Perforin hyperreleasability and depletion in cytotoxic T cells from patients with exacerbated atopic dermatitis and asymptomatic rhinoconjunctivitis allergica

Abstract

Background: As a plasma membrane pore-forming protein, perforin is essential for T-cell cytotoxicity mediated by lytic granules. Recent studies on the immune system of perforin knockout mice demonstrated striking similarities to the immunopathology of atopic diseases. Objective: We sought to investigate the perforin system of atopic patients. Methods: Monoclonal antibodies were used to characterize perforin-positive PBMCs of patients with exacerbated atopic dermatitis (AD) and asymptomatic rhinoconjunctivitis allergica (RCA) by means of immunoflow cytometry. In addition, a perforin release assay was developed to quantify the velocity of ionomycin and phorbol 12-myristate 13-acetate-induced secretion of lytic granules. Results: In atopic patients significantly fewer lymphocytes contained perforin-positive lytic granules compared with those of healthy control subjects (patients with AD: 14% ± 5%, n = 13, P < .0001; patients with RCA: 24% ± 5%, n = 9, P < .01; healthy control subjects: 33% ± 11%, n = 13). Of all CD8hi+ cytotoxic T lymphocytes (CTLs), only 18% ± 9% and 17% ± 12% were perforin-positive in patients with AD and RCA, respectively, compared with 44% ± 13% in control subjects (P < .0001). In addition, perforin-positive CD8hi+ CTLs of atopic patients released their perforin twice as fast and more completely than control CTLs. This means that 50% of initially perforin-positive CD8hi+ CTLs from patients with AD and RCA released their perforin completely within 32 ± 16 and 36 ± 19 minutes, respectively, and an over 85% release was reached within 113 ± 41 and 118 ± 60 minutes, respectively. In CTLs of healthy control subjects, however, it took 64 ± 40 minutes to achieve a 50% release of lytic granules, and an 85% depletion was not reached in 60 % of healthy control subjects, even after 180 minutes. Conclusion: The perforin hyperreleasability explains, at least in part, the decreased percentage of perforin-positive CD8hi+ CTLs in atopic patients. These distortions in the system of lyric granules of atopic patients may contribute to the functional defects observed in T-cell cytotoxicity in vivo and in vitro in patients with AD and RCA.