Cytokine Expression Profiles in Rabbit Models of Autoimmune Dacryoadenitis Induced by Autoadoptive Transfer of ex vivo Activated Lymphocytes

Abstract

Purpose: : When isolated and placed in primary culture, acinar cells from rabbit lacrimal glands appear to function as antigen-presenting cells, capable of presenting intracellular autoantigens to autologous peripheral blood lymphocytes in mixed cell reactions. We recently demonstrated that the most vigorously proliferating lymphocyte subset in such mixed cell reactions is CD4⁺ T cells. CD4⁺ cells purified from the autologous mixed cell reactions induce more severe lacrimal gland and ocular surface disease than either unfractionated lymphocytes or CD4-depleted lymphocytes when autoadoptively transferred to the donor animals’ remaining lacrimal gland. To test the hypothesis that the three different activated lymphocyte samples induced fundamentally different types of autoimmune pathophysiology, we used real time RT-PCR analysis to characterize cytokine and surface marker expression in the diseased lacrimal glands.

Methods: : One inferior lacrimal gland was surgically excised from each rabbit. Acinar cells were purified, cultured for 2 days, gamma-irradiated, and then co-cultured for 5 days with purified autologous PBL. Activated lymphocytes were either left unfractionated or stained with anti-rabbit CD4 and subjected to FACS to separate CD4-enirched and CD4-depleted samples before autoadoptive transfer. After 4 weeks, ocular surface status was assessed, and the rabbits were sacrificed. RNA was extracted from the inferior lacrimal glands and analyzed by real time RT-PCR using rabbit-specific primers and probes.

Results: : Tumor necrosis factor alpha and the CD80 co-stimulatory molecule were increased roughly 2 and 2.5-fold in all three groups. Neither IL-6 nor IL-10 changed markedly in any of the groups. Interferon gamma increased most markedly in glands injected with CD4-enriched lymphocytes, while both IL-2 and IL-4 decreased markedly in glands injected with CD4-depleted lymphocytes.

Conclusions: : Activated autoreactive CD4⁺ lymphocytes induce a T_H1-mediated pathophysiology. Fundamentally different pathophysiologies are induced by unfractionated lymphocytes and by CD4-depleted lymphocytes; these processes remain to be characterized.

Keywords: lacrimal gland • autoimmune disease