The Investigation of CD3 Subpopulation of T Lymphocytes in Canine Nodular Granulomatous Episcleritis
A.L. Andrade; S.S. Sakamoto; C.M. Silva; M.C.R. Luvizotto; H.F. Ferrari
There is a spectrum of idiopathic inflammatory diseases of the canine sclera and episclera that includes the entities diffuse and nodular episcleritis, diffuse and nodular scleritis and necrotizing scleritis. These disorders may be unilateral or bilateral, and are characterized by histopathologic changes of mononuclear inflammation, with or without vasculitis (Deykin et al. 1997). Episcleritis and nodular granulomatous episcleritis (NGE) lesions usually appear nonpainful, multiple, elevated, fleshy masses or as single mass arising at the limbus and infiltrating the adjacent corneal stroma (Gelatt 2007) and light microscopy examination will reveal a mixed inflammatory cellular infiltrate, episcleral and conjunctival vascular congestion, and edema (Fischer, 1982). The predominant cell types are histiocytes, lymphocytes, and plasma cells (Gelatt 2007). Despite the clinical importance and severity of the inflammatory diseases of the canine episclera and sclera, little is known of the immunopathogenesis of the lesions. In recent years it has become possible to further characterize the nature of canine inflammatory disease by identifying immunoglobulin depositation, and by phenotyping the tissue infiltrates using immunohistochemistry (Day et al. 2008). Then, the purpose of the present study was to use immunohistochemistry to investigate the expression of CD3 T lymphocytes in the pathogenesis of NGE.
Materials and Methods
The two cases reported here were dogs referred for surgical service of Veterinary Hospital (Unesp--campus de Araçatuba). Inclusion criteria included examination by veterinary ophthalmologist, confirmation of diagnosis by light microscopic evaluation of an episcleral biopsy, and follow-up examination by an ophthalmologist, medical therapy, duration of disease, diagnostic test results, physical and ophthalmic examination findings, and clinical outcomes were reviewed. Hematoxylin and eosin-stained canine episcleral biopsies were reviewed to evaluate morphology. Immunohistochemical staining was performed on formalin fixed, paraffin embedded biopsies using a streptavidin-biotin complex technique. Monoclonal antibody anti-CD3 (Anti-human T cell CD3 peptide, SIGMA--n.C7930) was used to identify T cells. Sections of normal canine lymph node were concurrently stained as a positive control, and additional episcleritis biopsy sections stained with omission of the primary antibody served as a negative control. Three x40 fields for each immunohistochemical stain were digitally photographed.
Two NGE biopsies met the inclusion criteria (one unilateral, one bilateral NGE). The mean age of the dogs was 6.8. The both animals were male. One dog was Beagle and other was mixed-Collie. The ocular signs were: simple mass arising at the limbus (lateral portion), elevated and fleshy. The conjunctival vessels were engorged. Corneal opacity was observed in both cases and uveitis signs. In addition to the mass lesion, other observed ocular signs included epiphora, mucopurulent ocular discharge and protrusion of the third eyelid. Fluorescein staining was negative bilaterally in two animals. Topical ophthalmic steroid (1% predinsone) and cyclosporine A preparation used before biopsy. Hematoxylin and eosin-stained revealed an inflammatory cell population dominated by lymphocytes and histiocytes with occasional plasma cells in both biopsies. There were numerous CD3 positive cells in cases studied.
Discussion and Conclusions
A breed predisposition for the American Cocker Spaniel for episcleritis, and the Collie for NGE has been documented previously (Paulsen et al. 1987, Deykin et al. 1997). The same was observed here for one of the cases. Although a sex predilection for female dogs has been suggested (Deykin et al. 1997), in this study, both cases occurred in males. Canine episcleritis is suspected to be an immune-mediated disease because of the failures to identify an etiologic agent, as well as the favorable response of clinical signs to immunosuppressive medications (Couto 1992, Carrie et al. 2007). It was observed here, because the animals were treated by topical corticosteroids and cyclosporine A. Medical therapy appears to be more effective than surgical excision combined with intralesional corticosteroid treatment, cryotherapy or beta irradiation (Pulsen et al. 1987). The ocular signs decrease after 30th day of the treatment. The light microscopic findings of canine episcleritis have been described (Deykin et al. 1997). A cell-mediated response dominated by T lymphocytes has been suggested based on findings from a single biopsy. However, the use of immunohistochemistry to specifically identify T cell is more specific. In this same way, Breaux et al (2008) investigated the immunopathologic aspects of the canine episcleral biopsies to identify macrophages, B and T cells, and they observed that there is a significant difference in the inflammatory cell population in episcleritis. These findings are also in keeping with immunophenotypic investigations of human sclerotic lesions, where there is a dominance of T lymphocytes that have been further identified as being of the CD4+ subpopulation. Unfortunately, it was not possible to define here in two cases, because we could have the antibody for it. The infiltrating T cells are likely of prime importance in regulating the immunopathologic processes that lead to the tissue destruction in granulomatous scleritis. Although T cells are a pivotal immunoregulatory population they not necessary directly mediate the tissue damage that characterizes scleritis (Day et al. 2008). In conclusion, there is participation of CD T cells in canine NGE. Future investigations should attempt to identify the type of immunoglobulin produced by infiltrating B cells, the types of T cells present, and identification and quantification of cytokines involved.
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