Insulin protein and proliferation in ductal cells in the transplanted pancreas of patients with type 1 diabetes and recurrence of autoimmunity

Abstract

Aim/hypothesis: We investigated whether beta cell neoformation occurs in the transplanted pancreas in patients with type 1 diabetes who had received a simultaneous pancreas-kidney transplant (SPK) and later developed recurrence of autoimmunity.

Methods: We examined pancreas transplant biopsies from nine SPK patients with or without recurrent autoimmunity or recurrent diabetes and from 16 non-diabetic organ donors. Tissues were analysed by immunohistochemistry and immunofluorescence.

Results: Numerous cytokeratin-19 (CK-19)(+) pancreatic ductal cells stained for insulin in six SPK recipients with recurrent autoimmunity, in five of whom diabetes requiring insulin therapy recurred. These cells also stained for the transcription factor pancreatic-duodenal homeobox-1 (Pdx-1), which is implicated in pancreatic development and beta cell differentiation. The number of insulin(+) ductal cells varied, being highest in the patient with the most severe beta cell loss and lowest in the normoglycaemic patient. In the patient with the most severe beta cell loss, we detected insulin(+)CK-19(+)Pdx-1(+) cells staining for the proliferation-related Ki-67 antigen (Ki-67), indicating proliferation. We were unable to detect Ki-67(+) beta cells within the islets in any SPK patient. Some insulin(+)CK-19(-) ductal cells contained chromogranin A, suggesting further endocrine differentiation. Insulin(+) cells were rarely noted in the pancreas transplant ducts in three SPK patients without islet autoimmunity and in six of 16 non-diabetic organ donors; these insulin(+) cells were never CK-19(+).

Conclusions/interpretation: Insulin(+) pancreatic ductal cells, some apparently proliferating, were found in the transplanted pancreas with recurrent islet autoimmunity/diabetes. Replicating beta cells were not detected within islets. The observed changes may represent attempts at tissue remodelling and beta cell regeneration involving ductal cells in the human transplanted pancreas, possibly stimulated by hyperglycaemia and chronic inflammation.

Figure 1. Insulin⁺ cells in pancreatic ducts

Insulin expression demonstrated by immunohistochemistry in pancreas transplant biopsies from 6 SPK patients with recurrent autoimmunity (A–N). Patients #1–5 (A–M), who had developed recurrence of diabetes, had the most ductal cells stained for insulin. Panel A: patient #1, first biopsy; infiltrating lymphocytes are seen surrounding a duct containing insulin⁺ cells. Panels B and C: patient#1, second biopsy, serial sections stained for insulin and isotype control, respectively. In B, staining is weak but distinct and clearly above background in C. Panel D: patient #1, second biopsy, larger duct stained for insulin; staining is present also on the non-luminal side in many cells. Panel E and F: patient #1, second biopsy, ducts with intense insulin staining. Panel G: patient #1, a few ducts and some smaller ductal-like structures (lower right corner) are stained for insulin. Panel H: biopsy of patient #2, showing larger ducts and some smaller ductal-like structures stained for insulin. Panel I: biopsy of patient #3, insulin staining in a duct (arrow) is weaker than in the nearby islet. Some weaker staining is seen in the acinar tissue, possibly involving smaller ductal-like structures. Panel J: pancreas transplant biopsy of patient #4, showing three islets, one of which was heavily infiltrated by lymphocytes; Panels K and L are cropped from J to help visualize ducts containing insulin⁺ cells (arrows). Some weaker and less distinct staining is seen in the acinar tissue, possibly involving smaller ductal-like structures. Panel M: pancreas transplant biopsy of patient #5; not all ductal cells stained for insulin (arrows); staining is present also on the non-luminal side in many cells. Panel N: Rare insulin⁺ cells (arrow) were seen in a minority of ducts in patient #6; he had not yet developed significant insulitis and beta cell destruction and was normoglycemic. Panel O: Rare insulin⁺ cells (arrow) were seen in control pancreata. Some single insulin⁺ cells are also seen in the acinar tissue. Objective lens: 63× (A, D, F, G), 100× (B, C, E), 40× (H, I, M, N, O), 20× (J; K and L are cropped from J).

Figure 2. Co-expression of insulin and CK-19 in ductal cells of an SPK patient with recurrent autoimmunity

Sections are from the biopsy of patient #1. Images were acquired using a confocal microscope with a 40× lens. Panel A, B and C show staining for insulin, CK-19 and merged, respectively. In C, note the cell marked by the right arrow, which stained for CK-19 but did not stain for insulin. The single cells marked by arrows in C are shown enlarged in D and E. Panel D shows an insulin⁺CK-19⁺ cell, panel E shows a cell expressing CK-19 only. Colocalization analysis was performed by confocal microscopy and is graphically displayed in F and G, corresponding to the cells shown in D and E, respectively, for the data points collected along the horizontal line (44 μm). Immunofluorescence is not detected when the line goes across the nucleus. Objective lens: 40× (A–C).

Figure 3. Co-expression of insulin, CK-19 and PDX-1 in ductal cells of SPK patients with recurrent autoimmunity

Panel A shows insulin/CK-19 and CK-19/PDX-1 co-expression in the ducts by double immunofluorescence using serial sections from the biopsy of patient #1. Panel B shows expression of PDX-1, insulin and CK-19 in 3 serial sections of the transplanted pancreas of patient #2. A small duct is stained for insulin, CK-19 and PDX-1 in the bottom right corner. A damaged islet with weak insulin staining is also seen. Panel C shows cropped images of the images in B to highlight the staining in the small duct. Panel D shows insulin/CK-19/PDX-1 colocalization by triple immunofluorescence for patient # 6. Objective lens: 40× (A–D).

Figure 4. Colocalization analysis of insulin, CK-19 and Ki-67

Panel A demonstrates absence of colocalization of insulin and CK-19 in control pancreata #15. A Ki-67⁺ cell is shown outside the duct. Panel B shows colocalization of insulin, CK-19 and Ki-67 in the pancreas transplant biopsy of patient #1. There is colocalization of insulin with CK-19 and one cell in the duct also expressed Ki-67. Objective lens: 20× (A) and 40× (B).

Figure 5. Insulin-positive ductal cells expressing CgA do not express CK-19

The figure illustrates the exclusive expression of CK-19 and CgA in insulin⁺ ductal cells (A, patient #1; B, patient # 5; C, patient #2). Some background staining is visible in the nuclei but clear cytoplasmic staining is seen in the cells co-expressing CgA. Weaker cytoplasmic staining but above nuclear background seems also visible in a few other cells that did not stain for CgA. Objective lens: 20× (A, 4× zoom), 40× (B), and 20× (C).