Co-incubation of pig islet cells with spleen cells from non-obese diabetic mice causes decreased insulin release by non-T-cell- and T-cell-mediated mechanisms

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).

Fig. 1

Marked decrease of dynamic insulin release from perifused pig islet cells (PIC) incubated with mouse whole spleen cells. (a and b) Insulin release of 100 000 PIC cultured (•) alone (n = 15), (×) with 1·5 × 10⁶ syngeneic pig splenocytes (n = 2), or with 1·5 × 10⁶ NOD mouse whole spleen cells (□) irradiated (n = 2) or (○) not (n = 15). Perifusion was performed after 48 h (a) or 7 days (b) of co-incubation. The grey area corresponds to the glucose stimulation period. Each point indicates the mean value ±s.e.m. P < 0·001 between insulin release from PIC cultured alone or with mouse spleen cells.

Fig. 2

Involvement of plastic-adherent cells in early impairment of pig islet cell (PIC) function by spleen cells from NOD mice. (a) Insulin release from 100 000 PIC cultured for 48 h alone (•), with 1·5 × 10⁶ whole mouse spleen cells (○), or with 1·5 × 10⁶ mouse spleen cells depleted of plastic-adherent cells (□). Each point indicates the mean value ±s.e.m. of 10 experiments. P < 0·01 between PIC cultured with whole spleen cells or spleen cells depleted of plastic-adherent cells. (b) Insulin release from PIC cultured for 48 h alone (•) or with 300 000 mouse plastic-adherent cells (○). Each point indicates the mean value ±s.e.m. of 10 experiments. P < 0·01 between PIC cultured alone or with plastic-adherent spleen cells. (c) Insulin release from PIC cultured for 48 h alone (•), with supernatant fluids from previous co-cultures of PIC and mouse spleen cells (○), or with plastic-adherent spleen cells (□). Each point indicates the mean value ±s.e.m. of three experiments. P < 0·01 between PIC cultured alone or with supernatant fluids. (d) Effects of 0·01 µg/ml (▪), 1 µg/ml (◊), or 100 µg/ml (□) of gadolinium chloride on insulin release from PIC co-cultured with mouse spleen cells for 48 h. Comparisons were performed with co-cultures of PIC and spleen cells without gadolinium chloride (○) or with PIC alone (•). Each point indicates the mean value ±s.e.m. of three experiments. P < 0·01 between PIC cultured with mouse spleen cells in the absence and presence of Gad. The grey area corresponds to the glucose stimulation period.

Fig. 3

In vitro production of interleukin-1β (IL-1β) by 300 000 plastic-adherent spleen cells from NOD mice cultured for 48 h alone (□) or with 100 000 pig islet cells (PIC, ▪). Columns indicate the mean values ±s.e.m. of three experiments. *P < 0·04 between IL-1β production from mouse cells cultured alone or with PIC.

Fig. 4

Alteration of insulin release from pig islet cells (PIC) by mouse T cells or subsets. Insulin release of 100 000 PIC cultured alone (•) or with mouse spleen cell subsets for 7 days. The grey area corresponds to the glucose stimulation period during perifusion. (a) PIC were co-incubated with 150 000 mouse plastic-adherent, antigen-presenting cells (APC) (n = 4; ○), 1·5 × 10⁶ mouse purified T cells, or T cells mixed with APC (n = 5, □, and n = 5, ▪, respectively). P < 0·01 between insulin release from PIC alone and PIC cultured with T cells mixed with APC. (b) PIC were co-incubated with APC (n = 4; ○), 1·5 × 10⁶ mouse CD4⁺ cells, or CD4⁺ cells mixed with APC (□ and ▪, respectively, n = 4 each). P < 0·01 between insulin release from PIC alone and PIC cultured with CD4⁺ cells mixed with APC. (c) Representative experiment in which PIC were co-incubated with APC (n = 1; ○), 1·5 × 10⁶ mouse CD8⁺ cells, or CD8⁺ cells mixed with APC (n = 1, □, and n = 1, ▪, respectively).