Fas ligand-mediated mechanisms are involved in autoimmune destruction of islet beta cells in non-obese diabetic mice

Aims/hypothesis. A mechanism implicated in pancreatic islet beta-cell destruction in autoimmune diabetes is the binding of the Fas ligand (FasL) on T cells to Fas receptors on beta cells, causing their destruction. Evidence for this mechanism is, however, controversial. The aim of this study was to find whether the Fas ligand contributes to beta-cell death in autoimmune diabetes. Methods. We transplanted syngeneic islets under the renal capsule in non-obese diabetic (NOD) mice and treated the mice with a neutralizing monoclonal antibody to the Fas ligand. Survival of beta cells in islet grafts and phenotypes of graft-infiltrating cells were investigated. Results. We found 58 % (7 of 12) of mice treated with anti-Fas ligand antibody were normoglycaemic at 30 days after islet transplantation compared with none (0 of 9) of the mice treated with control antibody. Immunohistochemical analysis of islet grafts showed that infiltration of leucocytes (CD4⁺ T cells, CD8⁺ T cells, macrophages and neutrophils) and apoptosis of beta cells in the grafts was significantly decreased in mice treated with anti-Fas ligand antibody. Expression of proinflammatory cytokines (interleukin 1 alpha, tumour necrosis factor alpha and interferon gamma) was not different in islet grafts of mice treated with anti-Fas ligand and control antibodies. Conclusion/interpretation. These findings indicate that Fas ligand-mediated mechanisms play a major part in promoting leucocytic infiltration of islets and beta-cell destruction in autoimmune diabetes. [Diabetologia (2000) 43: 1149–1156] Received: 31 March 2000 and in revised form: 5 June 2000     

Human beta cells are exceedingly resistant to streptozotocin in vivo

Streptozotocin (STZ) causes beta cell death in rodents via the mechanism of DNA damage precipitating poly(ADP-ribose) synthetase activation followed by lethal nicotinamide adenine dinucleotide depletion. It is unclear whether humans are susceptible to this mechanism. Islets were isolated from STZ-sensitive (CD1 mice and Lewis rats) and resistant [fish (tilapia)] species and from man and then were transplanted into diabetic nude mice under the kidney capsule. Normoglycemic recipients with normal glucose tolerance tests on d 30 were injected with increasing iv doses of STZ and their plasma glucose levels followed for 5 d; glucose tolerance tests were repeated on nondiabetic mice. Mice were then killed; grafts and native pancreata were examined. Based upon three criteria (i.e. nonfasting plasma glucose levels, glucose tolerance tests, and islet histology), the following observations were made: 1) Recipients of rat islets were resistant to 25 mg/kg but were uniformly diabetic at doses of 50 or 75 mg/kg. 2) Recipients of mouse islets were resistant to 75 mg/kg but were uniformly diabetic at 150 or 200 mg/kg. 3) Recipients of the fish islets were resistant to 300, 400, and 450 mg/kg. 4) Recipients of human islets were resistant to 100, 200, 300, 400, and 450 mg/kg. The results in recipient mice bearing long-term rat, mouse, or fish islet grafts were the same as previously published dose-response data for each donor species. We extrapolate from our results based on human islet grafts in mice that human beta cells are exceedingly resistant to STZ.     

Engineering of glucose-stimulated insulin secretion and biosynthesis in non-islet cells.

The high-capacity glucose transporter known as GLUT-2 and the glucose phosphorylating enzyme glucokinase are thought to be key components of the "glucose-sensing apparatus" that regulates insulin release from the beta cells of the islets of Langerhans in response to changes in external glucose concentration. AtT-20ins cells are derived from anterior pituitary cells and are like beta cells in that they express glucokinase and have been engineered to secrete correctly processed insulin in response to analogs of cAMP, but, unlike beta cells, they fail to respond to glucose and lack GLUT-2 expression. Herein we demonstrate that stable transfection of AtT-20ins cells with the GLUT-2 cDNA confers glucose-stimulated insulin secretion and glucose regulation of insulin biosynthesis and also results in glucose potentiation of the secretory response to non-glucose secretagogues. This work represents a first step toward creation of a genetically engineered "artificial beta cell."     

Phenotypic evolution of cells resistant to bromodeoxyuridine.

Variants resistant to bromodeoxyuridine (BrdUrd) and deficient in thymidine kinase (ATP:thymidine 5'-phosphotransferase; EC 2.7.1.21) have been obtained from V79 Chinese hamster cells by a combination of spontaneous and drug-induced change. Initial mutations take place in wild-type populations as a facilitating step to give partially resistant clones that can be isolated by one-step selection in BrdUrd. When these tolerant populations are maintained for extended periods in BrdUrd-containing medium, a gradual phenotypic transition occurs in which BrdUrd appears to act as an inductive as well as selective agent. Thymidine kinase activity declines logarithmically over an interval of 8-10 weeks as the growth rate rises and the cells become completely resistant to BrdUrd. Relative plating efficiency in hypoxanthine/aminopterin/thymidine medium also decreases, but the decrease is not coordinate with shifts in thymidine kinase activity. The potential for colony formation in hypoxanthine/aminopterin/thymidine continues to decrease exponentially for at least 18 weeks after thymidine kinase deficiency and complete resistance to BrdUrd have been established. These phenotypic modifications are continuous or multistep in character; by clonal analysis they are found to occur in most, if not all, cells maintained in the presence of BrdUrd. Populations in transition thus come to be complex mosaics of different phenotypes that are comparatively stable if isolated in drug-free medium. The progressive evolution of cells resistant to BrdUrd will require new models for an underlying explanation.     

Mammalian cells resistant to tumor suppressor genes.

Expression of p53 causes growth arrest or apoptosis in many normal and neoplastic cell types, but the relationship between these two effects has remained obscure. To begin to dissect the underlying mechanisms at a genetic level, we have generated mutant cells resistant to the action of wild-type p53. Rat embryo fibroblasts transformed with ras and a temperature-sensitive p53 (tsp53(135val)) gene were chemically mutagenized and selected for growth at a temperature at which p53 adopts a wild-type conformation (31.5 degrees C). Clones that grew exponentially at 31.5 degrees C were selected. Cell fusion experiments demonstrated that the mutations conferring resistance to p53-mediated growth arrest were dominant. The mutagenized clones were resistant not only to p53-mediated growth arrest, but also to the apoptosis induced by E1A in conjunction with p53, and partially resistant to the retinoblastoma tumor suppressor, pRB. The results suggest that a single downstream pathway can control the induction of growth arrest and apoptosis, and that both p53 and RB function through this pathway.     

Flat revertants isolated from Kirsten sarcoma virus-transformed cells are resistant to the action of specific oncogenes.

Two flat revertants have been isolated from mutagen-treated populations of Kirsten murine sarcoma virus (Ki-MuSV)-transformed NIH/3T3 cells. These revertants, which appear to be cellular variants resistant to transformation by the Ki-MuSV oncogene v-Ki-ras, contain Ki-MuSV-specific DNA, elevated levels of the v-Ki-ras gene product p21, and rescuable transforming virus. Cell hybridization studies indicated that the revertant phenotype is dominant in hybrids between revertant cells and cells transformed by Ki-MuSV or the closely related Harvey MuSV and BALB MuSV. Analysis of hybrid cells resulting from the fusion of these revertants to cell lines transformed by other retroviruses showed that the action of certain oncogenes structurally unrelated to v-Ki-ras also could be suppressed. Thus, there appear to be functional relationships and diversities among transforming genes (oncogenes) not readily apparent from their structural characteristics.     

Insulin-secreting INS-1E cells express functional TRPV1 channels

We have studied whether functional TRPV1 channels exist in the INS-1E cells, a cell type used as a model for β-cells, and in primary β-cells from rat and human. The effects of the TRPV1 agonists capsaicin and AM404 on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the INS-1E cells were studied by fura-2 based microfluorometry. Capsaicin increased [Ca²⁺]_i in a concentration-dependent manner, and the [Ca²⁺]_i increase was dependent on extracellular Ca²⁺. AM404 also increased [Ca²⁺]_i in the INS-1E cells. Capsazepine, a specific antagonist of TRPV1, completely blocked the capsaicin- and AM404-induced [Ca²⁺]_i increases. Capsaicin did not increase [Ca²⁺]_i in the primary β-cells from rat and human. Whole cell patch clamp configuration was used to record currents across the plasma membrane in the INS-1E cells. Capsaicin elicited inward currents that were inhibited by capsazepine. Western blot analysis detected TRPV1 proteins in the INS-1E cells and the human islets. Immunohistochemistry was used to study the expression of TRPV1, but no TRPV1 protein immunoreactivity was detected in the human islet cells and the human insulinoma cells. We conclude that the INS-1E cells, but not the primary β-cells, express functional TRPV1 channels.     

Immunologic mechanisms implicated in autoimmune destruction of beta cells in type I diabetes]

Lymphocytic infiltration of the islets of Langerhans results from activation of immunity effector cells during the period preceding clinical onset of diabetes. After escape from thymic selection, the autoreactive cell clones proliferate in the peripheral hemopoietic organs prior to invading the islets of Langerhans. The selection, expansion and activation of autoreactive cells are under genetic control. The development of quantitative tests of cellular immunity should allow determination of the importance of the anti-islet immunologic reaction, suspected at present from the presence of specific autoantibodies in genetically predisposed subjects at high risk of insulin dependence.     

Rabbits are not resistant to prion infection

The ability of prions to infect some species and not others is determined by the transmission barrier. This unexplained phenomenon has led to the belief that certain species were not susceptible to transmissible spongiform encephalopathies (TSEs) and therefore represented negligible risk to human health if consumed. Using the protein misfolding cyclic amplification (PMCA) technique, we were able to overcome the species barrier in rabbits, which have been classified as TSE resistant for four decades. Rabbit brain homogenate, either unseeded or seeded in vitro with disease-related prions obtained from different species, was subjected to serial rounds of PMCA. De novo rabbit prions produced in vitro from unseeded material were tested for infectivity in rabbits, with one of three intracerebrally challenged animals succumbing to disease at 766 d and displaying all of the characteristics of a TSE, thereby demonstrating that leporids are not resistant to prion infection. Material from the brain of the clinically affected rabbit containing abnormal prion protein resulted in a 100% attack rate after its inoculation in transgenic mice overexpressing rabbit PrP. Transmissibility to rabbits (>470 d) has been confirmed in 2 of 10 rabbits after intracerebral challenge. Despite rabbits no longer being able to be classified as resistant to TSEs, an outbreak of "mad rabbit disease" is unlikely.     

Mouse MSV transformed cells resistant to 8-azaguanine.

Mouse cells transformed by murine sarcoma virus were made resistant to 8-azaguanine. Resistant cells and cell clones isolated from them were deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity. They did not grow in HATG medium, did not incorporate labeled hypoxanthine, and had negligible HGPRT activity. The resistance was genetically stable. The resistant cells were hyperdiploid and contained telocentric chromosomes only. The resistant cells as well as the progenitor cells were slightly tumorigenic in mice, the plating efficiency in soft agar was very low. The parental cells and aza-G resistant cells produced C-type viral particles having RNA-dependent DNA polymerase activity. The resistance to aza-G did not influenced the expression of murine sarcoma virus genome in cells. The resistant cells are suitable for preparation of cell hybrids.     

Myeloid haemopoietic cells of patients with chronic granulomatous disease are relatively resistant to TNF

We have previously reported an altered surface marker expression and chemotaxis of G-CSF-induced neutrophils from patients with severe congenital neutropenia. However, effects of G-CSF and influence of the underlying disease on neutrophils could not be discerned. In this study we have evaluated the effects of G-CSF on neutrophil phenotype and function in patients under chemotherapy and in healthy test subjects. We found a significantly enhanced expression of FcγRI, CD14 and CD54 and a decrease in the level of FcγRIII during G-CSF treatment. In addition, motility of G-CSF-induced neutrophils was significantly decreased. The effects were seen in patients under cytotoxic chemotherapy and in healthy test subjects. Surface marker alterations and neutrophil motility were affected by G-CSF administration in a dose-dependent manner. Kinetic studies on neutrophils from healthy test subjects demonstrated that all effects could be seen after a single administration of 300 μg G-CSF and began to appear within 4 h. Release of partially immature neutrophils from the bone marrow and indirect activation of these cells by G-CSF are discussed as possible reasons for the findings presented. They demonstrate that G-CSF has profound effects on neutrophil phenotype and function in vivo which might have clinical implications.     

Unusual resistance of ALR/Lt mouse beta cells to autoimmune destruction: role for beta cell-expressed resistance determinants

Genetic analysis of autoimmune insulin-dependent diabetes mellitus (IDDM) has focused on genes controlling immune functions, with little investigation of innate susceptibility determinants expressed at the level of target beta cells. The Alloxan (AL) Resistant (R) Leiter (Lt) mouse strain, closely related to the IDDM-prone nonobese diabetic (NOD)/Lt strain, demonstrates the importance of such determinants. ALR mice are unusual in their high constitutive expression of molecules associated with dissipation of free-radical stress systemically and at the beta-cell level. ALR islets were found to be remarkably resistant to two different combinations of beta-cytotoxic cytokines (IL-1beta, tumor necrosis factor alpha, and IFN-gamma) that destroyed islets from the related NOD and alloxan-susceptible strains. The close MHC relatedness between the NOD and ALR strains (H2-Kd and H2-Ag7 identical) allowed us to examine whether ALR islet cells could survive autoimmune destruction by NOD-derived Kd-restricted diabetogenic cytotoxic T lymphocyte clones (AI4 and the insulin-reactive G9C8 clones). Both clones killed islet cells from all Kd-expressing strains except ALR. ALR resistance to diabetogenic immune systems was determined in vivo by means of adoptive transfer of the G9C8 clone or by chimerizing lethally irradiated ALR or reciprocal (ALR x NOD)F1 recipients with NOD bone marrow. In all in vivo systems, ALR and F1 female recipients of NOD marrow remained IDDM free; in contrast, all of the NOD recipients became diabetic. In conclusion, the ALR mouse presents a unique opportunity to identify dominant IDDM resistance determinants expressed at the beta cell level.     

Human microvascular endothelial cells are sensitive to IGF-I but resistant to insulin at the receptor level

Human microvascular endothelial cells (HMVEC) are sensitive to IGF-I but insulin resistant and express several times more IGF-I receptors (IGF-IR) than insulin receptors (IR). Our aim was to investigate the mechanism of this insulin resistance in cultured HMVEC by studying receptor activation and signal propagation downstream. The IGF-IR beta-subunit and the IR beta-subunit were detected and found to co-precipitate. IRA was the major IR isoform expressed in HMVEC. IGF-I 10(-9) to 10(-8)M phosphorylated its cognate receptor beta-subunit. IGF-I also phosphorylated the IR beta-subunit at 10(-9)M. Phosphorylation of insulin receptor substrate 1 was obtained by IGF-I 10(-9) to 10(-8)M. Akt was phosphorylated by IGF-I at 10(-8) to 10(-7)M and by insulin 10(-7)M. IGF-I at 10(-8) to 10(-6)M significantly increased DNA-synthesis. We conclude that microvascular endothelial cells are sensitive to IGF-I but resistant to insulin due to a preponderance of IGF-I receptors and sequestration of insulin receptors into insulin/IGF-I hybrid receptors.     

Central memory CD4 T cells are the predominant cell subset resistant to anergy in SIV disease resistant sooty mangabeys

OBJECTIVE: HIV infection in humans and experimental SIV infection in rhesus macaques leads to the loss of recall antigen responses, immunological anergy in CD4 T cells and AIDS. In contrast, natural infection in sooty mangabeys with SIV does not lead to disease despite high viral loads accompanied by resistance of their CD4 T cells to undergo immunological anergy. The objective of the study was to further elucidate the mechanisms that contribute to anergy resistance in CD4 T cells from sooty mangabeys and identify whether the anergy resistance is a function of a specific subset or the entire cell population. MATERIALS AND METHODS: Susceptibility or resistance to anergy was analyzed in antigen specific and defined CD4 T cell subsets from peripheral blood of sooty mangabeys and rhesus macaques by using an in vitro anergy inducing protocol; expression of the anergy associated gene GRAIL was measured. RESULTS: Antigen specific CD4 T cells from SIV disease resistant sooty mangabey are relatively resistant to the development of anergy. This resistance is associated with a lack of an upregulation of GRAIL. Conversely, rhesus macaque CD4 T cells are sensitive to anergy induction associated with upregulation of GRAIL. Furthermore the anergy resistant phenotype of sooty mangabey CD4 T cells predominantly resides in central memory cells defined either as CD4+CD45RA-CD62L+ or CD4+CD28+CD95+CCR7+. CONCLUSIONS: The maintenance of recall responses in sooty mangabeys is associated with the resistance of central memory CD4 T cells to the induction of anergy which may represent an important mechanism underlying SIV disease resistance in this species.     

Antibodies cytotoxic to bovine parathyroid cells in autoimmune hypoparathyroidism.

We utilized a recently developed long-term serum-free culture system for bovine parathyroid cells to detect antibodies in seven patients with autoimmune hypoparathyroidism (AHP). Antibodies were tested by indirect immunofluorescence methods and by cytotoxicity utilizing the chromium (51Cr) release technique. Seven AHP sera caused specific lysis [57 +/- 6% release of 51Cr vs. 5 +/- 1% for 56 controls (15 normal subjects and 41 patients with diverse other conditions associated with immune dysfunction)]. The least effect of any of the AHP sera on cell lysis exceeded the greatest effect of any of the control sera. Absorption of AHP sera (two cases) with bovine pituitary, thyroid, liver, or kidney cells did not affect lysis, but absorption with adrenal or parathyroid cells caused a marked decrease in specific lysis. Cytotoxicity determined by 51Cr release increased with antiserum concentration and time of incubation. Cytotoxicity was dependent on complement. Replicating parathyroid cells provide a uniform reproducible detection method for anti-parathyroid antibodies in AHP. The autoantibodies in AHP appear to be specific for tissue (parathyroid and adrenal cortex) but not for species.     

Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption

Osteopontin is one of the major noncollagenous bone matrix proteins produced by osteoblasts and osteoclasts, bone cells that are uniquely responsible for the remodeling of mineralized tissues. Osteoclasts express the alphavbeta3 integrin, which is one of the receptors for osteopontin. Recent knockout studies revealed that noncollagenous bone matrix proteins are functionally important in regulation of bone metabolism. However, the significance of the presence of osteopontin in in vivo has not been known. We report here that osteopontin knockout mice are resistant to ovariectomy-induced bone resorption compared with wild-type mice. Microcomputed tomography analysis indicated about 60% reduction in bone volume by ovariectomy in wild-type mice, whereas the osteopontin-deficient mice exhibited only about 10% reduction in trabecular bone volume after ovariectomy. Reduction in uterine weight was observed similarly in both wild-type and osteopontin-deficient mice, indicating the specificity of the effect of osteopontin deficiency on bone metabolism. We propose that osteopontin is essential for postmenopausal osteoporosis in women. Strategies to counteract osteopontin's action may prove effective in suppressing osteoporosis.     

Mononuclear cells from human neonates are partially resistant to the action of 1,25-dihydroxyvitamin D

1,25-Dihydroxyvitamin D [1,25-(OH)2D] inhibits mitogen-induced proliferation of lymphocytes by a receptor-mediated mechanism. Peripheral blood lymphocytes may serve as a model for detecting hereditary defects in the response of classical target organs to 1,25-(OH)2D. Delayed bone mineralization and deficient intestinal calcium absorption are common in low birth weight formula-fed infants. The defect in calcium absorption exists despite normal or even elevated serum 1,25-(OH)2D levels, suggesting partial end-organ resistance to the hormone. We assessed the response to 1,25-(OH)2D of activated mononuclear cells obtained from cord blood of fullterm and preterm infants and from peripheral blood of adults. We found that the inhibitory effect of 1,25-(OH)2D on mitogen-induced [3H]thymidine incorporation was significantly less [mean, 34 +/- 8% (+/- SE)] in mononuclear cells from neonates (independent of gestational age) compared to mononuclear cells from adults (66 +/- 5%; P less than 0.001). This difference in the inhibitory effect was not due to a smaller number of high affinity receptors for 1,25-(OH)2D in activated cord blood lymphocytes. We conclude that the coupling between the receptors for 1,25-(OH)2D and the biological response in neonates is less efficient than that in adults.