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* Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029;
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; and
Gene and Cell Medicine, Mount Sinai School of Medicine, New York, NY 10029
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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cells (rat insulin promoter (RIP)-M3 mice). RIP-M3 mice were normoglycemic and responded normally to glucose challenge but were remarkably resistant to diabetes induced by MLDS. Islets from MLDS-treated RIP-M3 mice had fewer inflammatory cells and expressed lower levels of chemokines than those from MLDS-treated controls. The role of M3 in chemokine blockade during insulitis was further supported by in vitro experiments demonstrating that multiple chemokines up-regulated during islet inflammation are high-affinity M3 ligands that can be simultaneously sequestered. These results implicate chemokines as key mediators of insulitis and suggest that their blockade may represent a novel strategy to prevent insulitis and islet destruction. |
Introduction |
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cells (1). The factors leading to the destruction of the islets are still not known, but it is well accepted that immune-based mechanisms involving macrophages and T cells are responsible for the death of the cells (2, 3).
Experimentally induced insulin-dependent diabetes mellitus in rodents with multiple low doses of streptozotocin (MLDS)3 is a widely used model that has clinical and histoimmunological features similar to those of human disease, with T cells and macrophages playing a major pathogenic role (4). When administered in animals at multiple low doses, the cell toxin streptozotocin is thought to induce initial cell damage by alkylating the DNA in islet cells (5) and/or by spontaneously releasing NO (6). Subsequently, as a result of a novel cell Ag expression, mononuclear cells will infiltrate the islets and start a multifactorial process (7) that will lead to islet destruction and diabetes.
The trafficking of macrophages and T cells has been shown to be regulated by chemokines, small m.w. chemoattractant proteins that interact with G protein-coupled receptors present at the cell surface of these leukocytes to regulate their migration, differentiation, and function (8, 9, 10). The migration of macrophages and T cells is controlled by multiple chemokines, including CCL2, CCL21, CCL19 (9, 11, 12), CXCL10, and CXCL9 (13). The migration of monocytes and macrophages is mediated by CCL2 and its receptor, CCR2 (14). Both homeostatic and inflammatory T cell trafficking are under the control of chemokines. The homeostatic migration of T cells is regulated by CCL21 and CCL19. Both CCL19 and CCL21 interact with a common receptor, CCR7, which is expressed by naive and memory T cells (15). The influx of effector T cells into inflamed areas is regulated by multiple chemokines, including the IFN-
-inducible chemokines CXCL10 and CXCL9 and their receptor CXCR3 (13).
Chemokines are not highly expressed by the normal islets, but chemokine expression has been detected in islets cultured in vitro. Primary cultures of murine and human pancreatic islets express and secrete CCL2 (16). CCL2 expression in the pancreas parallels disease progression in NOD mice (17, 18). A recent study has suggested an important role for CCL2 in the clinical outcome of islet transplantation in patients with type I diabetes (16). Low CCL2 secretion by islets before transplantation was the most relevant factor for predicting long-lasting insulin independence. CCL21 is not normally expressed by pancreatic islets, but its expression has been observed in the pancreas of NOD mice (19, 20). The expression of CCL21 by the islets has been proposed to be an important factor contributing to autoimmunity (21). Moreover, cells secrete CXCL9 and CXCL10 during virus-induced diabetes (13, 22).
Interference with the chemokine system has been shown to attenuate or prolong the development of experimental models of diabetes. For instance, animals lacking the chemokine receptor CXCR3 show a delayed onset of type 1 diabetes subsequent to a viral infection (13). In addition, Ab neutralization of a macrophage-derived chemokine (CCL22) causes a significant reduction of CCR4+ T cells within the pancreatic infiltrates and inhibits the development of insulitis and diabetes in NOD mice (23). Finally, the treatment of NOD mice with a neutralizing anti-CCR5 Ab inhibits cell destruction and diabetes (24). In none of these models, however, is there a complete abrogation of disease, which suggests that other factors (including additional chemokines) may be relevant for pathogenesis.
Testing a possible role for multiple chemokines in the pathogenesis of diabetes has been hampered by the lack of pharmacological agents with broad specificity and by the fact that many chemokine receptor genes are clustered in the genome, which precludes the generation of compound mutants by conventional breeding techniques. A novel tool for testing chemokine function is M3, a chemokine-binding protein encoded by the mouse herpesvirus 68 (MHV-68). M3 blocks chemokine receptor-binding interfaces and displaces and removes chemokines from inflammatory sites (25). Recent studies from our laboratory show that the expression of M3 by cells blocks lymphocyte recruitment induced by transgenic (tg) expression of CCL21 (26), CCL2, and CXCL13 (27).
In this study we show that chemokines are expressed in the islets of mice treated with MLDS before the development of diabetes. Furthermore, we show that the expression of the promiscuous chemokine decoy receptor M3 in the islets reduces the leukocyte infiltration and islet destruction induced by MLDS. These results indicate that chemokines are important determinants of diabetes and suggest that multichemokine blockade may be a novel approach to prevent insulitis and diabetes.
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Materials and Methods |
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Rat insulin promoter (RIP)-M3 tg mice were described previously (26). B6D2F1 mice were obtained from Charles River Laboratories. All mice were housed under specific pathogen-free conditions in individually ventilated cages at the Mount Sinai School of Medicine Animal Facility (New York, NY). All experiments were performed following institutional guidelines.
Streptozotocin treatment in vivo
To induce diabetes, mice (6–10 wk of age) were injected i.p. with streptozotocin (STZ) (40 mg/kg freshly dissolved in cold 0.1 M citrate buffer (pH 4.5); Calbiochem, EMD Biosciences) for 5 consecutive days as previously described (28). Blood glucose was monitored weekly over the following 35 or 70 days using an Ascensia Elite XL one-touch blood glucometer (Bayer). Animals were considered diabetic when their blood glucose levels were >250 mg/dl in two consecutive daily measurements. In some experiments mice were treated with a single i.p. of STZ (300 mg/kg body weight) and blood glucose was observed daily for 1 wk. Mice in the control group received a corresponding volume of sodium citrate buffer alone.
Histology
Tissues for light microscopic examination were fixed by immersion in 10% phosphate-buffered formalin and then processed for paraffin sections. Routinely, 5-µm sections were cut and stained with H&E. For immunohistochemical staining fresh-frozen sections were first fixed with ice-cold acetone for 20 min and dried and stored at –20°C. Slides were incubated for 1 h at room temperature with purified primary Abs followed by incubation with the appropriate labeled secondary Abs for 30 min. Primary Abs used were anti-CD45 (catalog no. 550539), CD3 (catalog no. 553058), CD11c (catalog no. 553799), CD11b (catalog no. 553308), and CD31 (catalog no. 550274) from BD Biosciences, rat anti-insulin (catalog no. MAB1417), anti-CCL1 (catalog no. MAB845), anti-CCL21 (catalog no. AF455) from R&D Systems, anti-CXCL9 (provided by J. Farber and H. Zhang, National Institute of Allergy and Infectious Diseases), and guinea pig polyclonal anti-insulin (catalog no. A0564) from DakoCytomation. The secondary Abs used were Alexa Fluor 488 goat anti-rat IgG (catalog no. A-11006), Alexa Fluor 594 goat anti-rat IgG (catalog no. A-11007), and Alexa Fluor 594 goat anti-rabbit IgG (catalog no. A-11037) from Molecular Probes and FITC anti-Guinea Pig IgG (catalog no. 127065160) from Jackson ImmunoResearch Laboratories.
To study the cellular changes promoted by STZ treatment we performed semiquantitative analysis on insulin/CD45-stained sections, assessing 20–80 islets per animal. Three grades of infiltration were based on the number of CD45+ cells in or around the islet: 1 (10–20 cells), 2 (20–50 cells), and 3 (>50 cells). At least 20 sections were evaluated per mouse and per day in a blinded fashion. Data are presented as mean insulitis score ± SD for the indicated experimental groups.
Intraperitoneal glucose tolerance test
After a 16-h fast, glucose (1.5 g/kg body weight in saline (0.9% NaCl)) was administered i.p. The blood glucose was monitored at 0, 30, 60, 120, and 240 min using an Ascensia Elite XL one-touch blood glucometer (Bayer).
Isolation of pancreatic islets of Langerhans
Islets of Langerhans were isolated as previously described (29). Briefly, the common bile duct was clamped distal to the pancreatic duct junction at its hepatic insertion. The proximal common bile duct was then cannulated using a 27-gauge needle, and the pancreas was infused by retrograde injection of 2 ml of ice-cold collagenase solution (1.0 mg/ml; Sigma-Aldrich) in HBSS (Invitrogen Life Technologies). Pancreatic tissue was recovered and subjected to a 12-min digestion at 37°C. Subsequently, ice cold HBSS was added and the suspension was vortexed at full speed for 10 s. Islets were hand picked under a dissection microscope. Islets were used immediately after isolation to obtain RNA or protein.
Western analysis
To analyze Glut-2 expression, islets from control and tg mice were sonicated in freshly prepared lysis buffer (5% SDS, 80 mmol/L Tris-HCl (pH 6.8), 5 mmol/L EDTA, and 0.5 mmol/L PMSF). The supernatants containing the cell lysate were separated by centrifugation and the protein concentrations were determined using the MicroBCA assay (Pierce). Forty micrograms of protein from each sample was added to loading buffer and analyzed using 10% SDS-polyacrylamide gels. Proteins were transferred from the gels to Immobilon-P membranes (Millipore) using standard techniques. Blots were incubated with Abs against Glut-2 (Santa Cruz Biotechnology) and actin (Sigma-Aldrich) and then with a peroxidase-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology) and a peroxidase-conjugated rabbit anti-mouse IgG (Abcam), respectively.
To analyze M3 expression, 200 islets from control and tg animals were incubated for 24 h in glucose-free medium and supernatants were collected. Twenty micrograms of protein from each sample was processed as described above. Blots were incubated with primary Abs against M3 (26) and a peroxidase-conjugated goat anti-rabbit IgG (Abcam). Chemiluminescence was detected using the Western Lightning Western Blot Chemiluminescence Reagent Plus (enhanced luminol) (PerkinElmer).
Islet glucose-stimulated insulin release
The insulin secretion assay was performed as described by Eizirik et al. with some modifications (30). Briefly, 20 islets from each group were set in quintuplicate in a 24-well plate and incubated in CMRL 1066 medium (Cellgro, Mediatech) supplemented with 1.7 mM glucose or with 16.7 mM glucose for 60 min at 37°C in an atmosphere of 95% O2 and 5% CO2. For insulin content, fresh islets were collected, washed with 1 ml of PBS, and sonicated in acid ethanol to extract insulin. All of supernatants and extractions were kept at –20°C. Insulin released into supernatants and insulin content was measured by ELISA (ALPCO Diagnostic).
Quantitative real-time PCR (Q-PCR)
Total RNA was extracted from pooled islets using the RNeasy maxi kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed for 3 µg of RNA. Q-PCR was conducted in duplicate from 25 ng of cDNA and with each primer at 0.4 µM in a 30-µl final reaction volumes of 1x SYBR Green PCR Master Mix (Applied Biosystems). PCR cycling conditions were 50°C for 2 min, 95°C for 15 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative expression levels were calculated as 2Ct ubiquitin – Ct gene (where Ct is cycle threshold; for details see ABI PRISM 7700 User Bulletin no. 2; Applied Biosystems) using ubiquitin RNA as endogenous control. The melt curve was performed and primer dimers were absent. The sequences of primers used to study chemokine mRNA expression are described in Table I. The results were graphed as fold increase in chemokine expression between days 0 and 10 and the experiments were repeated at least three times.
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SPR experiments were conducted using a Biacore 2000 biosensor at 25°C under conditions of 20 mM HEPES (pH 7.4), 150 mM sodium chloride, 0.005% Triton X-100. The M3 and human (h)CCL2 used in this analysis were produced as described elsewhere (J. M. Alexander-Brett and D. H. Fremont, submitted for publication). Murine (m)CXCL10, mCCL21, and hXCL1 were purchased from BioSource, and vascular endothelial growth factor (VEGF) was from PeproTech. For the two-site binding assay, M3 was pre-equilibrated with biotinylated CCL2 at a 10:1 ratio, and the mixture was injected over a NeutrAvidin-coated sensor chip to immobilize 
500 resonance units (RU) of material. M3 is captured via stable interaction with CCL2 and only the free second site is available for binding. The chemokines CXCL10 and CCL21 were injected over the sensor chip at 50 nM to assess their ability to bind the second M3 site, with XCL1 and VEGF (each at 50 nM) serving as positive and negative controls, respectively.
M3-chemokine binding affinities were measured using an SPR-based competition assay, which is described in full detail elsewhere (J. M. Alexander-Brett and D. H. Fremont, 2006, submitted for publication). Briefly, the competition assay facilitates the measurement of M3-chemokine binding affinities that are otherwise unobtainable by direct surface binding methods due to severe mass transport artifacts. The solution-surface competition assay uses a mutant form of M3 in which the s2b-s3 loop is mutated from 80EELGQ84 to 80SRRGR84, referred to as M3BBXB (where B represents a basic amino acid such as arginine and X is any other amino acid), which exhibits decreased chemokine binding on-rates and thus does not suffer from mass transport limitations. The solution-surface competition assay is conducted by measuring equilibrium (Req) chemokine binding to surface-bound M3BBXB as the amount of M3 solution competitor is increased. The experiment yields a competition titration curve that is mathematically fit to obtain the solution-based wild-type (wt) M3-chemokine binding affinity.
To perform this assay, the M3BBXB mutant was immobilized on a CM5 sensor chip to a level of 800 RU. CXCL10 (15 nM) and CCL21 (8 nM) were each pre-equilibrated with M3 over a range of concentrations from 1:0 to 1:2 molar ratio. Mixtures were injected over the flow cells at 60 µl/min, and the surface was regenerated with 1 M calcium chloride. Competition titration curves were analyzed using equations previously derived for solution-surface SPR competition using the program Scientist (Micromath Science Software) to yield KI, the solution affinity for M3 binding to each chemokine.
For the second-site binding assay, M3 (1 mg/ml) was pre-equilibrated with biotinylated CCL2 at a 10:1 ratio, and the mixture was injected over a neutravidin coated sensor chip to immobilize 500 RU of material. M3 is captured via a stable 2:1 interaction with CCL2 and only the free second site is available for binding to other chemokines. The chemokines CXCL10 and CCL21 were injected over the sensor chip at 50 nM to assess their ability to bind the second M3 site, with XCL1 and VEGF (each at 50 nM) serving as the positive and negative controls, respectively.
Statistical analysis
Data are expressed as means ± SD. An unpaired t test, one-way ANOVA, and repeated measures ANOVA were used to determine statistical significance. Differences were considered significant when p < 0.05.
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Results |
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MLDS initially induce cell damage (5), and mononuclear cells that infiltrate the islets start a multifactorial process that leads to destruction of the insulin-producing cells (31). To characterize the chemokines produced in the MLDS model, we performed Q-PCR on total RNA isolated from islets. We examined the expression of all known murine chemokine ligands in islets of Langerhans taken from control mice before (day 0, n = 40) and after MLDS treatment on days 3 (n = 6), 5 (n = 6), and 10 (n = 30). With the exception of CCL20 and CCL19, chemokines were expressed at very low levels before treatment (data not shown). No significant differences in chemokine expression were detected 3–5 days after the initiation of treatment (data not shown). However, there were striking differences in the expression levels of some chemokines, most notably CXCL9, CXCL10, and CCL1 (which were expressed at 31- to 178-fold higher levels than at day 0) (Fig. 1A).
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Expression of M3 does not affect the ability of cells to produce or secrete insulin
Next, we tested the hypothesis that an islet-specific chemokine blockade affected the development of diabetes induced by MLDS. To this end we used mice expressing the chemokine-binding protein M3 in the islets (RIP-M3 mice) (26). We have previously shown that mice expressing M3 in the islets develop normally and are normoglycemic. Furthermore, we found that the constitutive expression of M3 in the pancreas of the RIP-M3 mice did not affect the development of lymphoid or nonlymphoid tissues. To rule out that expression of M3 affected cell function we performed both in vitro and in vivo experiments.
First we confirmed that islets from tg animals secreted M3 in vitro. Isolated islets from tg RIP-M3 mice secreted immunoreactive 44-kDa M3 protein after 24 h of culture (Fig. 2A). No immunoreactivity was found in the medium from control islets. Densitometric scanning of the bands showed that islets from homozygous (tg/tg) mice released approximately double the amount of M3 as heterozygous (tg/wt) islets (data not shown, n = 4 experiments).
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cells affected glucose homeostasis, we analyzed the insulin content and the secretion of insulin after a glucose challenge in islets from controls and RIP-M3 tg mice. There was no significant difference in the insulin content between islets from control and RIP-M3 mice (n = 15 per group, Fig. 2B). As expected, higher levels of secreted insulin were detected in the supernatants of cultured islets from both control and RIP-M3 tg mice after they were exposed to high concentrations of glucose, but there was no difference between the groups (Fig. 2C, n = 3 experiments). Finally, the results of an i.p. glucose tolerance test on 8-wk-old control and RIP-M3 tg mice were identical, with plasma glucose reaching maximal levels 90 min after glucose administration (Fig. 2D). To evaluate whether cell integrity was altered by M3 expression we treated the mice with a high dose of STZ (300 mg/kg). Two days after treatment all control and RIP-M3 tg mice (n = 5 per group) became diabetic (Fig. 2E). Taken together these results indicate that the insulin synthesis and secretion are not altered by the expression of M3.
Expression of M3 in cells blocks the development of diabetes induced by MLDS
To test the effect of M3 on the development of diabetes we treated 6–10 wk-old male RIP-M3 mice and control littermates with either STZ (40 mg/kg) or vehicle for 5 consecutive days. As expected (32), Four weeks after the beginning of treatment 85% of the control mice treated with MLDS were diabetic. At this point only 35% of the RIP-M3 tg/wt mice and, remarkably, none of the homozygous RIP-M3 mice were diabetic (Fig. 3A). After 70 days all control mice were diabetic but only 60% of heterozygous mice and none of the homozygous RIP-M3 mice developed disease (n = 5 per group).
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Expression of M3 in cells prevents the recruitment of inflammatory cells
Recent studies from our laboratory show that islet-specific expression of M3 blocks leukocyte recruitment induced by islet-specific expression of CCL21 (26), CCL2, and CXCL13 (27).
To test the hypothesis that M3 expression blocked STZ-induced diabetes by preventing chemokine-induced recruitment of inflammatory cells, we treated mice (n = 3 per group) with MLDS and then sacrificed at 7, 14, and 21 days after the first injection. The infiltration of CD45+ cells into the islets of control mice was first seen on day 7 (Fig. 4, A and B), and the number of inflammatory cells and the frequency of infiltrated islets increased thereafter. By day 21 most control islets were infiltrated by mononuclear cells and had lost normal morphologic integrity. In contrast, a small number of infiltrating cells was found in RIP-M3 tg/wt mice only after 21 days of treatment and the morphological appearance of the islets was normal. Remarkably, none of the homozygous RIP-M3 mice showed infiltrating cells at day 21 (p = 0.035, one-way ANOVA), and the insulin-positive cells appeared normal (Fig. 4B).
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M3 binds multiple chemokines expressed during insulitis with high affinity
Inflammatory stimuli generally result in the up-regulation of groups of chemokines rather than a single species, as reflected by the elevated expression of numerous chemokines in the MLDS model described here. Previous studies have indicated that M3 binds to both murine and human chemokines from all four structural classes, thus acting as a broad-spectrum chemokine scavenger (34, 35). However, the M3 affinities for several chemokines that are relevant to the MLDS model have not been reported. To establish whether M3 binds to up-regulated inflammatory and homeostatic chemokines, we measured its affinity for mCCL21 and mCXCL10. Binding studies were conducted using a SPR-based competition assay (J. M. Alexander-Brett and D. H. Fremont, submitted for publication), which yielded affinities of 12 ± 1 and 320 ± 30 pM for mCCL21 and mCXCL10, respectively (Fig. 5A). It was also of interest to assess whether a single M3 dimer could simultaneously engage two distinct chemokines. This question was addressed using a two-site SPR binding assay in which M3 was immobilized to the sensor chip via interaction with the high-affinity chemokine hCCL2, and chemokine binding to the second site was demonstrated by injecting several chemokines over the M3-CCL2 coupled sensor chip (shown schematically in Fig. 5B). The chemokines mCCL21, mCXCL10, and hXCL1 all bind the available M3 second site, indicating that M3 is capable of simultaneously binding relevant chemokines of different structural classes at opposite ends of the dimer (Fig. 5B). The lack of binding for the negative control protein VEGF demonstrates the specificity of this interaction.
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Discussion |
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In diabetes, an accumulation of cells around the islets precedes the onset of clinical disease. We hypothesized that chemokines are critical determinants of the influx of leukocytes into the islets and, thus, constitute one of the major component in the pathogenesis of diabetes. To start addressing the role of chemokines in diabetes, we have examined in a comprehensive and quantitative fashion the expression of chemokines in the MLDS model. Our results show that of all the murine chemokine ligands, only two (CCL20 and CCL19) were expressed, albeit at low levels, in pancreatic islets of B6D2F1 mice before MLDS treatment. MLDS treatment induced the high expression of several chemokines, including CXCL9, CXCL10, and CCL2, before the onset of diabetes. The expression of these chemokines was likely triggered by IFN- and TNF-
(40), cytokines whose expression has been documented in islets before the onset of MLDS-induced diabetes (41). The early expression of these cytokines likely influenced the production of chemokines by resident inflammatory cells, endocrine cells, and endothelial cells and promoted the infiltration of the islets, first by macrophages and subsequently by lymphocytes (31). The infiltrating cells themselves represent a major source of chemokines, as suggested by our immunohistochemical analysis. We propose that this initial burst in the expression of chemokines contributes to the further recruitment of inflammatory cells and the destruction of the islets. Further studies focusing on the temporal analysis of chemokine mRNA and protein will be required to evaluate the role of chemokines in the onset of diabetes triggered by MLDS.
Although the changes in chemokine expression suggest a link with pathogenesis, they do not prove it. The results shown here indicate that chemokines are critical for pathogenesis. The expression of a chemokine scavenger in the islets dramatically reduced cellular infiltration and blocked the development of diabetes. This effect was more pronounced in the islets from homozygous RIP-M3 mice, which expressed higher levels of M3 than the islets of heterozygous mice. We believe that the blockade in cellular recruitment was caused by M3 inactivation of one or more chemokines expressed during the course of MLDS treatment. The crystal structure of M3 shows that M3 dimerizes and generates a binding site for the N-terminal region of chemokines that precludes the binding to their receptors (42). M3 also uses electrostatic mimicry to directly inhibit chemokine-glycosaminoglycan interactions for a diverse array of chemokines (D. H. Alexander and J. M. Fremont-Brett, 2006, submitted for publication). Glycosaminoglycan association is important for chemokine function, being a prerequisite for chemokine uptake, transcytosis to the apical side of the endothelial cell, and appropriate solid-phase presentation to the passing leukocytes (43). In vivo, M3 reduces mononuclear cellular responses after MHV-68-induced meningitis in mice (44) and blocks leukocyte recruitment to the pancreas induced by CCL21 (26), CXCL13, and CCL2 (27), respectively.
We suggest that these scavenging properties of M3 are essential for the reduced inflammation observed in the RIP-M3 tg mice. Many of the chemokines up-regulated in the MLDS model are known to bind M3 with high affinity (34, 35). In this study we show that M3 binds the murine chemokines CCL21 and CXCL10 with picomolar affinity (12 and 320 pM, respectively) (Fig. 5). Further, we show that M3 is able to engage either of these chemokines while simultaneously binding CCL2 at opposite ends of the dimer structure, suggesting that M3 can effectively inhibit mixed populations of chemokines as observed in the insulitis models. As such, M3 may disrupt the proper presentation and function of CCL2 (42), CXCL10, and CCL21, important players regulating the migration of monocytes and T cells that are critical for the development of diabetes (19, 23). Although our results suggest that the primary site of chemokine blockade induced by M3 is the endocrine pancreas, we cannot rule out that the trace amounts of M3 present in pancreatic lymph nodes and the bloodstream (not shown) may contribute to the effects seen here.
In conclusion, we demonstrate that chemokine expression is a hallmark of insulitis promoted by MLDS treatment. This altered expression is not limited to one or a few chemokines; several chemokines are expressed at the same time, a pattern already observed in other conditions (8, 45, 46, 47, 48). We also show that a chemokine blockade prevents mice from developing insulitis and diabetes induced by MLDS. Taken together, these results constitute direct evidence that chemokines are critical determinants of MLDS-induced diabetes. Future work will be directed to investigate whether M3 can block diabetes occurring in autoimmune settings and to test whether it can prevent the rejection of transplanted islets.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grants DK067381 (to S.A.L.) and AI051426 (to D.H.F.). 
2 Address correspondence and reprint requests to Dr. Sergio A. Lira, Immunobiology Center, Mount Sinai School of Medicine, 1425 Madison Avenue, Box 1630, New York, NY 10029. E-mail address: sergio.lira{at}mssm.edu
3 Abbreviations used in this paper: MLDS, multiple low-doses of streptozotocin; h, human; MHV-68, mouse herpesvirus 68; m, mouse; Q-PCR, quantitative real time PCR; RIP, rat insulin promoter; RU, resonance unit; SPR, surface plasmon resonance; STZ, streptozotocin; tg, transgenic; VEGF, vascular endothelial growth factor; wt, wild type.
Received for publication August 16, 2006. Accepted for publication January 18, 2007.
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References |
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cell destruction in type I (insulin-dependent) diabetes mellitus. J. Pathol. 152: 141-148. [Medline] cell destruction resulting from the collaboration between macrophages and T cells. Autoimmunity 27: 109-122. [Medline] T cells. Clin. Exp. Immunol. 109: 116-120. [Medline]-exposed human and rat islet cells. Diabetologia 44: 325-332. [Medline]-inducible protein of 10 kDa (CXC chemokine ligand (CXCL) 10) but not monokine induced by IFN- (CXCL9) imprints a pattern for the subsequent development of autoimmune disease. J. Immunol. 171: 6838-6845. herpesvirus 68. J. Virol. 77: 624-630. [Medline]-cell function. J. Clin. Invest. 90: 1263-1268. [Medline]
B1 (p50)-deficient mice are not susceptible to multiple low-dose streptozotocin-induced diabetes. J. Endocrinol. 173: 457-464. [Abstract] herpesvirus selective chemokine binding protein that inhibits chemokine action. J. Virol. 74: 6741-6747. -herpesvirus-induced lethal meningitis. J. Clin. Invest. 109: 905-914. [Medline]This article has been cited by other articles:
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  E. Masson, S. Koren, F. Razik, H. Goldberg, E. P. Kwan, L. Sheu, H. Y. Gaisano, and I. G. Fantus High {beta}-cell mass prevents streptozotocin-induced diabetes in thioredoxin-interacting protein-deficient mice Am J Physiol Endocrinol Metab, June 1, 2009; 296(6): E1251 - E1261. [Abstract] [Full Text] [PDF]
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 J. G Mabley, P. Pacher, K. G K Murthy, W. Williams, G. J Southan, A. L Salzman, and C. Szabo The novel inosine analogue, INO-2002, protects against diabetes development in multiple low-dose streptozotocin and non-obese diabetic mouse models of type I diabetes J. Endocrinol., September 1, 2008; 198(3): 581 - 589. [Abstract] [Full Text] [PDF]
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 N. Saederup, L. Chan, S. A. Lira, and I. F. Charo Fractalkine Deficiency Markedly Reduces Macrophage Accumulation and Atherosclerotic Lesion Formation in CCR2-/- Mice: Evidence for Independent Chemokine Functions in Atherogenesis Circulation, April 1, 2008; 117(13): 1642 - 1648. [Abstract] [Full Text] [PDF]
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 A. P. Martin, M. G. Grisotto, C. Canasto-Chibuque, S. L. Kunkel, J. S. Bromberg, G. C. Furtado, and S. A. Lira Islet Expression of M3 Uncovers a Key Role for Chemokines in the Development and Recruitment of Diabetogenic Cells in NOD Mice Diabetes, February 1, 2008; 57(2): 387 - 394. [Abstract] [Full Text] [PDF]
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 M. B. Ruiz-Arguello, V. P. Smith, G. S. V. Campanella, F. Baleux, F. Arenzana-Seisdedos, A. D. Luster, and A. Alcami An Ectromelia Virus Protein That Interacts with Chemokines through Their Glycosaminoglycan Binding Domain J. Virol., January 15, 2008; 82(2): 917 - 926. [Abstract] [Full Text] [PDF]
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 J. M. Alexander-Brett and D. H. Fremont Dual GPCR and GAG mimicry by the M3 chemokine decoy receptor J. Exp. Med., December 24, 2007; 204(13): 3157 - 3172. [Abstract] [Full Text] [PDF]
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H. I. Callewaert, C. A. Gysemans, L. Ladriere, W. D'Hertog, J. Hagenbrock, L. Overbergh, D. L. Eizirik, and C. Mathieu Deletion of STAT-1 Pancreatic Islets Protects Against Streptozotocin-Induced Diabetes and Early Graft Failure but Not Against Late Rejection Diabetes, August 1, 2007; 56(8): 2169 - 2173. [Abstract] [Full Text] [PDF]
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), RIP-M3 tg/tg (n = 5, •) and their non-tg (wt) littermates (n = 5, 
) (p = 0.78; one-way ANOVA).
