. The current studies were performed to determine aging-related changes in polymorphonuclear neutrophil (PMN) function after corneal infection with Pseudomonas aeruginosa. METHODS: Total PMN number, macrophage inflammatory protein (MIP)-2 mRNA and protein expression, and ocular bacterial load were determined in 8-week- and 12-month-old inbred BALB/c mice at various times after infection with P. aeruginosa. In addition, 12-month-old mice were treated systemically with the MIP-2 polyclonal antibody (pAb) to determine the effects of MIP-2 neutralization on ocular disease and PMN recruitment. RESULTS: Histologically, PMN infiltration into the cornea of 12-month-old mice was delayed initially and was associated with an inability to reduce bacterial load at later postinfection (PI) times. In addition, a significantly greater number of PMNs were found in the cornea of 12-month-old mice at later PI times. The increase in PMN number in 12-month-old mice correlated with a persistence of MIP-2 expression in cornea at these later times. Systemic treatment of 12-month-old mice with neutralizing MIP-2 pAb versus normal rabbit serum (NRS) resulted in reduced corneal PMN number and ocular disease. CONCLUSIONS: These data provide evidence that persistence of PMN in the cornea of 12-month-old mice contributes to corneal tissue destruction after P. aeruginosa challenge. Further evidence also is provided that the chemoattractant MIP-2 contributes to the altered PMN response in these animals.
Assuntos
Envelhecimento/imunologia , Úlcera da Córnea/imunologia , Infecções Oculares Bacterianas/imunologia , Neutrófilos/fisiologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/isolamento & purificação , Animais , Quimiocina CXCL2 , Úlcera da Córnea/microbiologia , Úlcera da Córnea/patologia , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/patologia , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos BALB C , Monocinas/genética , Monocinas/imunologia , Infiltração de Neutrófilos/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , RNA Mensageiro/metabolismoRESUMO
The kinetics of IL-1 (alpha and beta) production after Pseudomonas aeruginosa corneal infection was examined in susceptible (cornea perforates) C57BL/6J (B6) and resistant (cornea heals) BALB/cByJ (BALB/c) mice. IL-1alpha and -1beta (mRNA and protein) were elevated in both mouse strains, and levels peaked at 1 day postinfection (p.i. ). Significantly greater amounts of IL-1 protein were detected in B6 vs BALB/c mice at 1 and 3 days p.i. At 5 days p.i., IL-1alpha and -1beta (mRNA and protein) remained elevated in B6, but began to decline in BALB/c mice. To test the significance of elevated IL-1 in B6 mice, a polyclonal neutralizing Ab against IL-1beta was used to treat infected B6 mice. A combination of subconjunctival and i.p. administration of IL-1beta polyclonal Ab significantly reduced corneal disease. The reduction in disease severity in infected B6 mice was accompanied by a reduction in corneal polymorphonuclear neutrophil number, bacterial load, and macrophage inflammatory protein-2 mRNA and protein levels. These data provide evidence that IL-1 is an important contributor to P. aeruginosa corneal infection. At least one mechanism by which prolonged and/or elevated IL-1 expression contributes to irreversible corneal tissue destruction appears to be by increasing macrophage inflammatory protein-2 production, resulting in a prolonged stimulation of polymorphonuclear neutrophil influx into cornea. In contrast, a timely down-regulation of IL-1 appears consistent with an inflammatory response that is sufficient to clear the bacterial infection with less corneal damage.
Assuntos
Quimiocinas/biossíntese , Úlcera da Córnea/imunologia , Infecções Oculares Bacterianas/imunologia , Interleucina-1/metabolismo , Neutrófilos/imunologia , Infecções por Pseudomonas/imunologia , Animais , Quimiocina CXCL2 , Quimiocinas/antagonistas & inibidores , Quimiocinas/genética , Contagem de Colônia Microbiana , Úlcera da Córnea/microbiologia , Úlcera da Córnea/patologia , Suscetibilidade a Doenças , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/patologia , Feminino , Soros Imunes/administração & dosagem , Imunidade Inata , Interleucina-1/antagonistas & inibidores , Interleucina-1/genética , Interleucina-1/imunologia , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/patologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , RNA Mensageiro/metabolismo , Ruptura EspontâneaRESUMO
PURPOSE: To determine whether binding of Pseudomonas aeruginosa (P. aeruginosa) to the scarified mouse cornea depends on interaction with proteoglycans (PGs). METHODS: Scarified corneas were treated with anti-proteoglycan monoclonal antibodies (MAbs), glycosaminoglycans (GAGs), heparinase III or chondroitin ABC lyase before inoculation with P. aeruginosa strain ATCC 19660 or PAO1. Scanning electron microscopy (SEM) was used to quantitate adherent bacteria. Frozen sections of unwounded and wounded mouse cornea, the latter treated or not treated with heparinase III were stained to spatially localize perlecan [core protein or heparan sulfate (HS) side chains]. Anti-perlecan MAb against the heparan sulfate proteoglycan core protein and succinyl wheat germ agglutinin (sWGA), a lectin which recognizes N-acetyl-glucosamine in heparan sulfate, respectively were used. RESULTS: Anti-perlecan MAb, as well as heparan sulfate, heparin and heparinase III decreased the binding of both bacterial strains to cornea, and the decrease was concentration-dependent. Fluorescence microscopic analysis of sections of mouse cornea immunostained with anti-perlecan MAb showed that perlecan was localized to the epithelial basement membrane. Scarification of the mouse cornea exposed perlecan in the basement membrane and increased bacterial binding to this site was consistent with this exposure. Lectin staining revealed that heparinase treatment removed heparan sulfate side chains of perlecan from the exposed basement membrane, and this removal was consistent with a decrease in bacterial binding. CONCLUSIONS: These studies provide evidence that perlecan, core protein and its heparan sulfate side chains serve as a binding site for Pseudomonas aeruginosa when the basement membrane of the cornea is exposed.
Assuntos
Membrana Basal/metabolismo , Membrana Basal/microbiologia , Córnea/metabolismo , Córnea/microbiologia , Proteoglicanas de Heparan Sulfato/metabolismo , Pseudomonas aeruginosa/fisiologia , Animais , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/fisiologia , Condroitina ABC Liase/farmacologia , Epitélio/metabolismo , Feminino , Imunofluorescência , Heparina/farmacologia , Heparitina Sulfato/farmacologia , Camundongos , Camundongos Endogâmicos ICR , Polissacarídeo-Liases/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Coloração e Rotulagem , Aglutininas do Germe de TrigoRESUMO
PURPOSE: Mice favoring Th1 (C57BL/6, C57BL/10, and B10.D2/nSn) versus Th2 (BALB/c, BALB/cBy, BALB.B, and BALB.K) response development were evaluated for their response to infection with Pseudomonas aeruginosa. This study addresses the question of whether Th1 versus Th2 response propensity affects the pathogenesis of bacterial keratitis in mice. METHODS: Ocular disease was determined by mean clinical score, slit lamp, plate counts, and histopathology, and antigen-specific cellular responses were assessed by immunostaining and measurement of delayed type hypersensitivity (DTH). RESULTS: Strains of mice favoring Th1 (B6, BL10, and B10.D2) versus Th2 (BALB/c, BALB/cBy, BALB.B, and BALB.K) responsiveness were infected with P. aeruginosa. Mice favoring Th1 response development exhibited a similar course of disease and the infected eyes of all mice perforated by 7 days postinfection (p.i.). Strains (BALB/c, BALB/cBy, BALB.B, and BALB.K) favoring Th2 response development exhibited a milder course of disease, and none of the infected corneas perforated at 7 days p.i. In a Th1-responsive strain (B10.D2), positive immunostaining for CD4+ and CD8+ T cells was observed in the cornea by 3 days p.i. and by 5 days p.i., respectively, some cells stained positively for IL2-R, indicating that the cells were activated. In contrast, in a Th2 responder strain (BALB/c), there was no detectable positive immunostaining in cornea for any of the T-cell markers tested and DTH was significantly elevated in B10.D2 versus BALB/c mice. CONCLUSIONS: These studies are the first to provide evidence that in P. aeruginosa ocular infection, mouse strains favoring development of a Th1-type response are susceptible (cornea perforates), whereas strains favoring Th2 response development are resistant (no corneal perforation).
Assuntos
Infecções Oculares Bacterianas/imunologia , Ceratite/imunologia , Infecções por Pseudomonas/imunologia , Células Th1/imunologia , Células Th2/imunologia , Animais , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Córnea/imunologia , Córnea/microbiologia , Córnea/patologia , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/patologia , Feminino , Hipersensibilidade Tardia/imunologia , Técnicas Imunoenzimáticas , Ceratite/metabolismo , Ceratite/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa , Receptores de Interleucina-2/metabolismo , Ruptura Espontânea , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5-7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.
Assuntos
Infecções Oculares Bacterianas/imunologia , Infecções Oculares Bacterianas/patologia , Monocinas/fisiologia , Infiltração de Neutrófilos/imunologia , Animais , Quimiocina CXCL2 , Contagem de Colônia Microbiana , Suscetibilidade a Doenças , Infecções Oculares Bacterianas/metabolismo , Soros Imunes/administração & dosagem , Imunidade Inata , Injeções Intraperitoneais , Ceratite/imunologia , Ceratite/metabolismo , Ceratite/patologia , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Monocinas/genética , Monocinas/imunologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/citologia , Pseudomonas aeruginosa/imunologia , RNA Mensageiro/metabolismo , Proteínas Recombinantes/administração & dosagemRESUMO
PURPOSE: To determine the biological significance of individual endogenous tissue inhibitors of metalloproteinases (TIMPs) in protection against tissue destruction using a Pseudomonas aeruginosa-induced model of corneal ulceration. METHODS: Corneal TIMP-1, -2, and -3 mRNA levels were compared between young adult (resistant) and aged (susceptible) mice challenged with P. aeruginosa. Resistant mice that demonstrated greater amounts of an individual TIMP were treated with polyclonal antibody (pAb) to that TIMP. To determine whether TIMP neutralization exacerbated P. aeruginosa-induced corneal disease, TIMP pAb- and normal rabbit serum (NRS)- (control) treated mice were examined macroscopically and histopathologically after infection. Corneal neutrophil (PMN) myeloperoxidase (MPO) levels also were examined in these mice. RESULTS: Greater amounts of TIMP-1 mRNA only were found in corneas of resistant versus suscep tible mice after P. aeruginosa challenge. Systemic treatment of resistant mice with TIMP-1 pAb resulted in corneal perforation by 5 to 7 days after infection (PI). Histopathologic evaluation of corneal tissues from TIMP-1 pAb- versus NRS-treated mice confirmed that TIMP-1 pAb treatment resulted in extensive stromal dissolution. This treatment also was associated with loss of epithelium within the central cornea. Both the histopathology and PMN MPO enzyme assays also showed an increase in corneal PMN number following TIMP-1 pAb treatment. CONCLUSIONS: These studies provide evidence that, after P. aeruginosa infection, adequate endogenous expression of TIMP-1 in cornea protects against extensive corneal tissue destruction. The protective effects of TIMP-1 may be multifactorial. In addition to directly protecting extracellular matrix components from active matrix metalloproteinases, TIMP-1 may either directly or indirectly influence recruitment of PMNs into infected cornea. Finally, TIMP-1 also may affect wound healing and resurfacing of the corneal epithelium.
Assuntos
Úlcera da Córnea/prevenção & controle , Infecções Oculares Bacterianas/prevenção & controle , Infecções por Pseudomonas/prevenção & controle , Inibidor Tecidual de Metaloproteinase-1/fisiologia , Animais , Córnea/efeitos dos fármacos , Córnea/metabolismo , Córnea/patologia , Úlcera da Córnea/metabolismo , Úlcera da Córnea/microbiologia , Úlcera da Córnea/patologia , Infecções Oculares Bacterianas/metabolismo , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/patologia , Imunoglobulina G/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/enzimologia , Peroxidase/metabolismo , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , RNA Mensageiro/metabolismo , Coelhos , Proteínas Recombinantes/imunologia , Ruptura Espontânea/metabolismo , Ruptura Espontânea/microbiologia , Ruptura Espontânea/patologia , Ruptura Espontânea/prevenção & controle , Inibidor Tecidual de Metaloproteinase-2/fisiologia , Inibidor Tecidual de Metaloproteinase-3/fisiologiaRESUMO
The cornea is a transparent and avascular tissue that functions as the major refractive structure for the eye. A wide variety of growth factors, chemokines, cytokines and their receptors are synthesized by corneal epithelial and stromal cells, and are found in tears. These molecules function in corneal wound healing and in inflammatory responses. Proteoglycans and glycoproteins are essential for normal corneal function, both at the air-epithelial interface and within the extracellular matrix. The ocular MUC mucins may play roles in forming the mucus layer of the tear film, in regulating tear film spread, and in inhibiting the adhesion of pathogens to the ocular surface. Lumican, keratocan and mimecan are the major keratan sulfate proteoglycans of the corneal stroma. They are essential, along with other proteoglycans and interfibrillar proteins, including collagens type VI and XII, for the maintenance of corneal transparency. Corneal epithelial cells interact with a specialized extracellular matrix structure, the basement membrane, composed of a specific subset of collagen type IV and laminin isoforms in addition to ubiquitous extracellular matrix molecules. Matrix metalloprotein-ases have been identified in normal corneal tissue and cells and may play a role in the development of ulcerative corneal diseases. Changes in extracellular matrix molecule localization and synthesis have been noted in other types of corneal diseases as well, including bullous keratopathy and keratoconus.
Assuntos
Córnea/metabolismo , Proteínas do Olho/fisiologia , Olho/patologia , Animais , Quimiocinas/metabolismo , Quimiocinas/fisiologia , Córnea/citologia , Citocinas/metabolismo , Citocinas/fisiologia , Matriz Extracelular/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Substâncias de Crescimento/metabolismo , Substâncias de Crescimento/fisiologia , Humanos , Proteoglicanas/metabolismoRESUMO
PURPOSE: The role of complement in phagocytosis and killing of P. aeruginosa was examined using serum from aged vs young donor mice. METHODS: Phagocytosis, complement hemolytic and microbicidal assays were used. RESULTS: Serum from young donor mice contained a heat-labile factor which significantly enhanced phagocytic activity of cells from young mice compared with similarly treated aged donor serum. Use of cobra venom factor (CVF) to destroy C3 and the terminal complement components in serum from young or aged donor mice also significantly decreased the phagocytic activity of young cells. EGTA treatment of young or aged donor serum, to activate the alternative pathway and selectively inhibit activation of the classical pathway, resulted in a significant decrease in phagocytosis by young cells in the presence of donor serum from either group. Alternative pathway mediated hemolysis also was measured and was significantly reduced in aged vs young donor serum. PMN microbicidal activity was tested using cells from young mice in the presence of aged vs young donor serum, but no significant differences were noted. CONCLUSION: These data provide evidence that defects in the alternative pathway of complement in the serum of aged animals lead to decreased phagocytic activity of cells from young mice, but not impaired bacterial killing.
Assuntos
Envelhecimento/fisiologia , Proteínas do Sistema Complemento/fisiologia , Fagocitose/fisiologia , Pseudomonas aeruginosa/fisiologia , Animais , Sangue/efeitos dos fármacos , Atividade Bactericida do Sangue/fisiologia , Fenômenos Fisiológicos Sanguíneos , Complemento C3/fisiologia , Ácido Egtázico/farmacologia , Feminino , Hemólise/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Neutrófilos/fisiologiaRESUMO
PURPOSE: To compare neodymium:YAG (Nd:YAG) laser effects on acrylic, silicone, and poly(methyl methacrylate) (PMMA) intraocular lens (IOL) polymers. METHODS: Ten Nd:YAG laser exposures were produced in each of 6 implantation-quality acrylic (Alcon MA60BM), silicone (Staar AQ1016), and PMMA (Alcon MC60BM) IOLs under identical conditions. Each polymer type was irradiated at 6 power settings (0.3, 0.5, 1.0, 1.5, 2.0, and 3.0 mJ) and at 2 focal points (midpoint of lens optic and on the posterior surface to which a cellophane membrane was affixed). The linear extent of the damage was measured using light microscopy. Specimens exposed to 1.0 mJ were processed for scanning electron microscopy. RESULTS: The damage threshold (> or = 5 microns depth) was 0.3 mJ for silicone and 1.0 mJ for acrylic and PMMA IOLs. At the clinically relevant power levels, 1.0 to 2.0 mJ, the depth of damage in the acrylic polymer was 11.9 to 30.5 times less than the depth in the silicone polymer. Similarly, the depth of damage in the PMMA polymer was 5.4 to 52.6 times less than the depth in the silicone polymer. The morphologic pattern of damage in the silicone IOL showed a deep, irregularly configured trough with meandering tendrils. Acrylic IOL damage morphology consisted of an ameboid-shaped entry site without radiating fractures and mild posterior penetration. Poly(methyl methacrylate) IOL damage consisted of a shallow focal trough with radiating fractures. CONCLUSIONS: The silicone IOL polymer had the lowest threshold for laser-induced damage and greater linear extension of damage than the PMMA and acrylic IOL polymers. Poly(methyl methacrylate) and silicone polymers exhibited collateral damage or ejected particulates adjacent to the entry site, whereas the acrylic polymer showed a discrete locus of damage.
Assuntos
Resinas Acrílicas , Terapia a Laser/efeitos adversos , Lentes Intraoculares , Polimetil Metacrilato , Elastômeros de Silicone , Implante de Lente Intraocular , Microscopia Eletrônica de VarreduraRESUMO
In this study, the role of intercellular adhesion molecule 1 (ICAM-1) in the pathogenesis of Pseudomonas aeruginosa keratitis was examined by using inbred ICAM-1-deficient knockout mice. These mice had significantly less (P = 0.02) ocular disease than wild-type mice, suggesting that ICAM-1 contributes to a more severe disease response following P. aeruginosa infection.
Assuntos
Molécula 1 de Adesão Intercelular/fisiologia , Ceratite/microbiologia , Infecções por Pseudomonas/etiologia , Animais , Olho/imunologia , Olho/patologia , Ceratite/imunologia , Ceratite/patologia , Camundongos , Camundongos Knockout , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/patologiaRESUMO
Pseudomonas aeruginosa is the most common pathogen among contact lens-associated infections. This study investigated the response of the murine cornea to infection with an ocular strain of P. aeruginosa isolated from a subject with an inflammatory adverse response to contact lens wear termed CLARE. Although this bacterium was isolated in confluency (greater than 2000 cfu lens-1) from the lens at the time of the inflammatory episode, no infection of the cornea subsequently developed. Male C57BL/6J mice (20 per strain) had their corneas scratched with a 26 gauge needle (3 parallel 1.0 mm wounds in the left eye only). The incisions were centered over the pupillary axis and penetrated the epithelial cell basal lamina and into the superficial stroma. The CLARE strain was found to persist (viable bacteria could be cultured from corneal homogenates) up to 8 hr, as did the virulent control strain ATCC 19660. At 24 hr, only ATCC 19660 could be cultured, indicating an inability of the strain isolated from CLARE, Paer1, to persist in the eye consistent with the human inflammatory episode. Histological examination of the mouse tissue showed further differences between infection by the two strains. Infection with ATCC 19660 resulted in tissue necrosis and a large population of polymorphonuclear leukocytes (PMNs) recruited to the wound site. In contrast, during infection with the CLARE strain, PMN recruitment was reduced and temporally delayed. The CLARE strain grew as well as ATCC 19660 in vitro but produced less protease activity, in particular less elastase. The decreased PMN response and decreased protease production by the CLARE strain may have been responsible for the lack of ocular damage and apparent healing of the wound. P. aeruginosa strains are considered to be invasive or cytotoxic to corneal tissue, however this strain may represent a third inflammatory type consistent with its differing pathology.