Laura Crisa

The Class I HLA Repertoire of Pancreatic Islets Comprises the Nonclassical Class Ib Antigen HLA-G

Selective expression of the human class Ib HLA molecule HLA-G in immunologically protected sites and its function in the inhibition of NK and T-cell effector functions support an important role of this molecule in immunoregulation. Here, we demonstrate that HLA-G is constitutively expressed in the endocrine compartment of the human pancreas. Surface expression of this HLA determinant in endocrine cells is regulated in response to growth and inflammatory stimuli. Furthermore, we provide evidence that HLA-G expressed in this tissue may associate with a subset of insulin-containing granules and may be shuttled to the cell surface in response to secretory stimuli. Thus, HLA-G presentation by endocrine cells may be regulated in concert with their secretory activity. These results identify the expression of a major histocompatibility complex locus with putative regulatory functions in human pancreatic islets, a finding with potentially important implications for the progression of autoimmunity as well as for the establishment of transplant tolerance to this tissue.

β1 integrin is a crucial regulator of pancreatic β-cell expansion

Development of the endocrine compartment of the pancreas, as represented by the islets of Langerhans, occurs through a series of highly regulated events encompassing branching of the pancreatic epithelium, delamination and differentiation of islet progenitors from ductal domains, followed by expansion and three-dimensional organization into islet clusters. Cellular interactions with the extracellular matrix (ECM) mediated by receptors of the integrin family are postulated to regulate key functions in these processes. Yet, specific events regulated by these receptors in the developing pancreas remain unknown. Here, we show that ablation of the β1 integrin gene in developing pancreatic β-cells reduces their ability to expand during embryonic life, during the first week of postnatal life, and thereafter. Mice lacking β1 integrin in insulin-producing cells exhibit a dramatic reduction of the number of β-cells to only ∼18% of wild-type levels. Despite the significant reduction in β-cell mass, these mutant mice are not diabetic. A thorough phenotypic analysis of β-cells lacking β1 integrin revealed a normal expression repertoire of β-cell markers, normal architectural organization within islet clusters, and a normal ultrastructure. Global gene expression analysis revealed that ablation of this ECM receptor in β-cells inhibits the expression of genes regulating cell cycle progression. Collectively, our results demonstrate that β1 integrin receptors function as crucial positive regulators of β-cell expansion.

Endothelium-derived Netrin-4 supports pancreatic epithelial cell adhesion and differentiation through integrins α2β1 and α3β1.

Background Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1–3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified. Methodology/Principal Findings Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins α2β1and α3β1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins α2β1 and α3β1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and up-regulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated. Conclusions/Significance Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors α2β1 and α3β1 integrins.

Switching-on survival and repair response programs in islet transplants by bone marrow-derived vasculogenic cells

OBJECTIVE—Vascular progenitors of bone marrow origin participate to neovascularization at sites of wound healing and transplantation. We hypothesized that the biological purpose of this bone marrow–derived vascular component is to contribute angiogenic and survival functions distinct from those provided by the local tissue-derived vasculature. RESEARCH DESIGN AND METHODS AND RESULTS—To address this hypothesis, we investigated the functional impact of bone marrow–derived vascular cells on pancreatic islets engraftment using bone marrow–reconstituted Id1+/−Id3−/− mice, a model of bone marrow–derived vasculogenesis. We show that, in this model, bone marrow–derived vasculogenic cells primarily contribute to the formation of new blood vessels within islet transplants. In contrast, graft revascularization in a wild-type background occurs by tissue-derived blood vessels only. Using these distinct transplant models in which bone marrow–and tissue-derived vasculature are virtually mutually exclusive, we demonstrate that bone marrow–derived vasculogenic cells exhibit enhanced angiogenic functions and support prompt activation of islets survival pathways, which significantly impact on islets engraftment and function. Moreover, gene profiling of vascular and inflammatory cells of the grafts demonstrate that neovascularization by bone marrow–derived cells is accompanied by the activation of a genetic program uniquely tuned to downregulate harmful inflammatory responses and to promote tissue repair. CONCLUSIONS—These studies uncover the biological significance of bone marrow–derived vasculogenic cells in the response to injury during transplantation. Enhancing the contribution of bone marrow–derived vasculogenic cells to transplantation sites may help to overcome both limited angiogenic responses of the adult tissue-derived vasculature and untoward effects of inflammation on transplant engraftment.

An RT6a Gene Is Transcribed and Translated in Lymphopenic Diabetes-Prone BB Rats

T-cells expressing the RT6 surface alloantigen appear to perform important immunoregulatory functions in the rat. Diabetes-prone BB rats lack circulating RT6+ T-cells and spontaneously develop autoimmune diabetes mellitus and thyroiditis. The coisogenic diabetes-resistant BB rat does circulate RT6+ T-cells and is free of disease. Transfusions leading to engraftment of RT6+ T-cells prevent both diabetes and thyroiditis in the diabetes-prone rat. To investigate the absence of this subset in the lymphopenic BB rat, we used both molecular and biochemical procedures and made the following observations: 1) an mRNA encoding RT6 protein is present in diabetes-prone spleen cells; 2) nucleotide sequencing of this transcript reveals an intact coding sequence for the RT6.1 alloantigen; 3) sensitive chemiluminescent assay of diabetes-prone lymph node cell detergent extracts shows that diabetes-prone RT6 mRNA is translated in vivo; 4) quantitatively, diabetes-prone lymph node cells express ≤ 10% of the RT6.1 protein found on similar numbers of diabetes-resistant BB cells; and 5) finally, we obtained evidence of an intact phosphatidylinositol linkage of the molecule to the cell surface and successfully immunoprecipitated the phosphatidylinositol-linked protein with DS4.23 monoclonal antibody, indicating that the RT6.1 antigen is correctly processed and folded in diabetes-prone lymph node cells. We conclude that the near total absence of RT6+ T-cells in the diabetes-prone BB rat is unlikely to be because of a defect in RT6 gene expression per se. Defects in RT6 gene regulation or other cellular defects leading to premature cell death in the T-cell lineage, alone or in combination, may instead be responsible.

Biochemical Studies of RT6 Alloantigens in BB/Wor and Normal Rats: Evidence for Intact Unexpressed RT6a Structural Gene in Diabetes-Prone BB Rats

Lymphocytes bearing the T-lymphocyte differentiation antigen RT6 play an important immunoregulatory role in the development of autoimmune diabetes in BB rats. Immunofluorescence studies suggest that diabetesprone (DP)- but not diabetes-resistant (DR)-BB rat lymphocytes fail to express RT6 antigen during ontogeny. Two alloantigenic forms of the molecule exist, i.e., RT6.1 and RT6.2; both are linked to cell membranes by a phosphatidylinositol (PI) linkage. In these studies, Pl-phospholipase C (PLC) treatment of lymphocytes from BB and normal rats followed by immunoabsorption and sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of released proteins with anti-RT6 allotype-specific monoclonal antibodies was performed. RT6.1 in several nondiabetic rat strains was found to consist of a family of nonglycosylated and variably glycosylated molecules: an N-Glycanase–resistant 24,000- to 26,000- Mr peptide and four N-Glycanase–sensitive peptides of 29,000, 31,000, 33,000, and 34,000 Mr In contrast, RT6.2 was found to be a 24,000- to 26,000-Mr nonglycosylated polypeptide. The electrophoretic pattern of RT6.1 was observed to be the same when the antigen was extracted from W3/25+ (CD4+) versus W3/25- T lymphocytes or from resting versus mitogen-activated cells. A pattern of bands characteristic of the RT6.1 antigen found in normal rat strains was detected after PLC treatment or detergent solubilization of lymphocytes obtained from DR rats. In contrast, no evidence of either RT6 species was found after PLC or detergent treatment of comparable numbers of T lymphocytes from DP-BB rats. Interestingly, T lymphocytes from Wistar-Furth (RT6.2+) × DP (RT6−) F1 crosses were observed to coexpress both RT6.2 and RT6.1 molecules, with the electrophoretic pattern of RT6.1 being similar to that obtained in DR and other rat strains. This study provides biochemical evidence that DP rats may have an intact RT6a structural gene.

Circulating Anti-Immunoglobulin Antibodies in Recent-Onset Type I Diabetic Patients

Human sera from 51 recent-onset insulin-dependent (type I) diabetic patients and 47 unrelated control subjects were screened for the possible presence of circulating factors reacting with several anti-pancreatic islet monoclonal antibodies (MoAb.ISL) in solid-phase radioimmunoassay methods (the original goal being the detection of anti-idiotypic islet cell antibodies and/or specific islet cell antigen–bearing immune complexes). MoAbs from the parental myeloma cell line and purified immunoglobulins (Igs) from different animal species were controls. Type I diabetic sera showed significantly increased binding to MoAb.ISL-coated wells compared with normal subjects (P < .001). However, the same sera also tended to show a higher binding to the control (non–islet-related) MoAb. Sera from type I diabetic patients also reacted with horse, bovine, pig, rabbit, and goat IgG. Displacement of the binding has been obtained by F(ab′)2 and/or Fc fragments of IgG. Evidence has been obtained regarding a similar reaction with human IgM. All the sera were negative when tested for rheumatoid factor by nephelometry. The circulating antibodies described have been proven to be different from islet cell autoantibodies. An anti-Ig antibody is thus present in the sera of recent-onset diabetic patients and represents an additional immunological phenomenon with possible physiopathological and clinical significance.

Enhancement of circulating immune complexes in episodic cluster headache: interaction with lymphocyte and monocyte subsets

Abnormalities have already been reported in the phenotypic frequency of the major histocompatibility complex antigens and in the relative proportions of the leukocyte subsets in patients affected by episodic cluster headache (ECH). Circulating immune complexes (CIC) and leukocyte subsets have been studied in 19 patients affected by episodic cluster headache during and in the absence of periods of crisis. The presence of CIC was determined in another five patients outside periods of crisis. CIC were evaluated using two techniques based on different principles revealing different types of CIC: the solid phase Clq method, and the conglutinin radioimmune assay. Monoclonal antibodies have been used to phenotype total T, helper/suppressor, K and NK leukocyte subsets, and monocytes. The findings demonstrate an increased incidence of CIC in the 19 ECH patients followed with time (37%) with respect to control subjects (10%), and an increase in Leu7-positive cells (K and NK cells) and in LeuM3-positive cells (monocytes). Interestingly, when patients were grouped according to the presence of CIC, only CIC-positive patients showed the previously described abnormalities. This would suggest that the cell-mediated immune alterations present in these patients may interact with the presence of CIC.

αE-Catenin Is a Positive Regulator of Pancreatic Islet Cell Lineage Differentiation

SUMMARY The development and function of epithelia depend on the establishment and maintenance of cell-cell adhesion and intercellular junctions, which operate as mechanosensor hubs for the transduction of biochemical signals regulating cell proliferation, differentiation, survival, and regeneration. Here, we show that αE-catenin, a key component of adherens junctions, functions as a positive regulator of pancreatic islet cell lineage differentiation by repressing the sonic hedgehog pathway (SHH). Thus, deletion of αE-catenin in multipotent pancreatic progenitors resulted in (1) loss of adherens junctions, (2) constitutive activation of SHH, (3) decrease in islet cell lineage differentiation, and (4) accumulation of immature Sox9+ progenitors. Pharmacological blockade of SHH signaling in pancreatic organ cultures and in vivo rescued this defect, allowing αE-catenin-null Sox9+ pancreatic progenitors to differentiate into endocrine cells. The results uncover crucial functions of αE-catenin in pancreatic islet development and harbor significant implications for the design of β cell replacement and regeneration therapies in diabetes.

A CCR2+ myeloid cell niche required for pancreatic β cell growth

Organ-specific patterns of myeloid cells may contribute tissue-specific growth and/or regenerative potentials. The perinatal stage of pancreas development marks a time characterized by maximal proliferation of pancreatic islets, ensuring the maintenance of glucose homeostasis throughout life. Ontogenically distinct CX3CR1+ and CCR2+ macrophage populations have been reported in the adult pancreas, but their functional contribution to islet cell growth at birth remains unknown. Here, we uncovered a temporally restricted requirement for CCR2+ myeloid cells in the perinatal proliferation of the endocrine pancreatic epithelium. CCR2+ macrophages are transiently enriched over CX3CR1+ subsets in the neonatal pancreas through both local expansion and recruitment of immature precursors. Using CCR2-specific depletion models, we show that loss of this myeloid population leads to a striking reduction in β cell proliferation, dysfunctional islet phenotypes, and glucose intolerance in newborns. Replenishment of pancreatic CCR2+ myeloid compartments by adoptive transfer rescues these defects. Gene profiling identifies pancreatic CCR2+ myeloid cells as a prominent source of IGF2, which contributes to IGF1R-mediated islet proliferation. These findings uncover proproliferative functions of CCR2+ myeloid subsets and identify myeloid-dependent regulation of IGF signaling as a local cue supporting pancreatic proliferation.