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Cell Biology International (2006) 30, 1054–1056 (Printed in Great Britain)
Mast cells accumulate in the renal capsule adjacent to transplanted pancreatic islets in rats
Caroline Kampfa* and Leif Janssonb
aDepartment of Genetics and Pathology, Rudbeck Laboratory, C5:3, Uppsala University, SE-751 85 Uppsala, Sweden
bDepartment of Medical Cell Biology, Uppsala University, SE-751 85 Uppsala, Sweden


Abstract

Mast cells are important mediators of normal angiogenesis, and participate in normal would healing, i.e. processes involved in pancreatic islet engraftment. The aim of the study was to evaluate if mast cells are present in islet grafts. For this purpose, male normoglycaemic Wistar–Furth rats were either untreated or syngeneically implanted with 250 islets under the renal capsule. The animals were killed 1 month later, and the kidneys and endogenous pancreas were removed, fixed and embedded in paraffin. The distribution of mast cells was studied in Alcian Blue stained sections. Mast cells were rarely encountered in endogenous islets, but were frequent in the renal capsule adjacent to islet grafts. Mast cells interspersed between graft endocrine cells were as rare as in the endogenous pancreas. We conclude that mast cells may contribute to the engraftment after islet transplantation.


Keywords: Pancreatic islets, Mast cells, Transplantation.

*Corresponding author. Tel.: +46 18 4715032; fax: +46 18 552739.


1 Introduction

Mast cells are ubiquitously present in connective tissues in the body, and constitute an important component in the innate immune system, especially in the initial host defence against bacteria (Galli and Nakae, 2003; Malaviya and Abraham, 2001). Furthermore, mast cells accumulate adjacent to sites of angiogenesis, and tissue repair. Several mast cell mediators are angiogenic and products such as tryptase and chymase help to degrade the matrix to provide space for neovascular sprouts (Hiromatsu and Toda, 2003).

Transplantation of isolated pancreatic islets necessitates a revascularization of the graft soon after implantation to achieve survival of the endocrine cells (Menger et al., 2001). This process normally occurs through an angiogenic process during the first week post-transplantation, but recent studies suggest that there is a vascular dysfunction in islet grafts, partially dependent upon a decreased vascular density in the transplant (Jansson and Carlsson, 2002). The decrease in vascular density seems to be due to a lack of capillaries interspersed among the endocrine cells, whereas microvessels located in the connective tissue between the islets are abundant (Mattsson et al., 2002). The reasons for this impaired revascularization are at present unknown, but in view of the importance of mast cells in tissue repair processes we deemed it of interest to evaluate the presence of mast cells in grafted islets.

2 Materials and methods

Male Wistar–Furth rats (Møllegaard-Bomholtdgaard; Ry, Denmark) aged 3 months were used in all experiments. Pancreatic islets were isolated by a collagenase digestion method, as previously described (Sandler et al., 1987). The islets were cultured free-floating in groups of 150 for 3–4 days in 5ml culture medium consisting of RPMI 1640 (Sigma–Aldrich, St. Louis, MO, USA) supplemented with L-glutamine (Sigma–Aldrich), benzylpenicillin (100U/ml; Roche Diagnostics Scandinavia, Bromma, Sweden), streptomycin (0.1mg/ml; Sigma–Aldrich) and 10% (vol/vol) fetal calf serum (Sigma–Aldrich). Culture medium was changed every second day. A total of 250 islets were implanted under the left renal capsule of six syngeneic rats anaesthetized with an intraperitoneal injection of ekviticine (chloral hydrate and pentobarbital; Apoteksbolaget, Umeå, Sweden). After 1 month the graft-bearing kidney and the endogenous pancreas were removed and fixed in 10% buffered formalin overnight, before being histoprocessed and embedded in paraffin.

Sections were deparaffinized in xylene rehydrated in graded alcohols and stained with alcian blue (Merck, Darmstadt, Germany) at pH 0.5. The tissue sections were incubated in solution A (10ml HCl and 90ml 70% alcohol) and thereafter solution B (10ml HCl and 90ml 70% alcohol and 1g alcian blue) for 15min. The incubation was followed by a 5min wash in solution A and 10min in tap water. The nuclei were counterstained with Kernecht rot (Merck) and the sections were dehydrated and mounted in Pertex mounting medium (HistoLab, Göteborg, Sweden).

3 Results

In endogenous islets of both transplanted and non-transplanted rats mast cells were sparse, and were seen only occasionally. They were more abundant in the exocrine tissue, especially in connective tissue, around major ducts and blood vessels (Fig. 1). Mast cells were frequently found in islet grafts under the renal capsule, and were consistently seen in the border zone between the renal capsule and the endocrine cells (Figs. 2 and 3). Mast cells interspersed among the graft endocrine cells were very rare, and no mast cells were seen in other parts of the renal capsule or parenchyma than those adjacent to the graft.


Fig. 1

Mast cells (arrows) in connective tissue around interlobular blood vessels in the rat pancreas. Magnification: scale bar 50μm.


Fig. 2

Several mast cells (arrows) can be seen between the renal capsule and the islet graft. Magnification: scale bar 50μm.


Fig. 3

Higher magnification of a mast cell staining (arrow) in the connective tissue adjacent to an islet graft under the renal capsule. Magnification: scale bar 20μm.




4 Discussion

The present study shows that mast cells are frequently found in islet grafts in greater abundance than in endogenous islets. Approximately one mast cell per 10 islets has been reported in the normal pancreas (Hahn von Dorsche, 1984; Westermark, 1971). This is in line with the present findings, since only occasional mast cells were seen in endogenous islets. The number of intra-islet mast cells is doubled in type-2 diabetes, especially when associated with amyloidosis, for unknown reasons (Mlac et al., 1975; Westermark, 1971).

The reasons for the increased number of mast cells in grafted islets are also unknown. At the time-point chosen for the study, i.e. 1 month post-transplantation, angiogenesis should be finished (Jansson and Carlsson, 2002). Nevertheless, mast cells were found in the renal capsule overlying the graft. Released mast cell tryptase is mitogenic for fibroblasts and smooth muscle cells in other organs (Brown et al., 1995; Ruoss et al., 1991). These findings are of particular interest, since islet grafts usually contain a lot of connective tissue (Mattsson et al., 2002) and have a lower blood perfusion rate than endogenous islets (Jansson and Carlsson, 2002). Thus, it may well be that mast cells are involved in this graft dysfunction, which is under further investigation.

One concern is that mast cells are known to participate in chronic rejections in kidneys (Pardo et al., 2000), hearts (Koskinen et al., 2001; Li et al., 1992), lungs (Minami et al., 1995), livers (O'Keeffe et al., 2002) and also in xenogeneic rejection of islets (Mandel et al., 1995). However, no other signs of chronic or acute rejection were seen, so we deem it unlikely that this was the cause of mast cell infiltration.

Acknowledgements

Birgitta Bodin is gratefully acknowledged for her skilled technical assistance. Financial support was received from the Swedish Research Council (72X-109, 72XD-15043), the Swedish Diabetes Association, the Juvenile Diabetes Research Foundation, the EFSD/Novo Nordisk for Type 2 Diabetes Research Grant, Barndiabetesfonden, Åke Wibergs Foundation, and the Family Ernfors Fund.

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Received 21 December 2004/11 August 2005; accepted 19 April 2006

doi:10.1016/j.cellbi.2006.09.016


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)