Abstract
In vitro studies were conducted in the non-obese diabetic (NOD) mouse, prone to Type 1 autoimmune diabetes, to investigate the mechanisms involved in cell-mediated rejection of pig islet xenografts. Our previous work concerning the mechanisms of proliferation of xenogeneic lymphocytes to pig islet cells (PIC) was not indicative of PIC impairment. Consequently, a test was developed based on perifusion analysis of the alteration of basal and stimulated insulin release from adult PIC incubated with mouse splenocytes or subsets. Compared with PIC incubation alone or with syngeneic pig splenocytes, co-incubation with mouse whole spleen cells resulted in a decrease of basal and stimulated insulin release (P < 0.001). Two components of this alteration were detected separately: PIC impairment was decreased (P < 0.01) after removal of plastic-adherent cells from spleen cells, but maintained (P < 0.01) when plastic-adherent cells alone were co-incubated with PIC. The increase of murine interleukin-1 beta when mouse plastic-adherent spleen cells were cultured with PIC (P < 0.04) was indicative of macrophage activation. Soluble factors produced during co-incubation of mouse splenocytes or plastic-adherent cells with PIC were involved in the impairment process, since supernatant fluids collected during previous PIC-mouse cell co-incubations directly altered (P < 0.01) insulin release from PIC. Moreover, impairment of PIC by mouse spleen cells was abolished (P < 0.01) by gadolinium chloride (which inhibits macrophages), but not by cyclosporin A. Another mechanism was apparent, since co-incubation of PIC with purified mouse T cells or CD4+ T cells, re-mixed with antigen-presenting cells, led to a decrease (P < 0.01) of insulin release. This model, based on the alteration of dynamic basal and stimulated insulin release, is indicative of in vitro cell-mediated alteration of PIC in the NOD mouse. The effect of whole spleen cells was rapid, and a crucial role was played by plastic-adherent cells. Two mechanisms were responsible for the behaviour of these cells: an early direct effect (at least in part via soluble products); and the indirect presentation of PIC xenoantigens (leading to impairment by CD4+ T lymphocytes).