RESUMO
The purpose of the present study was to test the potential of mouse bone marrow-derived mesenchymal stem cells (BD-MSCs) in improving tear production in a mouse model of Sjögren's syndrome dry eye and to investigate the underlying mechanisms involved. NOD mice (n = 20) were randomized to receive i.p. injection of sterile phosphate buffered saline (PBS, control) or murine BD-MSCs (1 × 106 cells). Tears production was measured at baseline and once a week after treatment using phenol red impregnated threads. Cathepsin S activity in the tears was measured at the end of treatment. After 4 weeks, animals were sacrificed and the lacrimal glands were excised and processed for histopathology, immunohistochemistry, and RNA analysis. Following BD-MSC injection, tears production increased over time when compared to both baseline and PBS injected mice. Although the number of lymphocytic foci in the lacrimal glands of treated animals did not change, the size of the foci decreased by 40.5% when compared to control animals. The mRNA level of the water channel aquaporin 5 was significantly increased following delivery of BD-MSCs. We conclude that treatment with BD-MSCs increases tear production in the NOD mouse model of Sjögren's syndrome. This is likely due to decreased inflammation and increased expression of aquaporin 5.
RESUMO
PURPOSE: Chronic inflammation of the lacrimal gland results in changes in the composition of the extracellular matrix (ECM), which is believed to compromise tissue repair. We hypothesized that increased production/activity of matrix metalloproteinases (MMPs), especially MMP-2 and -9, in inflamed lacrimal glands modifies the ECM environment, therefore disrupting tissue repair. METHODS: The lacrimal glands from female MRL/lpr and male NOD mice along with their respective control strains were harvested and divided into three pieces and processed for histology, immunohistochemistry, zymography, Western blotting, and RNA analyses. In another study, MRL/lpr mice were treated for 5 weeks with a selective MMP2/9 inhibitor peptide or a control peptide. At the end of treatment, the lacrimal glands were excised and the tissue was processed as described above. RESULTS: There was a 2.5- and 2.7-fold increase in MMP2 gene expression levels in MRL/lpr and NOD mice, respectively. Matrix metalloproteinase 2 and 9 enzymatic activities and protein expression levels were significantly upregulated in the lacrimal glands of MRL/lpr and NOD mice compared to controls. Treatment with the MMP2/9 inhibitor resulted in decreased activity of MMP-2 and -9 both in vitro and in vivo. Importantly, MMP2/9 inhibitor treatment of MRL/lpr mice improved aqueous tear production and resulted in reduced number and size of lymphocytic foci in diseased lacrimal glands. CONCLUSIONS: We conclude that MMP2/9 expression and activity are elevated in lacrimal glands of two murine models of Sjögren's syndrome, suggesting that manipulation of MMP2/9 activity might be a potential therapeutic target in chronically inflamed lacrimal glands.
Assuntos
Regulação da Expressão Gênica , Aparelho Lacrimal/enzimologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , RNA/genética , Síndrome de Sjogren/genética , Lágrimas/enzimologia , Animais , Western Blotting , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Masculino , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos NOD , Reação em Cadeia da Polimerase em Tempo Real , Síndrome de Sjogren/enzimologiaRESUMO
Multiple-use dental bib clips are considered to present relatively low risks for transmitting infections and, thus, are thought to only require disinfection between patient visits. This study was designed to: 1) determine the presence and composition of bacterial contaminants on reusable rubber-faced metal bib clips after dental treatment at the hygiene clinic at Tufts University School of Dental Medicine and 2) evaluate the effectiveness of the disinfection for this clip type. Aerobic and anaerobic bacterial contaminant loads on the surfaces of the clips were investigated immediately after hygiene treatments were rendered and again after clips were disinfected. The species and strains of bacterial isolates were identified using 16S rDNA sequencing and Human Oral Microbe Identification Microarray analyses. The results demonstrated that although the use of disinfection proved to be significantly effective, some clips retained at least one bacterium on their surfaces after disinfection. Although the bacterial species present on disinfected clips were typical skin or environmental isolates, some were oral in origin. In the study's settings, bacterial presence on the clips did not indicate an infectious disease problem. The different bacterial loads on clips suggest that cross-contamination risks may not be the same for all clinics, and that this difference may be related to the type of treatments and services performed.
Assuntos
Bactérias Aeróbias/isolamento & purificação , Bactérias Anaeróbias/isolamento & purificação , Higiene Bucal , Roupa de Proteção , Bactérias Aeróbias/classificação , Bactérias Anaeróbias/classificação , HumanosRESUMO
Although multiple-use dental napkin holders have a relatively low risk of transmitting infection, they do require disinfection between patients. This study sought to: 1) determine the presence of bacterial load on two types of clips of reusable bib chains after dental procedures at the Endodontics and Orthodontics clinics at Tufts University School of Dental Medicine; and 2) evaluate the effectiveness of disinfecting the clips. These specialty clinics represent a wide spectrum of patients, procedures, and appointment times. Bacterial load on the bib clips was determined immediately following dental treatments-both before and after their disinfection-during morning and afternoon sessions. The results revealed that, after treatments, there was a statistically significant difference when comparing the two clinics for bacterial burden on the clips. Furthermore, there was a statistically significant difference in bacterial load on the two types of clips. Disinfection of the bib clips was highly effective in both clinics. Clinically, the results suggest that due to the nature of the treatment, the demographic population, and the type of bib clips used, patients in different clinics may be exposed to varying bacterial concentrations on the bib clips, and thus to different possible cross-contamination risks. Future analyses will be performed to identify the bacterial species in samples from both pre- and post-disinfected clips, and to determine if they harbor disease-causing bacterial species that can pose a potential, yet undetermined risk for cross-contamination.
Assuntos
Infecção Hospitalar/transmissão , Equipamentos Odontológicos/microbiologia , Contaminação de Equipamentos , Controle de Infecções Dentárias/métodos , Infecção Hospitalar/prevenção & controle , Desinfecção , Humanos , Metais , Estatísticas não ParamétricasRESUMO
The purpose of the current study was to determine if saliva contains biomarkers that can be used as diagnostic tools for Sjögren's syndrome (SjS). Twenty seven SjS patients and 27 age-matched healthy controls were recruited for these studies. Unstimulated glandular saliva was collected from the Wharton's duct using a suction device. Two µl of salvia were processed for mass spectrometry analyses on a prOTOF 2000 matrix-assisted laser desorption/ionization orthogonal time of flight (MALDI O-TOF) mass spectrometer. Raw data were analyzed using bioinformatic tools to identify biomarkers. MALDI O-TOF MS analyses of saliva samples were highly reproducible and the mass spectra generated were very rich in peptides and peptide fragments in the 750-7,500 Da range. Data analysis using bioinformatic tools resulted in several classification models being built and several biomarkers identified. One model based on 7 putative biomarkers yielded a sensitivity of 97.5%, specificity of 97.8% and an accuracy of 97.6%. One biomarker was present only in SjS samples and was identified as a proteolytic peptide originating from human basic salivary proline-rich protein 3 precursor. We conclude that salivary biomarkers detected by high-resolution mass spectrometry coupled with powerful bioinformatic tools offer the potential to serve as diagnostic/prognostic tools for SjS.
Assuntos
Biomarcadores/análise , Informática Odontológica , Saliva/química , Proteínas Salivares Ricas em Prolina/análise , Síndrome de Sjogren/diagnóstico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Idoso , Algoritmos , Sequência de Aminoácidos , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Precursores de Proteínas/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Síndrome de Sjogren/metabolismo , Glândula Submandibular/metabolismoRESUMO
PURPOSE: Dry eye syndromes affect a significant proportion of the population worldwide with reported prevalence ranging from 6% to more than 34%. Patients with dry eye can experience intense pain due to eye irritation, gritty/scratchy feeling in the eyes, blurry vision, and light sensitivity. Available treatments for dry eye syndromes remain mainly palliative. The purpose of the present study was to test the hypothesis that inhibiting sodium absorption via the epithelial sodium channel (ENaC) will increase ocular hydration in both normal as well as in animals with experimentally induced dry eye. METHODS: ENaC inhibitors were dissolved in an aqueous buffer that mimics the composition of tears and were applied topically to the ocular surface of isoflurane-anesthetized mice. The effect of ENaC inhibitors was compared with that of the secretagogue uridine triphosphate (UTP; 1%), a purinergic receptor agonist which was shown to increase tear volume in animals. Tear production was measured for 10 s using phenol red-impregnated cotton threads. Fluorescein staining that assesses ocular surface damage was performed at baseline and then at days 1, 2, and 3 after the induction of dry eye in mice. RESULTS: Our data show that the inhibition of ENaC led to a time- and concentration-dependent increase in tear volume in normal mice. The effect of ENaC inhibition after a single application outperformed UTP, as it was long-lasting with tear volume still above baseline values 8 h postdosing. ENaC inhibition, which led to increased tear production, improved fluorescein scores in our dry eye model, when compared with nontreated or animals treated with buffer or UTP. CONCLUSION: We conclude that the inhibition of ENaC provides long-lasting increases in ocular surface hydration and that ENaC blockers could provide an effective new therapy for chronic dry eye.
Assuntos
Síndromes do Olho Seco/tratamento farmacológico , Bloqueadores dos Canais de Sódio/uso terapêutico , Lágrimas/metabolismo , Administração Tópica , Amilorida/farmacologia , Animais , Corantes , Diuréticos/farmacologia , Cães , Síndromes do Olho Seco/patologia , Canais Epiteliais de Sódio/metabolismo , Feminino , Fluoresceína , Camundongos , Camundongos Endogâmicos BALB C , Antagonistas de Receptores Purinérgicos P1/farmacologia , Bloqueadores dos Canais de Sódio/administração & dosagem , Bloqueadores dos Canais de Sódio/farmacocinética , Lágrimas/química , Lágrimas/efeitos dos fármacos , Uridina Trifosfato/farmacologiaRESUMO
PURPOSE: Ongoing studies demonstrate that the murine lacrimal gland is capable of repair after experimentally induced injury. It was recently reported that repair of the lacrimal gland involved the mobilization of mesenchymal stem cells (MSCs). These cells expressed the type VI intermediate filament protein nestin whose expression was upregulated during the repair phase. The aim of the present study was to investigate the roles of vimentin, a type III intermediate filament protein and a marker of epithelial-mesenchymal transition (EMT) in repair of the lacrimal gland. METHODS: Injury was induced by direct injection of interleukin (IL)-1 into the exorbital lacrimal gland. MSCs were prepared from injured glands using tissue explants. Expression of vimentin and the transcription factor Snai1, a master regulator of EMT, was determined by RT-PCR, Western blotting analysis, and immunofluorescence. RESULTS: These data show that vimentin expression, at both the mRNA and the protein levels, was upregulated during the repair phase (2-3 days postinjury) and returned to the control level when repair ended. Temporal expression of Snai1 mirrored that of vimentin and was localized in cell nuclei. Cultured MSCs isolated from injured lacrimal glands expressed Snai1 and vimentin alongside nestin and alpha smooth muscle actin (another biomarker of EMT). There was a strong positive correlation between Snai1 expression and vimentin expression. CONCLUSIONS: It was found that EMT is induced during repair of the lacrimal gland to generate MSCs to initiate repair, and that mesenchymal-epithelial transition is then activated to form acinar and ductal epithelial cells.
Assuntos
Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/fisiologia , Traumatismos Oculares/fisiopatologia , Aparelho Lacrimal/lesões , Cicatrização/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Separação Celular , Células Cultivadas , Traumatismos Oculares/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica/fisiologia , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMO
The purpose of the present study was to determine if the lacrimal gland contains 5-bromo-2'-deoxyuridine (BrdU)-label retaining cells and if they are involved in tissue repair. Animals were pulsed daily with BrdU injections for 7 consecutive days. After a chase period of 2, 4, or 12 weeks, the animals were sacrificed and the lacrimal glands were removed and processed for BrdU immunostaining. In another series of experiments, the lacrimal glands of 12-week chased animals were either left untreated or were injected with interleukin 1 (IL-1) to induce injury. Two and half days post-injection, the lacrimal glands were removed and processed for BrdU immunostaining. After 2 and 4 weeks of chase period, a substantial number of lacrimal gland cells were BrdU(+) (11.98 ± 1.84 and 7.95 ± 1.83 BrdU(+) cells/mm(2), respectively). After 12 weeks of chase, there was a 97% decline in the number of BrdU(+) cells (0.38 ± 0.06 BrdU(+) cells/mm(2)), suggesting that these BrdU-label retaining cells may represent slow-cycling adult stem/progenitor cells. In support of this hypothesis, the number of BrdU labeled cells increased over 7-fold during repair of the lacrimal gland (control: 0.41 ± 0.09 BrdU(+) cells/mm(2); injured: 2.91 ± 0.62 BrdU(+) cells/mm(2)). Furthermore, during repair, among BrdU(+) cells 58.2 ± 3.6 % were acinar cells, 26.4 ± 4.1% were myoepithelial cells, 0.4 ± 0.4% were ductal cells and 15.0 ± 3.0% were stromal cells. We conclude that the murine lacrimal gland contains BrdU-label retaining cells that are mobilized following injury to generate acinar, myoepithelial and ductal cells.
Assuntos
Bromodesoxiuridina/análise , Aparelho Lacrimal/química , Aparelho Lacrimal/fisiologia , Cicatrização/fisiologia , Animais , Bromodesoxiuridina/administração & dosagem , Bromodesoxiuridina/farmacocinética , Feminino , Aparelho Lacrimal/citologia , Aparelho Lacrimal/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Distribuição TecidualRESUMO
PURPOSE: Previously, it was reported that the murine lacrimal gland is capable of repair after experimentally induced injury and that the number of stem/progenitor cells was increased during the repair phase (2-3 days after injury). The aim of the present study was to determine whether these cells can be isolated from the lacrimal gland and propagated in vitro. METHODS: Lacrimal gland injury was induced by injection of interleukin (IL)-1, and injection of saline vehicle served as control. Two and half days after injection, the lacrimal glands were removed and used to prepare explants or acinar cells for tissue culture. Cells derived from the explants and the acinar cells were grown in DMEM supplemented with 10% fetal bovine serum. Cells were stained for the stem cells markers, nestin, vimentin, ABCG2, and Sca-1. Cell proliferation was measured using an antibody against Ki67 or a cell-counting kit. The adipogenic capability of these cells was also tested in vitro. RESULTS: Results show that nestin-positive cells can be isolated from IL-1-injected, but not saline-injected, lacrimal glands. A population of nestin-positive cells was also positive for vimentin, an intermediate filament protein expressed by mesenchymal cells. In addition, cultured cells expressed two other markers of stem cells, ABCG2 and Sca-1. These cells proliferated in vitro and can be induced to form adipocytes, attesting to their mesenchymal stem cell property. CONCLUSIONS: Murine lacrimal glands contain mesenchymal stem cells that seem to play a pivotal role in tissue repair.
Assuntos
Aparelho Lacrimal/citologia , Células-Tronco Mesenquimais/citologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adipogenia , Animais , Antígenos Ly/metabolismo , Biomarcadores/metabolismo , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células , Separação Celular/métodos , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Filamentos Intermediários/metabolismo , Antígeno Ki-67/metabolismo , Aparelho Lacrimal/metabolismo , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/metabolismo , Nestina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vimentina/metabolismoRESUMO
PURPOSE: The authors recently reported that a severe inflammatory response resulting in substantial loss of acinar cells was induced by a single injection of interleukin-1alpha into the lacrimal gland and that this effect was reversible. The purpose of the present study was to determine the mechanisms involved in lacrimal gland injury and repair. METHODS: Inflammation was induced by direct injection of recombinant human interleukin-1alpha (IL-1alpha, 1 microg in 2 microL) into the exorbital lacrimal glands of anesthetized female BALB/c mice. Animals were killed 1, 2, 3, 4, 5, 6, or 7 days after injection. Exorbital lacrimal glands were then removed and processed for measurement of protein secretion, histology, immunohistochemistry, and Western blotting. RESULTS: The results show that lacrimal gland acinar cells are lost through programmed cell death (apoptosis) and autophagy. They also show that the number of nestin (a stem cell marker)-positive cells increased 2 to 3 days after injury and that some of these cells were also positive for Ki67 (a cell proliferation marker) and alpha-smooth muscle actin (a marker of myoepithelial cells). Finally, they show that the amount of phosphorylated Smad1/5/8 (effector molecules of bone morphogenetic protein 7 [BMP7]) increased 2 to 3 days after injury and could also be detected in nestin-positive cells. CONCLUSIONS: The lacrimal gland contains stem/progenitor cells capable of tissue repair after injury. Programmed cell death after injury triggers proliferation and differentiation of these cells, presumably through activation of the BMP7 pathway.
Assuntos
Dacriocistite/metabolismo , Aparelho Lacrimal/metabolismo , Cicatrização/fisiologia , Animais , Apoptose/efeitos dos fármacos , Autofagia , Western Blotting , Diferenciação Celular , Proliferação de Células , Dacriocistite/induzido quimicamente , Dacriocistite/patologia , Modelos Animais de Doenças , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Marcação In Situ das Extremidades Cortadas , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Interleucina-1alfa/farmacologia , Proteínas de Filamentos Intermediários/metabolismo , Antígeno Ki-67/metabolismo , Aparelho Lacrimal/efeitos dos fármacos , Aparelho Lacrimal/ultraestrutura , Proteínas de Membrana Lisossomal/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Nestina , Proteínas Recombinantes/farmacologia , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , Células-Tronco/fisiologiaRESUMO
PURPOSE: The purpose of the present study was to investigate the roles of caspase 1 and extracellular signal-regulated kinase (ERK) in inflammation-induced inhibition of lacrimal gland secretion. METHODS: Lacrimal gland inflammation was induced by injection of lipopolysaccharide (LPS; to study the role of caspase 1) or IL-1beta (to study the role of ERK). Lacrimal gland protein secretion was measured using a spectrofluorometric assay. Caspase 1 and ERK activities in the lacrimal gland were measured by immunohistochemistry, Western blotting, or both. Aqueous tear production was measured using phenol red-impregnated cotton threads. RESULTS: Injection of LPS into the lacrimal gland inhibited neurally and adrenergic agonist-induced protein secretion by 77% and 54%, respectively, and activated caspase 1. The degree of inhibition achieved by LPS was similar to that obtained with injection of IL-1beta. Inhibition of caspase 1 alleviated the inhibitory effect of LPS on lacrimal gland secretion. IL-1beta activated ERK in the lacrimal gland in vitro and in vivo, and this effect was blocked by UO126, an inhibitor of MEK, the ERK-activating enzyme. IL-1beta injection into the lacrimal gland inhibited aqueous tear production by 52% and inhibited neurally and adrenergic agonist-induced protein secretion by 80% and 55%, respectively. UO126 alleviated the inhibitory effect of IL-1beta on aqueous tear production and lacrimal gland protein secretion. CONCLUSIONS: LPS inhibits lacrimal gland secretion by activating caspase 1, and IL-1beta activates the ERK pathway to inhibit lacrimal gland protein secretion and aqueous tear production.
Assuntos
Caspase 1/metabolismo , Dacriocistite/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas do Olho/metabolismo , Aparelho Lacrimal/efeitos dos fármacos , Animais , Western Blotting , Butadienos/farmacologia , Dacriocistite/induzido quimicamente , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Inflamação/induzido quimicamente , Inflamação/enzimologia , Interleucina-1beta/farmacologia , Aparelho Lacrimal/enzimologia , Lipopolissacarídeos , Camundongos , Camundongos Endogâmicos BALB C , Nitrilas/farmacologia , Proteínas Recombinantes/farmacologia , Espectrometria de Fluorescência , Lágrimas/metabolismoRESUMO
Emerging studies from our laboratory demonstrate that interleukin-1 (IL-1) family members play a major role in impairing lacrimal gland functions. Here we have extended our investigations to observe the effects of IL-1 on aqueous tear production, lacrimal gland secretion, lacrimal gland histology, and acinar and ductal cell proliferation. We demonstrate that a single injection of IL-1 into the lacrimal glands inhibited neurally- as well as agonist-induced protein secretion resulting in decreased tear output. Meanwhile, IL-1 injection induced a severe, but reversible (7-13 days), inflammatory response that led to destruction of lacrimal gland acinar epithelial cells. Finally, we demonstrate that as the inflammatory response subsided and lacrimal gland secretion and tear production returned to normal levels, there was increased proliferation of acinar and ductal epithelial cells. Our work uncovers novel effects of IL-1 on lacrimal gland functions and the potential regenerative capacity of the mouse lacrimal gland.
Assuntos
Dacriocistite/induzido quimicamente , Síndromes do Olho Seco/induzido quimicamente , Interleucina-1alfa/toxicidade , Aparelho Lacrimal/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Dacriocistite/patologia , Síndromes do Olho Seco/patologia , Células Epiteliais/efeitos dos fármacos , Feminino , Aparelho Lacrimal/metabolismo , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peroxidase/metabolismo , Especificidade da Espécie , Lágrimas/metabolismoRESUMO
Sjögren's syndrome, an inflammatory disease affecting the lacrimal and salivary glands, is the leading cause of aqueous tear-deficient type of dry eye. We previously showed that interleukin-1beta (IL-1beta) protein is up regulated in the lacrimal gland of a murine model of Sjögren's syndrome and that exogenous addition of this cytokine inhibits neurotransmitter release and lacrimal gland protein secretion. In the present study we investigated the role of c-Jun NH2-terminal kinase (JNK) in IL-1beta-mediated inhibition of lacrimal gland secretion and tear production. In vitro, IL-1beta induced a time-dependent activation of JNK with a maximum 7.5-fold at 30 min. SP600125, a JNK inhibitor, inhibited, in a concentration-dependent manner, IL-1beta-induced activation of JNK with a maximum of 87% at 10(-4) m. In vivo, IL-1beta stimulated JNK and the expression of the inducible isoform of nitric oxide synthase (iNOS). IL-1beta inhibited high KCl and adrenergic agonist induced protein secretion by 85% and 66%, respectively. SP600125 alleviated the inhibitory effect of IL-1beta on KCl- and agonist-induced protein secretion by 79% and 47%, respectively, and completely blocked the expression of iNOS. Treatment for 7 days with SP600125 increased tear production in a murine model of Sjögren's syndrome dry eye. We conclude that JNK plays a pivotal role in IL-1beta-mediated inhibition of lacrimal gland secretion and subsequent dry eye.
Assuntos
Interleucina-1/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Aparelho Lacrimal/metabolismo , Animais , Antracenos/farmacologia , Western Blotting , Depressão Química , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Feminino , Imuno-Histoquímica , Isoenzimas/biossíntese , Isoenzimas/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Cinética , Aparelho Lacrimal/efeitos dos fármacos , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Síndrome de Sjogren/tratamento farmacológico , Síndrome de Sjogren/metabolismo , Lágrimas/metabolismoRESUMO
Several studies investigated the effect of aging on rat and human lacrimal gland physiology. However, in most of these studies, only two age groups were investigated. Furthermore, those studies did not correlate the age-related histological changes that occur in the lacrimal gland to the functional changes (nerve activity and protein secretion) that might occur with aging. Thus, the purpose of the present study was to investigate the effect of aging on lacrimal gland structure, innervation and function using BALB/c mice at different ages. Exorbital lacrimal glands were removed from 3, 8, 12, 24, and 32-month-old, male BALB/c mice, fixed, embedded and processed for histology and immunohistochemistry. Sections were stained with hematoxylin and eosin to determine morphological changes and lymphocytic infiltration; giemsa to identify mast cells; and Kinyoun's carbol fucsin solution to indicate lipofuscin-like inclusions. Parasympathetic and sympathetic nerves were identified by immunofluorescence techniques. To measure acetylcholine release and protein secretion, lacrimal gland pieces were incubated in Krebs Ringer buffer containing 5 mM KCl (control), 75 mM KCl (depolarizing buffer which activates nerves), carbachol (a cholinergic agonist, 10(-4) M), or phenylephrine (an alpha1-adrenergic agonist, 10(-4) M) for 20 min. The media were collected and analysed for acetylcholine and peroxidase using a spectrofluorometric assay. KCl-, carbachol- and phenylephrine-stimulated peroxidase secretion decreased in lacrimal glands from 8, 12, and 24-month-old mice when compared to 3-month-old animals. Both the density and distribution of parasympathetic and sympathetic nerves surrounding the acini decreased with increasing age. Acetylcholine release from lacrimal gland nerves decreased in 24-month-old mice compared to 3- and 12-month-old animals. Similarly, progressive morphological changes, including increased numbers of lipofuscin-like inclusions, mast cells and lymphocytic infiltration occurred in an age-dependent manner. We conclude that structural alterations of mouse lacrimal gland, including increased accumulation of lipofuscin-like inclusions, chronic inflammation and functional alterations including decreased acetylcholine release and protein secretion occurred with aging.