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

SUMMARYIn 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, coincubation 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-1b 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 1 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 1 T lymphocytes).

Sensitivity of insulin production from encapsulated islets to endotoxin‐stimulated macrophage inflammatory mediators

“…Substantiating this observation, we noted that much lower levels of nitrite correlated with complete blockage of the insulin response when the NO was produced by LPS and mrIFN‐γ‐activated macrophages in direct contact or immediately beneath the islets in coculture. This confirmation of enhanced sensitivity of islets to coculture macrophage‐generated NO over that of chemically induced NO agrees with that of previously published coculture studies of islets with different immune cell preparations 3, 6, 12, 17, 50–52…”

“…In regards to other coculture studies with pig islets, Lalain et al demonstrated that coincubation of adult pig islets for 7 days with human peripheral blood mononuclear cells (PBMC) caused loss of insulin response to high glucose, as mediated both by macrophage and T‐lymphocyte mechanisms 51. Another study by You et al arrived at a similar conclusion when coincubating adult pig islets with mouse splenocytes or subsets taken from diabetic mice 52. Again, both non‐T‐cell and T‐cell‐mediated mechanisms were detected that caused decreased insulin release by the pig islets.…”

“…This confirmation of enhanced sensitivity of islets to coculture macrophage-generated NO over that of chemically induced NO agrees with that of previously published coculture studies of islets with different immune cell preparations. 3,6,12,17,[50][51][52] Alginate-encapsulated rat islets cocultured for 24 h with resident peritoneal macrophages has previously been shown by Wiegand et al to be lysed when those macrophages are activated by in vivo exposure to Cornyebacterium parvum. 12 Resident unactivated macrophages did not lyse the encapsulated rat islets.…”

Rapid functional evaluation of beta-cells by extracellular recording of membrane potential oscillations with microelectrode arrays

“…MEAs should be useful for investigating co-cultures, e.g. how co-culturing pig islets with spleen cells affects insulin secretion [21] or how bone marrow increases the survival time of human islets [16]. MEAs could be used to study co-cultures of macrophages and islets and determine the action of macrophage-released substances on beta-cell electrical activity.…”

Microchimerism and transmission of porcine endogenous retrovirus from a pig cell line or specific pathogen-free pig islets to mouse tissues and human cells during xenografts in nude mice

“…The primers used for PCR amplification of PERV sequences or pig mt DNA were synthesised by the Life Tech-During xenograft, there is a risk of transmitting infectious agents from pig to man. The risk of conventional zoonosis led us to isolate islets from specific pathogen-free (SPF) pigs [1,2,3,4,5,6,7,8]. However, there is also a risk of transmitting porcine endogenous retroviruses (PERV) [9,10,11,12].…”