Distinct cell clusters touching islet cells induce islet cell replication in association with over-expression of Regenerating Gene (REG) protein in fulminant type 1 diabetes.

BACKGROUND: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs), extracellular matrix (ECM), and possible cell clusters, are unclear. PROCEDURES: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG) Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells

Cell-cell interaction between acinar-like cells and islet endocrine cells encapsulated by common BMs.

<p><b>A:</b> A desmosomal junction (arrowhead) is observed between acinar-like cells (AC) and alpha cells (A), which are encapsulated by common BMs and ECM. <b>B</b>: Coated-pit-like structure is observed between an acinar-like cell (AC) and an alpha cell (A) touching directly and covered by common BMs (arrows). Inset shows a magnified view of the coated-pit-like structure. <b>C</b>: Excretion of vesicles from acinar-like cells (AC) to a beta cell (B). Note the vesicular membrane of the AC is dissolved (arrow), and the vesicular content is released to the beta cell (B) touching it. <b>D1</b>: Exocytotic features of vesicles in acinar-like cells (AC) to beta cells (B), which are in contact with each other. Arrowhead indicates BMs/ECM encapsulating acinar-like cells and beta cells, V: vasculature. <b>D2:</b> Higher magnified view of D1. The vesicle is internalized to the beta cell. AC: acinar-like cell, B: beta cell. <b>E:</b> Vesicles of acinar-like cells (AC) are internalized to touching alpha cells (A) and beta cell (B) shown by arrows. Arrowheads indicate BMs/ECM surrounding beta cell (B), alpha cell (A), and acinar-like cell (AC). Inset shows magnified view of (E). LB: lipofuscin body, ER: endoplasmic reticulum.</p

Replicating islet cells in fulminant type 1 diabetes (FT1DM).

<p><b>A</b>: Ki67-positive cells are increased in the pancreas of FT1DM. The cell composition of increased Ki67⁺ cells (red nuclei) is mainly REG Iα positive cells (green: arrowheads), islet non-beta cells (arrows), and islet beta cells (*, blue). <b>B</b>: The percentage of islets positive for Ki67 is increased in the pancreas of FT1DM. The number in parentheses indicates the total number of islets studied in FT1DM and control. See the detailed characterization of the subjects in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095110#s2" target="_blank">Research Design and Methods</a>. **P<0.0001 vs. controls, mean ± SEM. <b>C</b>: The number of islets positive for Ki67 in non-beta islet cells is increased in FT1DM. Non-beta islet cells were stained by mixed antisera for glucagon, somatostatin, and pancreatic polypeptide, and the values are expressed as percentage of islets positive for Ki67. The number in parentheses indicates the total number of islets studied in FT1DM and controls. **P = 0.02 vs. controls, mean ± SEM. <b>D</b>: The number of islets positive for Ki67 in beta cells tends to increase, but it is not significant due to the markedly decreased number of beta cells in individual islets in FT1DM. The number in parentheses indicates the total number of islets studied in FT1DM and controls. <b>E</b>: The number of Ki67-positive beta cells is increased in FT1DM. The numbers in parentheses indicate the total number of beta cells counted in the islets of FT1DM and controls. See a detailed characterization in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095110#s2" target="_blank">Research Design and Methods</a>. *P = 0.043 vs. controls, mean ± SEM.</p

REG Iα-positive cell cluster contacting with beta cell cluster and surrounded by common BMs and ECM.

<p><b>A–D</b>: Immunostaining for REG Iα (green) (<b>A</b>), acinar-like cell marker, amylase (blue). (<b>B</b>), and double immunostaining with insulin (brown) and fibronectin (red) (<b>C</b>). Auto-fluorescence of collagen fiber surrounding islet is observed (<b>A</b>, arrowheads). The merged image (<b>D</b>) shows that amylase-positive acinar-like cells that are in contact with beta cells express REG Iα protein (light blue: arrowheads), and the acinar-like cell cluster is surrounded by BMs/ECM (red, fibronectin). <b>E</b>: Electron-immunostaining with immuno-gold for REG Iα (20 nm: arrowheads) in acinar-like cell touching a beta cell containing insulin (5 nm: arrows). REG Iα is mainly localized in the center of the vesicle that is near the beta cell wall. <b>F</b>: Immuno-electron microscopy with immunogold for REG Iα (25 nm: arrowheads) and insulin (5 nm: arrows). Densely stained REG Iα vesicle (*) is just beside the cell wall touching a beta cell. Dissolved vesicles positive for REG Iα (arrowheads) are observed in insulin-containing beta cells.</p

Pathological features of the pancreas affected by fulminant type 1 diabetes (FT1DM).

<p><b>A:</b> CD8 + T cells (red) infiltrate from outside the islet, disrupting vascular BMs (green, arrows) through the interstitial space between the vasculature and islets. Arrowheads indicate BMs (green) of exocrine pancreatic cells. <b>B:</b> BMs and ECM surrounding islets (green) are markedly disrupted and punctuated (arrows) in FT1DM. The vasculatures of the islets show marked dilation, and the vascular BMs have lost the human-specific double membrane profile <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095110#pone.0095110-Virtanen1" target="_blank">[15]</a> (arrowheads). <b>C:</b> Acinar-like cell cluster touching Langerhans islets with thin interstitial surrounding (ATLANTIS) shows marked expression of REG Iα (green) in FT1DM. BMs (red, arrows) encapsulating the islet beta cells (blue) and ATLANTIS (green) are disrupted and discontinuous in some parts. <b>D:</b> Double immunostaining for amylase (red) and REG Iα (green) shows that amylase expression in the ATLANTIS (in circle) in inflamed FT1DM becomes faint in inverse relation to REG Iα over-expression. I: Islet, PAC: pancreatic acinar cells. <b>E:</b> Triple immunostaining for REG Iα (green), glucagon + somatostatin (SS) + pancreatic polypeptide (PP) (red), and insulin (blue) demonstrates that REG Iα-positive cells are not beta, glucagon, SS, or PP cells. <b>F:</b> Serum levels of REG Iα are increased in the patients with FT1DM of duration less than 2 weeks. **p<0.01 vs. controls.</p

Reconstructed feature of islet cell cluster, acinar-like cell cluster, and BMs and ECM.

<p><b>A–H</b>: Overview of the architecture reconstructed by serial pancreatic beta cell clusters (insulin: blue) and acinar-like cell clusters (amylase: red) surrounded by continuous BMs and ECM (fibronectin: green). <b>I, J</b>: Relationship between the beta cell area (X-axis) and the capsular area (Y-axis), which contains the beta cell area and the acinar-like cell area. A clear linear correlation between beta cell area and capsular area was observed (p<0.001, n = 517). See the schematic definition in (J). <b>J 1–3</b>: Schematic definition of capsular area and acinar-like cell area. The capsular area (b: dashed line) is composed of the acinar-like cell area (red in the dashed line) and the islet cell area (a: blue) (mostly beta cell area). A good correlation between the capsular area and islet cell area (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095110#pone-0095110-g003" target="_blank">Figure 3-I</a>) indicates that a constant proportion of islet cell clusters are accompanied by acinar-like cell clusters irrespective of the size of beta cell clusters.</p