Mesenteric lymph nodes as alternative site for pancreatic islet transplantation in a diabetic rat model

Xenotransplantation has recently emerged as a promising alternative to contrast the increasing demand for organ transplantation, and this resulted in many potential clinical applications [1], including the treatment of type 1 diabetes. The dramatic impact on the health of the world’s population has stimulated the need to develop alternative strategies for the treatment of the type 1 diabetes [2]. Pancreatic islet transplantation is becoming an increasingly important strategy for type 1 diabetes mellitus treatment, with a growing number of new procedures performed worldwide, and is able to normalize blood glucose or HbA1c levels in most recipients with diabetes [3‐9]. Transplantation of deceased human donor islets into the portal vein in patients with type 1 diabetes mellitus has had encouraging results [3‐11], but is associated with an immediate loss of 60–80% of islets through an inflammatory response known as the instant blood-mediated inflammatory reaction (IBMIR) [12, 13]. The long-term results of islet transplantation, therefore, could be significantly increased by improving the quality of the islet isolation technique to increase the yield from the donor pancreas, or reducing the number of islets required to improve hyperglycemia. In recent years, many studies explored alternative islet transplantation sites to reduce the number of islets required to mitigate hyperglycemia [14‐18].

The gastric sub-mucosal space (GSMS) is a promising alternative site for islet implantation [14‐16]: islets are transplanted through an endoscopic placement, and they would benefit from a rich blood supply and a high oxygen tension. Preclinical trials using a porcine model showed reduced early islet loss and insulin requirements when GSMS islets were compared to intraportal islets [14, 15], and islets were easily accessible for endoscopic monitoring of graft rejection [16].

As alternative sites to the liver, Komori et al [19] demonstrated the surprising potential of lymph nodes (LN) as transplant sites for multiple tissues, among which they also tested pancreatic islets. The goal of this study was to conduct a pilot experiment of feasibility of islet transplantation in the rat model. Following the results of the study by Komori et al [19], where LN was successfully used for islet engraftment in a mouse model, we tested, as a proof of concept, that successful engraftment could be achieved in a larger rodent model.

A total of 7 islet transplantations in mesenteric lymph nodes have been performed. Two rats died 24 and 36 h after transplantation due to complications.

Three groups of rats were considered:

Five transplanted rats survived until euthanasia through CO₂ inhalation. No transplanted rat acquired normal glucose blood levels and insulin independence after the transplantation. However, the mean blood levels of glycemia were significantly lower in transplanted rats compared with diabetic rats (470.4 mg/dl vs 605 mg/dl, p 0.04). Interestingly, transplanted rats have a significant weight increase after transplantation compared to diabetic rats (mean value 295 g in transplanted rats vs 245 g in diabetic rats, p < 0.05) (Fig. 5), with an overall improvement of social activities and health. There was no significant correlation between the number of transplanted islets and levels of glycemia.

As expected, there was no evidence of islet in mesenteric lymph-nodes in group A and B rats.

Immunohistochemical analysis of the 5 mesenteric lymph nodes of transplanted rats demonstrated the presence of living islets in one lymph node (Fig. 6), as confirmed by the presence of insulin-positive cells (Fig. 7) and anti-sinaptofisina, clone SY38 and anti-chromogranin A analyses (Fig. 8). In other transplanted lymph-nodes, there was no evidence of intact islet cells, but only macrophage elements positive for insulin, suggesting the existence of necrotic or apoptotic islets (Fig. 9).

The research for alternative transplant sites plays a fundamental role in the field of pancreatic islet transplantation and, in general, of cellular transplantation. The results reported by several previous studies show the gastric submucosal space is one of the most promising sites that could be clinically used in the near future [14‐16]. However, this assumption does not appear to be completely valid for the lymph nodes, whose use in islet transplantation is possible but it may be intended to better characterize the mechanism of allo- and xeno-transplantation of pancreatic islets in animal models.

The present study allowed obtaining more information about some aspects of the islet transplantation already highlighted by previous studies. The intent to prove whether the anatomical location of the lymph nodes affects islet survival and function was inspired by Komori’s work [19], who hypothesized and evaluated the surprising potential of lymph nodes as transplantation sites for multiple tissues. In their work, Komori et al [19],underlined the interesting properties of the lymph nodes: their specific environment protects and enhances survival of lymphocytes, they are close to blood vessels and they also contain fibroblastic reticular cells and other stromal cells that secrete chemokines. They engrafted three distinct healthy cell types, hepatocytes, thymic tissue and pancreatic islets, in different lymph nodes of mice and they observed their ability to promote survival and growth of transplanted cells [19]. Syngeneic mouse hepatocytes marked with green fluorescent protein were injected into the single large jejunal lymph node – it was selected because it is easily accessible and it is the largest in the mouse; one week after injection, the cells were retained mainly in the subcapsular sinus of the lymph node rather than the follicles or germinal centres and, additionally, they formed patches of tissue accompanied by remodelling of blood vessels and growth [19, 22]. Similarly, they injected thymic tissue into the same lymph node of athymic mice and ten months after they observed its long-term engraftment, confirmed by demonstrating that a T-cell-dependent immunity had been acquired by the recipients [19, 22]. Concerning pancreatic islets, the study showed successful islet transplantation in a diabetic syngeneic mouse model, by the infusion of 300 mouse islets mixed with Matrigel into the jejunal lymph node of mice treated with streptozotocin; as well as the hepatocytes, the islets were found in the subcapsular sinus of the lymph node and the expression of C-peptide was detected. Six weeks after transplantation the authors observed the normalization of blood glucose in the recipients, demonstrating the survival of pancreatic islets and the ability of the lymph nodes to sustain long-term normoglycaemia [19, 22]. Then, according to the immunological role of the lymph nodes, they hypothesize that an immune response might interfere with the function of the engrafted cells, so they induced an inflammatory reaction in the intraperitoneal cavity of normoglycaemic recipients with an injection of lipopolysaccharide: they observed a temporary reduction in mouse weight and blood glucose levels, but any increase in glucose blood levels above normal levels was observed, which meant functioning grafts [19, 22]. These data suggested that the lymph nodes did not have negative effects on grafted islets. Their analysis included two other important findings: they observed many endothelial cells expressed by grafted islet around the areas of the engraftment, suggesting that extensive vascular remodelling takes place during the engraftment [19]; additionally, they detected markers of neovascular remodelling in each of the engrafted lymph nodes, suggesting that blood vessels surrounding the lymph nodes also contribute to the neovascularization [19]. These observations allow the establishment of the lymph node as a new potential site for functional cellular transplant, taking account of other properties in addition to those already mentioned: its accessibility and the possibility to monitor cells’ function by its biopsy or even its excision; moreover, in the islet transplantation, another advantage could be the secretion of insulin directly into the portal circulation by using the lymph nodes of the abdominal cavity. The results of the present study, at least in part, confirm the hypothesis by Komori and colleagues [19], considering the following evidences: a) the blood glucose levels in transplanted rats were significantly lower than those in diabetic rats; b) transplanted rats’ weight was increased after islet transplantation; c) transplanted rats’ social activities and health were improved after transplantation; d) living islets were immunohistochemically demonstrated in one mesenteric lymph node. However, although the studies by Komori’s group [19] suggest that the lymph nodes are a suitable site for islet transplantation, it is at the same time possible that the lymph node may represent an adverse and more immunogenic site and that in the absence of immunosuppression allogeneic islets may be rejected very rapidly, and this is probably independent by the site used. This possibility would explain a fifth evidence of the present study: e) why in some transplanted lymph-nodes there was no evidence of liable islet cells, but only macrophage elements positive for insulin, suggesting the islet apoptosis. If this proves to be the case, nevertheless syngeneic islets show prolonged survival and function, we will still progress to studies in cynomolgus monkeys where immunosuppressive therapy will be administered, in contrast to rats which do not tolerate well immunosuppression.

Our study demonstrated that, although islet engraftment in lymph nodes is possible, islet transplantation in lymph nodes in rats resulted in few improvements of glucose parameters. Nevertheless, this study provided a step forward in the knowledge of the mechanisms of islet engraftment.