Topical cyclosporine inhibits conjunctival epithelial apoptosis in experimental murine keratoconjunctivitis sicca.

PURPOSE: Increased apoptosis in the conjunctival epithelium has been observed in experimental murine keratoconjunctivitis sicca (KCS). Topical cyclosporine (CsA) has been noted to reduce conjunctival epithelial apoptosis in chronic canine and human KCS. The purpose of this study is to determine if topical CsA treatment inhibits conjunctival epithelial apoptosis in a murine model of KCS. METHODS: Dry eye was induced in 3 groups of C57BL6 mice by subcutaneous injection of scopolamine TID and exposure to an air draft and low-humidity environment for 16 hours per day for 12 days. The dry eye control group received no topical treatment; a second group received 1 microL of 0.05% CsA topically TID (dry eye + CsA); and the third group received 1 microL of the castor oil vehicle of CsA topically TID (dry eye + vehicle). Normal mice were used as untreated controls. After 12 days, the mice were killed, and the right eyes and eyelids were excised, frozen, and cryosectioned. Transmission electron microscopy (TEM) was performed on conjunctival and corneal samples taken from the left eyes. Apoptosis was detected in frozen sections with the ApopTag (ISOL) In Situ Oligo Ligation Kit, which specifically detects DNA fragmentation. Immunohistochemical staining was performed to detect activated caspase-3. Conjunctival goblet cell number was counted in tissue sections stained with period acid Schiff (PAS) reagent. These assays were performed on 2 separate sets of mice. RESULTS: Compared with untreated controls and dry eye mice receiving CsA, the number of ISOL-positive epithelial cells in the bulbar and tarsal conjunctiva was significantly greater in the dry eye control and dry eye mice + vehicle groups (P < 0.01 for both groups). There was no significant difference in the number of ISOL-positive conjunctival epithelial cells between the dry eye control and dry eye + vehicle mice. There was no significant difference in ISOL-positive cells in the corneal epithelium between the untreated controls and the 3 treatment groups. Dry eye + CsA mice showed less activated caspase-3 staining than the dry eye control and the dry eye + vehicle groups. TEM showed loss of superficial differentiated cells and extensive nuclear fragmentation characteristic of apoptosis in the dry eye control and dry eye + vehicle groups but not in the dry eye + CsA group. There was significant loss of goblet cells in the bulbar and tarsal conjunctivae of the dry eye control and the dry eye + vehicle groups compared with untreated controls and the dry eye + CsA group. CONCLUSIONS: Topical CsA significantly reduced conjunctival epithelial apoptosis and protected against goblet cell loss in experimental murine KCS. Inhibition of apoptosis appears to be a key mechanism for the therapeutic effect of CsA for KCS.

Avaliação de células T regulatórias e pefil de ativação de linfócitos T na ceratoconjuntivite seca associada ao HTLV-1 / Regulatory T cells evaluation and T lymphocyte activation profile in keratoconjunctivitis sicca associated with HTLV-1

O HTLV-1 é o agente etiológico da leucemia /linfoma de células T do adulto (ATLL), da paraparesia espástica tropical/ mielopatia associada ao HTLV-1 (HAM/TSP) e da uveíte. Além destas, a ceratoconjutivite seca (CCS), doença multifatorial da lágrima e da superfície ocular, tem sido descrita com maior frequência em indivíduos infectados pelo HTLV-1. Assim como em outras doenças associadas, a CCS tem sido relacionada a uma elevada carga proviral. As células T regulatórias (Treg) são importantes na manutenção da homeostase do sistema imunológico e um comprometimento da imunorregulação exercido por elas pode contribuir para o ambiente inflamatório observado na CCS. Este estudo objetivou avaliar os linfócitos Treg de pacientes com CCS associada à infecção pelo HTLV-1. Foram realizados ensaios de imunofenotipagem por citometria de fluxo para avaliar a frequência de linfócitos T ativados (HLA-DR+) e de células T CD4+ e CD8+ regulatórios (FOXP3+), bem como a produção de IL-10 e TGF-β por estas células. Foram avaliados 37 pacientes infectados pelo HTLV-1 e assintomáticos para HAM/TSP, sendo 27 com diagnóstico positivo para a manifestação ocular (CCS), 10 com diagnóstico negativo (ASS), além de 17 voluntários não infectados pelo vírus (NI). As frequências de linfócitos T CD4+FOXP3+, CD8+FOXP3+, CD4+HLA-DR+ e CD8+HLA-DR+ foram significativamente maiores nos grupos CCS e ASS, quando comparados aos indivíduos não infectados. Quanto à produção das citocinas imunossupressoras, foi observada uma maior frequência de linfócitos T CD4+FOXP3+ duplo produtores de IL-10 e TGF-β no grupo CCS quando comparado ao grupo ASS. Com relação aos linfócitos CD8+FOXP3+, o grupo CCS apresentou uma maior frequência de células mono produtoras de IL-10 quando comparado ao ASS. Nossos resultados sugerem que a menor frequência de células Treg CD8+ produtoras de TGF-β em indivíduos infectados pelo HTLV-1 com CCS, pode contribuir para a intensificação da ativação celular e fisiopatologia da doença.

HTLV-1 is the causative agent of leukemia/lymphoma adult T-cell (ATLL), tropical spastic paraparesis / myelopathy associated with HTLV-1 (HAM / TSP) and uveitis. In addition, keratoconjunctivitis sicca (KCS), a multifactorial disease of the tear and of the ocular surface, has been more frequently reported in patients infected with HTLV-1. As for other HTLV-1-associated diseases, KCS has been related to a high proviral load. Regulatory T (Treg) cells are important in maintaining the homeostasis of the immune system. An impairment in the immunoregulation function of Treg may contribute to the inflammatory environment observed in the KCS. This study aimed to evaluate the Treg cells of patients with KCS associated with HTLV-1. Frequency of activated T cells (HLA-DR+) and CD4+ and CD8+ Treg cells (FOXP3+), as well as IL-10 and TGF-β production by Treg were quantified using flow cytometry. Thirty-seven HTLV-1 individuals were included (27 asymptomatic for HAM/TSP with positive diagnosis of ocular manifestation (KCS), 10 with negative diagnosis (ASS - asymptomatic). Seventeen non-infected individuals were included as controls (NI). The frequencies of CD4+ FOXP3+ T cells, CD8+FOXP3+, CD4+HLA-DR+ and CD8+HLA-DR+ were significantly higher in KCS and ASS groups when compared to non-infected individuals. As the production of immunosuppressive cytokines, a higher frequency of CD4+ FOXP3+ double producers of IL-10 and TGF-β in the KCS group was observed when compared to group ASS. Regarding the CD8+FOXP3+ lymphocytes, the KCS group had a higher frequency of mono cells producing IL-10 when compared to the ASS. Our results suggest that the lower frequency of Treg cells CD8+ TGF-β-producing in individuals infected with HTLV-1 with KCS, may contribute to the intensification of cellular activation and pathophysiology of the disease.

The tear film and ocular mucins.

Abstract The trilaminar tear film, composed of the lipid, aqueous and mucin layers, has many functions including defending the ocular surface. The aqueous layer has several soluble antimicrobial factors that protect the ocular surface. Ocular mucins have recently been studied with regard to their role in the defense of the eye as well as in dry eye syndromes. To date, 15 mucin genes have been identified, and six of these mucin genes are localized to or secreted by ocular glands or epithelia. Understanding the production, secretion and function of ocular mucins will aid in the treatment of dry eye syndromes and ocular surface microbial infections.