islet leukocyte infiltration
Quantitative analyses revealed significantly reduced mean fluorescence intensity of the peri-islet BM pan-laminin staining at sites of leukocyte invasion of islets and loss of insulin+ cells and that this correlates with disease progression, with a higher incidence of insulitis when more islets showed penetration of the peri-islet BM. Diabetes 2013;62:531-542 The IM and BM influence immune cell migration into or within inflamed tissues (14). Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. tween mega-islet formation and leukocyte infiltration. (A high-quality digital representation of this figure is available in the online issue.). Immunofluorescence staining of a whole mount (A) and thin sections (B; boxed areas show higher magnification) of leukocyte-invaded islets for pan-laminin (PLM) as a marker of the peri-islet BM, and the pan-leukocyte marker CD45 illustrates the correlation between the loss of the peri-islet BM and leukocyte infiltration into the pancreatic islet. leukocyte infiltration. 1 B). Bethesda, MD 20894, Copyright Sections from the pancreas head, body, and tail were analyzed by immunofluorescence staining. In Type 1 Diabetes Mellitus (T1DM), leukocyte infiltration of the pancreatic islets and the resulting immune-mediated destruction of beta cells precede hyperglycemia and clinical disease symptoms. Endothelial cells have also been used to coat the islets; transplantation of these coated islets has reduced coagulation, complement activation, and leukocyte infiltration [53, 54]. C: The peri-islet BM is further surrounded by a dense network of IM, represented here by collagen type III. Comment on: Korpos et al. Indeed, abundant infiltration has been described in several experimental models that never or rarely progresses to disease (6,7), indicating that penetration of the pancreatic islet is a crucial step in disease progression. (I) Mean identity of insulin mass. Privacy, Help 8600 Rockville Pike A 3-dimensional projection of a whole mount of a healthy mouse pancreas and visualization of the peri-islet capsule by immunofluorescence staining and electron microscopy are shown. 2D). Healthy islets with intact BMs were recorded when no inflammatory cells were found around the islet, peri-insulitis was scored when inflammatory cells were found around the islet but the peri-islet BM was intact, and islets showing disrupted pan-laminin staining at sites coincident with CD45+ cells and loss of insulin+ cells were scored as invaded islets (300–400 islets were counted per mouse). Endothelial cells have also been used to coat the islets; transplantation of these coated islets has reduced coagulation, complement activation, and leukocyte infiltration [53, 54]. 1A), are associated with the peri-islet BM (Fig. The authors thank Hartmut Halfter (Department of Neurology, University of Münster, Münster, Germany) for use of the LCM; Friedemann Kiefer, Stefan Volkery, and Ruben Böhmer (Max Planck Institute for Molecular Medicine, Münster, Germany) for assistance in the use of the LSM510 microscope; Karin Loser (Department of Dermatology, University of Münster, Münster, Germany) for help with quantitative real-time PCR; Horst Robenek and Karin Schlattmann (Arteriosclerosis Research, University of Münster, Münster, Germany) for help in the use of the electron microscope; and Frank Arfuso (Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Münster, Germany) for critical reading of the manuscript. 4 wk is about the first time that the first infiltrating T cells can be identified; 8 wk represents a time when leukocyte infiltration is prominent in most islets, still with no evidence of dysglycemia; and 15 wk is just before the time when clinical diabetes becomes evident. Infiltrating leukocytes retained a peri-islet location, even 215 days following suspension of antibody treatment, potentially forming a barrier to the entry of active, autoantigen-reactive T cells. We therefore used immunofluorescence microscopy to investigate whether cathepsins C, S, and W colocalized with the islet-invading leukocytes. Hyaluronan (HA) is an extracellular matrix (ECM) component that is present in mouse and human islet ECM. Penetration of the peri-islet BM is a critical step in disease progression in NOD mice. 2006 Sep;178(1-2):1-8. doi: 10.1016/j.jneuroim.2006.05.030. In 21-week-old NOD pancreata, 80% of the islets showed loss of peri-islet BM and IM staining (Fig. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-capture microdissection (LCM) material revealed upregulation of cathepsin S, W, and C in leukocyte-infiltrated islets compared with noninvaded pancreatic islets. The number of CD3-positive T lymphocytes infiltrating into pancreatic islets (>100 μm in diameter) and per section was 1.1 ± 0.2 cells/islet (67 ± 41 islets) and 31.1 ± 4.9 cells/mm 2, which was more than 15 times and 8 times higher than that of the control group (0.07 ± 0.10 cells/islet [16 ± 12 islets] and 3.5 ± 3.0 cells/mm 2), respectively. Here, we completely blocked adoptive transfer of diabetes and reduced spontaneous disease incidence from 71% to 17% by simultaneously administering a combination of antibodies directed against alpha4, beta2, and beta7 integrins and their ligands VCAM-1, MAdCAM-1, and ICAM-1 for 52 and 28 days, respectively. Several members of the cathepsin protease family, including cathepsin C, H, S, and W, showed a four- to sixfold increase in mRNA expression in infiltrated islets (Table 2), which was confirmed by quantitative real-time PCR (Supplementary Fig. The number of islets showing disruption of the BM was compared with the number of islets with an intact BM, as defined by fluorescence intensity of pan-laminin staining, in NOD mice aged 5 (presymptomatic stages), 14 (early onset), and >21 weeks (diabetic stage). These isoforms are differentially expressed in different BM types where they perform different functions (10) mediated by direct binding to a variety of different integrin and nonintegrin receptors (11). Similarly, clinical data on autologous islet transplantation on patients with CP showed elevated levels of proinflammatory cytokines immediately after transplantation . Scale bars are 100 μm. Mean fluorescence values of peri-islet BM staining for laminin were therefore used to define healthy versus invaded pancreatic islets. Jarpe AJ, Hickman MR, Anderson JT, Winter WE, Peck AB. In addition, several members of the MMP family, including MMP-2, MMP-7, and MMP-14, were upregulated at the mRNA level in infiltrated islets. 6; Supplementary Video 3). CMV-infected recipients demonstrated accelerated islet graft failure compared to noninfected controls. This was attributed to inhibition of leukocyte infiltration, production of pro-inflammatory cytokines and islet cell apoptosis at day 14 of MLD-STZ. Oral probiotic administration induces interleukin-10 production and prevents spontaneous autoimmune diabetes in the non-obese diabetic mouse. To investigate whether leukocyte infiltration into the human pancreatic islets is also associated with loss of peri-islet ECM components, human type 1 diabetic samples were analyzed (Supplementary Tables 2 and 3). LCM was used to isolate healthy islets from 4-week NOD pancreata, and islets showing early stages of immune cell penetration were isolated from >12-week NOD pancreata; mRNA was purified, and gene expression patterns were compared (Table 2; Supplementary Fig. CMV infection of the graft was only observed prior to complete graft failure. 2). Our data suggest that the ECM milieu influences the mode used by immune cells to infiltrate into tissues and raises novel possibilities for tissue-specific immune modulatory therapies. (F) The numbers of islets counted in H&E‐stained sections. Immunofluorescence staining of NOD 21-week-old pancreas sections for cathepsin C (A), cathepsin S (B), and cathepsin W (C) shows localization at sites where peri-islet BM staining is lost (arrows), as shown here by laminin (LM) γ1 staining. For histochemical staining, sections were re-fixed in cold acetone and stained with haematoxylin and eosin (H&E) to monitor islet evaluated the effectiveness of combinational treatment in in- infiltration. Stereological analyses reveal a correla-tion between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step. provided advice on immunology, contributed to discussion, and reviewed the manuscript. In vivo staining for cathepsin S, C, and W showed colocalization with some macrophages at the inflammatory front, suggesting their potential secretion from a macrophage subpopulation but not excluding other sources. Frozen pancreata from 5-week-old (normoglycemic) and 14-week-old NOD mice were sectioned completely and stained with antibodies to pan-laminin to label BMs, CD45, a pan-leukocyte marker, and insulin. No potential conflicts of interest relevant to this article were reported. Menu en zoeken; Contact; My University; Student Portal However, once autoaggressive T cells are activated, several steps are crucial for disease progression, including extravasation of the CD4+, CD8+ T lymphocytes from the postcapillary venules surrounding the islets of Langerhans and their subsequent infiltration of the islets, leading to β-cell destruction and onset of disease symptoms. Diabetologia. Recent studies have reported the existence of laminins, collagen type IV, and perlecan surrounding the pancreatic islets and the absence of staining for these antigens in pancreata of NOD mice, however, without correlation to disease progression in mice or with type 1 diabetes in humans (23,24). In addition, cathepsins may be associated with degradation of endogenous inhibitors, resulting in increased activity of other proteases (49). At 14weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. MLD-STZ in wild type mice induced islet leukocyte infiltration, cytokine production, beta-cell apoptosis, and hyperglycemia. 1D). To identify the immune cell-type(s), and … However, an indirect involvement of cathepsins by initiating a proteolytic cascade resulting in activation of other proteases that degrade the ECM is also possible. Electron microscopy confirmed that the pancreatic islet is ensheathed by a BM and an underlying IM composed of fibrillar collagens (Fig. However, the endothelial BM of blood vessels from the islet periphery and the acinar BMs remained intact (Fig. On the one hand, the effects of … The boxed areas are shown at higher magnification to the right. This ratio changed in 14-week-old NOD pancreata, and <50% of the islets were invaded by leukocytes and showed degraded BMs. Immunofluorescence staining revealed some colocalization of F4/80+ macrophages with cathepsin C staining (Fig. Optical sectioning throughout entire islets, defined by insulin+ staining, revealed a continuous BM encasing each islet that contained all major BM components (collagen IV, perlecan, nidogens, and laminins; Table 1) (23). Thank you for your interest in spreading the word about Diabetes. Treatment with anti-alpha4 integrin antibody retained some therapeutic benefit when administered from days 7, 40, or 70 of age. Following full immunological activation, there was a progressive enhancement of inflammatory signatures, culminating in the destruction of the islet PLOS ONE | www.plosone.org 1 March 2013 | Volume 8 | Issue 3 | e59701. Samples for electron microscopy were prepared according to standard protocols and analyzed with an EM-410 (Philips). L.S. (A) Total number of CD45 positive cells isolated from islet allografts seven days after transplant. 1F). A noninvasive means of directly monitoring the evolution of islet infiltrates would have important research and clinical applications. IL-10 is essential for disease protection following intranasal peptide administration in the C57BL/6 model of EAE. É.K. At 14weeks, 40% of the small islets, but more than 80% of large islets, showed leukocyte infiltration. The results have implications for the treatment of diabetes and provide a unique insight into the fate of disease-forming leukocytes following anti-CAM therapy. Scale bars are 100 μm and 50 μm in insets (P < 0.0001). In opposition to this proposal. performed the quantitative real-time PCR experiments. Clipboard, Search History, and several other advanced features are temporarily unavailable. This suggests that the cells responsible for formation of the peri-islet BM are not lost due to the inflammation and can regenerate the BM once the inflammation has subsided. The boxed area is shown at higher magnification to the right. At least 10 healthy and 10 invaded pancreatic islets were analyzed per mouse in 5- and 14-week-old NOD mice (n = 3/group). After sacrifice, the islet grafts were retrieved for analysis of infection and leukocyte infiltration. ***P < 0.0007. Macrophages and DCs are among the first cells recruited to pancreatic islets (34,35), and their localization was shown here to correlate with sites of loss of peri-islet BM (Fig. E: Collagen type III staining visualizes the IM of the peri-islet capsule that underlies the peri-capsular BM, marked by pan-laminin (PLM) staining. More recently, developmental studies in the mouse have shown that β-cell adhesion to the BM components laminins α4 or α5 is essential for growth and strongly promotes insulin secretion via integrin-mediated processes (28). Interestingly, in samples from humans with long-term type 1 diabetes, we found insulin− islets surrounded by an intact peri-islet BM and underlying IM. Human healthy pancreas and type 1 diabetic samples were provided by the Network for Pancreatic Organ Donors with Diabetes (nPOD) organ bank. In contrast, the group with IL-33 treatment showed well-preserved islet morphology with reduced/minimal leukocyte infiltration (Fig 1F). IFN-I is also a potent inducer of human leukocyte antigen I (HLA-I) molecules, a hallmark of T1D . The IM layer, which in most tissues underlies the BM, is composed of fibrillar collagens, such as collagen type I and III, nonfibrillar collagens, like collagen type VI, and noncollagenous glycoproteins, like fibronectin, tenascins, vitronectin, and chondroitin, or dermatan sulfate proteoglycans (12,13). Histology in PBS‐treated control mice showed that islet architecture was lost, with only a few remaining insulin‐positive cells and massive leukocyte infiltration. 5I and J). During NOD diabetes, there is an entry of additional leukocytes into islets that increases in number and complexity over time ( 14 ). questions about whether a mild leukocyte infiltration (e.g., 2-fold increase) into the pancreas and targeted towards islets is responsible for reductions in β-cell mass and function. Consistent with this possibility was the upregulation of several members of α-1 antitrypsin family (Table 2), known to inhibit cysteine proteases as well as serine proteases (33). (A high-quality digital representation of this figure is available in the online issue.). NCI CPTC Antibody Characterization Program. Cytokine Secretion, Leukocyte Infiltration, and Beta-Cell Damage. 4). Upon MLDS induction, Robo4‐deficiency resulted in increased pancreatic vascular permeability, leukocyte infiltration to the islets and islet apoptosis, associated with reduced insulin levels and faster diabetes development. N.K. 2A and B; Table 1; Supplementary Video 2) and loss of insulin staining within islets (Fig. On the one hand, the effects … Glucagon+ cells are organized along the peri-islet BM, whereas the insulin+ β-cells fill the inner part of the islet. Analysis of Inflammatory Leukocyte Infiltration Into IFN Receptor–Deficient Mice In normal mice, macrophages are the major leukocyte throughout the life of the mouse ( 13 ). (H) The number of apoptotic β cell per islet. The unpaired t test was applied for statistics, and P values < 0.05 were considered to be statistically significant. Additionally, FTY720 stimulated insulin secretion from isolated islets by approximately twofold under both normoglycemic and hyperglycemic conditions. By comparison, thalidomide was not effective in improving graft survival in immunocompromised nude mice. The strong upregulation of the serpins shown here is also consistent with the reported anti-inflammatory effects in NOD mice (42) and its recent use in clinical trials for treatment of type 1 diabetes (http://clinicaltrials.gov/ct2/show/NCT01319331). In contrast, Jnk1-/- mice were substantially protected from a loss of insulin producing cells and hyperglycemia in the MLD-STZ model despite a marked islet T cell and macrophage infiltrate. We show that the peri-capsular ECM is composed of a peri-islet BM embedded in an IM layer that interconnects with the surrounding blood vessels and exocrine tissue. C: A 3-dimensional projection of a whole-mount staining shows that glucagon+ cell clusters are individually ensheathed by a peri-islet BM, shown by perlecan staining. Protease- and protease inhibitor–specific microarray analyses (CLIP-CHIP) of laser-dissected leukocyte infiltrated and noninfiltrated pancreatic islets and confirmatory quantitative real time PCR and protein analyses identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. In vitro studies showed that cultured islets from Mkk3-/- and WT mice are equally susceptible to STZ and cytokine-induced apoptosis. The boxed area is shown at higher magnification to the right. The widespread disappearance of IM and BM components from the mouse and human islet capsule indicates that leukocyte infiltration into the pancreatic islet is fundamentally different from the mainly nonproteolytic mode of leukocyte extravasation from postcapillary venules . Diabetes 2013;62:531–542 - July 01, 2013, http://clinicaltrials.gov/ct2/show/NCT01319331, http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db12-0432/-/DC1, http://creativecommons.org/licenses/by-nc-nd/3.0/. Sign In to Email Alerts with your Email Address. Scale bars are 100 μm for A–E, 50 μm for insets in C, and 1 μm in F. (A high-quality digital representation of this figure is available in the online issue. Scale bars are 100 μm in A–C and in B insets and are 50 μm in A insets. Islets were identified by vascular morphology and islet infiltration state was characterized as mild (0.1–30% of islet volume infiltrated by T cells) or advanced (30–60% of islet volume infiltrated by T cells) as previously described . H: Glucagon-producing α-cells become evenly distributed throughout the islet in type 1 diabetic CD45−/insulin− samples and are enclosed within an intact peri-islet BM, as shown by laminin α5 staining. The peri-islet basement membrane, a barrier to infiltrating leukocytes in type 1 diabetes in mouse and human . supervised the work done in the laboratory and participated in writing the manuscript. 1A and C). relation to their size was compared in NOD and. 5H), which has been shown to be a ligand for β-cells, interaction with which enhances insulin production (28). Unable to load your collection due to an error, Unable to load your delegates due to an error. In this context, the role of the pancreatic endothelium as a barrier for autoimmunity- and inflammation-related Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. 1B and C). (G) PCNA + islet cells. of Langerhans. In marked contrast, a marginal-to-modest islet destruction and leukocyte infiltration were found in the pancreatic islets of NOD mice treated with catenarin at 4 mg/kg and over (Figure 2 (b)). © 2013 by the American Diabetes Association. The study received support from the European Foundation for the Study of Diabetes/Juvenile Diabetes Research Foundation (JDRF)/Novo Nordisk A/S (BD21070), JDRF (1-2005-903). In many cases, extravasated leukocytes can reside around the islets of Langerhans without induction of disease symptoms. HLA-I ... Islet T cell infiltration was quantified using machine learning and automated software analysis (4276 islets from nondiabetic donors, 1549 islets from AAb + donors, and 1815 islets from T1D donors), and the T cell density was calculated for each islet and donor. The composition of the peri-islet BM is biochemically distinct to BMs of blood vessels surrounding or within the islet, with characteristic expression of laminin α4 in the mouse and of laminin α5 in human tissue (21,22). ACKNOWLEDGMENTS. Leukocyte infiltration toward the islet graft was evidenced by CD11b immunostaining (Figure 3G). 1C and D) together with laminin γ1 and β1 chains. Because of the natural composition of islet‐like extracellular matrix (ECM) and its role in preventing leukocyte infiltration of pancreatic islets in the native pancreas, 18 we replaced ALG with a commercially available reagent, Matrigel (or MG), which resembles the composition of islet ECM. Laminins are composed of an α, β, and γ chain, and 5α, 4β, and 3γ laminin chains exist that combine to form at least 18 different isoforms. 1A–D. Although our data highlight the α-cells as a potential source of peri-islet BM components because of their tight association with the peri-islet BM in the reconstituted islets, other peri-islet cells, such as myofibroblasts or Schwann cells, which can synthesize BM and IM components, may also contribute to peri-islet BM restoration and represent appropriate support cells for islet transplantation. The mean fluorescence intensity per micrometer of degraded peri-islet BMs is significantly lower than that of intact peri-islet BMs. 2 ). The pro-inflammatory TNF-a and VEGF content in islet grafted kidneys was significantly lowered by the treatment. In pancreatic islets showing disrupted panlaminin staining at sites coincident with CD45+ cells and loss of insulin+ cells, the mean fluorescence value per micrometer was significantly reduced (P < 0.0007; Supplementary Fig. COVID-19 is an emerging, rapidly evolving situation. By implanting insulin capsules into diabetic NOD mice, we were able to observe the same phenomena and also identify the early formation of α-cell, glucagon+ clusters that were surrounded by a BM of the same composition as the peri-islet BM. After sacrifice, the islet grafts were retrieved for analysis of infection and leukocyte infiltration. The Peri-islet Basement Membrane, a Barrier to Infiltrating Leukocytes in Type 1 Diabetes in Mouse and Human. D: Stereological analyses of pancreatic sections from 5- and 14-week-old normoglycemic and 21-week-old diabetic NOD mice (n = 3/each group) were used to quantify the number of islets with intact peri-islet BMs (healthy islets and islets in peri-insulitis stage) vs. invaded islets showing loss of peri-islet BM staining.