Decreased In Vitro Type 1 Immune Response Against Coxsackie Virus B4 in Children With Type 1 Diabetes
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糖尿病学杂志 2006年第4期
1 Division of Pediatrics and Diabetes Research Centre, Department of Molecular and Clinical Medicine, Linkping University, Linkping, Sweden
2 Laboratory for Immunobiology, Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland
3 Enterovirus Laboratory, Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland
4 Turku Immunology Centre and Department of Virology, University of Turku, Turku, Finland
ABSTRACT
Enteroviruses, particularly Coxsackie virus B4 (CVB4), are considered to be involved in the pathogenesis of type 1 diabetes. We wanted to compare the characteristics of T-cell immune response to CVB4 in children with type 1 diabetes and healthy children with and without HLA risk-associated haplotypes (HLA-DR3-DQ2 or HLA-DR4-DQ8) for type 1 diabetes. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured with CVB4 and analyzed for cytokine and chemokine receptors by flow cytometry and for expression of transcription factors Tbet and GATA-3 by RT-PCR and Western blot. Culture supernatants were analyzed for secretion of -interferon (IFN-). In children with type 1 diabetes, a decreased percentage of T-cells expressed CCR2, CXCR6, interleukin (IL)-18R, and IL-12R2-chain after in vitro stimulation with CVB4 in comparison with healthy children with or without HLA risk genotype. Moreover, we found that children with type 1 diabetes had decreased IFN- secretion and expression of Tbet, both on mRNA and protein level, in CVB4-stimulated PBMCs. Accordingly, children with type 1 diabetes show an impaired type 1 immune response against CVB4 compared with healthy children. This may lead to a delayed clearance of the virus and, at least partly, explain why children with type 1 diabetes may be more prone to CVB4 infections and related complications, such as -cell damage.
Type 1 diabetes is an autoimmune disease caused by destruction of the insulin producing -cells in pancreas, which is probably mediated by autoreactive T-cells. The risk of developing type 1 diabetes is to some extent genetically determined, but environmental factors are also involved in the autoimmune process. This is demonstrated, e.g., by the rapid increase of the disease incidence during the last decades (1).
Enteroviruses, in particular Coxsackie virus B4 (CVB4), have been proposed to be an environmental trigger of -cell autoimmunity and type 1 diabetes. An increased number of enterovirus infections have been shown in pre-diabetic individuals in a number of prospective studies using serological tests and enterovirus RNA detection (2eC5). Enterovirus infections during pregnancy have also been suggested to be associated with an increased risk of developing type 1 diabetes in the offspring (5eC7); however, the results are contradictory (8,9).
Several case-control studies have reported serological evidence for recent enterovirus infection (10eC13) and enterovirus RNA (14eC17) in the peripheral circulation in patients with type 1 diabetes. Moreover, cellular and humoral immune responses to enteroviruses have been reported to be aberrant in children with type 1 diabetes carrying risk genotype for type 1 diabetes (18eC21). Despite these findings, more studies and especially prospective follow-up studies are still needed to determine whether there is a causal association of CVB4 infection and type 1 diabetes (22).
Several mechanisms for the induction of -cell destruction by viruses have been suggested. Viral antigens may show molecular mimicry with -cell antigens and induce autoreactivity, viruses may also cause a direct cytolysis of infected -cells or induce bystander activation of autoreactive T-cells due to the inflammatory mediators released in infected islets.
The balance of type 1 and type 2 T-cells plays a major role in the regulation of cellular and humoral immune responses to give rise to an appropriate immune response against foreign antigens, e.g., viruses. Nave T-cells express the chemokine receptors CXCR4 and CCR7 (23). Type 1 T-cells are characterized by expression of the cytokine receptors interleukin (IL)-12R2-chain and IL-18R (24) and the chemokine receptors CXCR3, CCR5, CCR2 (23), and CXCR6 (25). The transcription factor Tbet is associated with type 1 response, such as IFN- activation (24). The chemokine receptors CCR3 and CCR4 (23) and the transcription factors GATA-3 and c-maf are associated with type 2 T-cells (24).
To study whether the immune response to CVB4 differs between children with type 1 diabetes and healthy children, we stimulated peripheral blood mononuclear cells (PBMCs) with inactivated CVB4 and studied expression of intracellular transcription factors, secretion of IFN-, and the expression of cytokine and chemokine receptors on T-cells.
RESEARCH DESIGN AND METHODS
Cell culture.
After informed consent from the participants, venous blood was collected into Cell Preparation Tubes (Becton Dickinson, Stockholm, Sweden) from children with type 1 diabetes (mean for duration of type 1 diabetes was 20.2 months, range 1eC48 months; mean HbA1c [A1C] 6.0%) (n = 15, mean age 11 years; range 6eC17 years) and healthy children with (n = 13, mean age 11 years; range 10eC18 years) or without (n = 14, mean age 12 years; range 10eC16 years) HLA risk genes. The month for collection of blood samples did not differ between the groups. Selected HLA-DQB1 and -DQA1 alleles were typed as described previously (26,27) to detect the presence of type 1 diabetes risk-associated DR3-DQ2 (DQA105-DQB102) and DR4-DQ8 (DQAB10302) haplotypes. The healthy children without HLA risk-associated haplotypes were negative for autoantibodies against GAD, tyrosine phosphatase, and insulin. The healthy children with HLA risk genotype were also negative for autoantibodies, except for two individuals, one with tyrosine phosphatase autoantibodies and the other with insulin autoantibodies.
PBMCs were isolated from the blood samples by centrifugation at 1,500 x g for 20 min, and then the cells were washed three times with RPMI 1640 (Gibco&/Life Technologies, Paisley, U.K.) supplemented with 5% inactivated human AB+ serum (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). The cells were then diluted to 1 x 106 cells&/ml with RPMI 1640 supplemented with inactivated human AB+ serum (5%), L-glutamine (2 mmol&/l; Invitrogen, Stockholm, Sweden), and gentamicin (25 e蘥&/ml; Sigma-Aldrich, Stockholm, Sweden). The PBMCs were cultured in wells with 2 x 106 cells per well.
The PBMCs were stimulated on day 0 with heat-inactivated (+56°C for 30 min) CVB4 strain J.V.B. (1 e蘥&/ml; obtained from the American Type Culture Collection, Manassas, VA). On day 3, IL-2 was added (32 pg&/ml; BD PharMingen, Stockholm, Sweden). Supernatants from the cultures were collected on day 7 and stored at eC70°C for further analysis of cytokine profiles. On day 7, the cells were analyzed using flow cytometry. Lysed cells for mRNA and cell pellets for Western blot analysis were collected and saved at eC70°C and in liquid nitrogen, respectively, for further analysis.
Flow cytometry.
The cells were stained with 5 e蘬&/300,000 cells allophycocyanin-anti-CD4, peridinin chlorophyll protein-anti-CD8, phycoerythrin-anti-IL-12R-2-chain, phycoerythrin-anti-CCR4, fluorescein isothiocyanate (FITC)-anti-CCR5 as well as isotype-matched controls (BD PharMingen, San Josee, CA), FITC-anti-CXCR4, FITC-anti-CXCR3, phycoerythrin-anti-IL-18R, FITC-anti-CCR7, phycoerythrin-anti-CCR2, FITC-anti-CCR3, and phycoerythrin-anti-CXCR6 (R&D Systems, Minneapolis, MN) for 30 min. After being washed with PBS (Medicago, Uppsala, Sweden) supplemented with 0.5% BSA (Difco Laboratories, Detroit, MI), the cells were resuspended in 160 e蘬 PBS supplemented with 0.5% BSA. The labeled cells were analyzed with four-color flow cytometry using a FACSCalibur and CellQuest software (Becton Dickinson, San Josee, CA).
The lymphocytes were gated on forward and side scatter. Cells (15,000) were acquired, and two parameter dot plots were created. For isotype controls, 5,000 cells were acquired. Compensation was performed to adjust for spectrally adjacent dye pairs. The quadrants in the dot plots were placed according to the staining of the unstained cells and isotype controls.
Autoantibodies.
Levels of autoantibodies against GAD and thyrosine phosphatase were analyzed in plasma by immunoprecipitation described previously (28). Positivity for GAD and thyrosine phosphatase autoantibodies was determined as antibody levels above the 98th percentile.
Diabetes Autoantibody Standardization Program 2003 was used to set the specificity and sensitivity levels. For GAD autoantibody assay, the specificity was 98% and sensitivity was 78%, and for thyrosine phosphatase autoantibody assay, the specificity was 100% and the sensitivity was 48%.
Autoantibodies against insulin were analyzed by a radio binding assay according to Williams et al. (29). The cutoff for positivity was determined as antibody levels above the 98th percentile. In the Diabetes Autoantibody Standardization Program 2003, we had specificity for autoantibodies against insulin of 100%, whereas the sensitivity was 24%.
Standard curves and interpolated values of samples were performed using GraphPad Prism 4 (GraphPad Software, San Diego, CA).
Enzyme-linked immunosorbent assay for IFN-.
The concentration of IFN- in supernatant samples was studied according to the method described previously (30).
Real-time RT-PCR.
For the mRNA expression measurements, the total RNA were isolated from the samples by using Genelute total RNA isolation kit (Sigma-Aldrich, St. Louis, MO). cDNA was prepared using Applied Biosystems TaqMan cDNA transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer’s protocol. Elimination of the genomic DNA was done by additional DNAse I (Roche Diagnostics, Mannheim, Germany) treatment before the cDNA synthesis. Random Hexamers (Applied Biosystems) was used to prime the first-strand synthesis, and the reaction was carried out in a total volume of 20 e蘬 with Multiscribe Reverse transcriptase enzyme according to manufacturer’s protocol (Applied Biosystems). A total of 1.8 e蘬 cDNA was used for each TaqMan measurement triplicate.
6-carboxyfluoresceineClabeled TaqMan Gene Expression assays were used to measure transcription levels of the selected genes (T-bet, Hs00203436_m1; GATA-3, Hs00231122_m1; c-maf, Hs00193519_m1; IL-12R, Hs00234651_m1; IL-4R, Hs00166237_m1; and ribosomal 18s, Hs99999901_s1).
Real-time PCR was carried out according to manufacturer’s protocols using TaqMan Universal PCR master mix (Applied Biosystems). ABI Prism 7700 sequence detector instrumentation was used for signal detection. Sequence detector was programmed to an initial step of 2 min at 50°C and 10 min at 95°C, followed by 50 thermal cycles of 15 s at 95°C and 1 min at 60°C.
The quantitative value obtained from the TaqMan run is a threshold cycle (Ct), which indicates the number of PCR cycles at which the amount of amplified target molecule exceeds a predefined threshold value. The difference value (Ct) is the normalized quantitative value of the expression level of the target gene achieved by subtracting the Ct value of the housekeeping gene (18s) from the Ct value of the target gene.
In vitro-transcribed cDNA pool denoted as a calibrator was considered as an interassay standard and to which all the other samples were compared. Calculations are expressed as follows: Ct(x1) = Ct(x1) eC Ct (calibrator). The difference in expression level is given by 2eCCt.
Western blot analysis for Tbet and GATA-3.
Total protein (5 e蘥) isolated from CVB4-stimulated PBMCs was run on a 10% SDS-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and transferred onto polyvinylidene difluoride nitrocellulose blotting membrane (Amersham Biosciences UK, Buckinghamshire, U.K.). The membrane was blocked for 1 h in PBS-Tween (Medicago) supplemented with 5% nonfat dried milk (Bio-Rad Laboratories) and 0.2% sodium fluoride (Merck, Damstadt, Germany). The membrane was incubated with anti-T-bet (final dilution 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GATA-3 (final dilution 1:1,000; Santa Cruz Biotechnology) followed by washes and incubation with horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody (final dilution 1:15,000; Bio-Rad Laboratories). The membrane was blocked again as previously described. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (final dilution 1:1,000; Santa Cruz Biotechnology) was then incubated with the membrane followed by washes and incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (final dilution 1:15,000; Bio-Rad Laboratories). The proteins were detected with ECL Plus kit (Amersham Biosciences) and chemiluminiscence films (Amersham Biosciences). The immunoblots were semiquantified by densiometric scanning (Gel-PRO analyzer; Media Cybernetics, Silver Spring, MD).
Micorneutralization test for CVB4 antibodies.
CVB4 antibodies were determined by a microneutralization assay in green monkey kidney cells, a continuous cell line from green monkey kidneys. Pretitrated virus (75 e蘬) corresponding to 30eC300 TCID_50 units was mixed with 75 e蘬 fourfold dilutions of plasma specimens on microtiter plates (96-well Nunclon Microtest plates). The mixture was incubated for 1 h at 36°C and then left overnight at 4°C. Then, 30,000 cells were added to each well, and the plates were incubated at 36°C for 6 days before staining with crystal violet. The highest dilution with complete inhibition of virus-induced cytopathic effect was considered the end point titer.
Statistical analysis.
For comparison among three groups, Kruskal-Wallis was used as pretest and Mann-Whitney U test was used for comparison of two unrelated groups. For correlation analysis of variables, Spearman’s rank order correlation coefficient test was used. P values below 0.05 were considered significant. All statistical analyses were made using GraphPad Prism 4.
Ethical considerations.
The study was approved by the Regional Ethics Committee for Human Research at the Faculty of Health Sciences, Linkping University.
RESULTS
Flow cytometry analysis of cytokine- and chemokine-receptor expression on T-cells after CVB4 stimulation of PBMCs.
Children with type 1 diabetes showed decreased response to CVB4 when compared with healthy children with or without HLA risk-associated haplotypes as seen in the percentage of CD4 and CD8 T-cells expressing CCR2 (medians for CD4 T-cells 1.50, 5.98, and 7.36, respectively, and for CD8 T-cells 3.56, 7.50, and 13.97, respectively) and CXCR6 (medians for CD4 T-cells 49.53, 67.01, and 67.11, respectively, and medians for CD8 T-cells 49.68, 79.58, and 70.26, respectively) (Figs. 1A and B). This was also seen in the intensity of CCR2 (medians for CD4 T-cells 3.02, 4.10, and 4.13, respectively, and medians for CD8 T-cells 3.22, 4.22, and 4.55, respectively) and CXCR6 (medians for CD4 T-cells 10.00, 13.70, and 13.78, respectively, and medians for CD8 T-cells 10.00, 20.35, and 16.58, respectively) on CD4 and CD8 T-cells (Figs. 2A and B).
The percentage of CD4 and CD8 T-cells expressing IL-18R (medians for CD4 T-cells 24.30, 29.31, and 27.29, respectively, and medians for CD8 T-cells 18.18, 24.71, and 27.45, respectively, Figs. 3A and B) and the intensity of IL-18R (medians for CD4 T-cells 5.42, 6.32, and 6.16, respectively, and medians for CD8 T-cells 4.61, 6.15, and 6.44, respectively; Figs. 4A and B) on CD4 and CD8 T-cells were also lower in children with type 1 diabetes in comparison with healthy children with or without risk-associated HLA haplotypes. The percentage of CD8 T-cells expressing IL-12R2-chain and the intensity of IL-12R2-chain on CD8 T-cells tended to be lower in children with type 1 diabetes in comparison with healthy children with or without HLA risk haplotypes (median percentage 1.07, 2.61, and 2.60, respectively, and median intensity 2.55, 3.16, and 2.84, respectively) (Figs. 3B and 4B). There were no differences in expression of CXCR3, CXCR4, CCR3, CCR4, CCR5, or CCR7 between the groups.
Cytokine secretion of PBMCs stimulated with CVB4.
The secretion of IFN- by PBMCs after 7 days of CVB4 stimulation was significantly lower in children with type 1 diabetes in comparison with healthy children with or without HLA risk haplotypes (medians 10,441, 36,680, and 79,080 pg&/ml, respectively) (Fig. 5).
Real-time RT-PCR analysis of Tbet, GATA-3, c-maf, IL-12R, and IL-4R in CVB4-stimulated PBMCs.
The CBV4-induced mRNA levels of Tbet were significantly lower in children with type 1 diabetes in comparison with both healthy children with and without type 1 diabeteseCassociated risk haplotypes (medians 16.89, 36.52, and 36.39, respectively) (Fig. 6A). There were no differences in mRNA levels of GATA-3, c-maf, IL-12R, and IL-4R between the three groups.
Western blot analysis of Tbet and GATA-3 in CVB4-stimulated PBMCs.
A tendency of lower ratio of Tbet to GAPDH measured as optical density was observed in children with type 1 diabetes in comparison with healthy children without HLA-associated risk genotype (medians 0.83 and 1.12, respectively) (Fig. 6B). There were no differences in the ratio of GATA-3 to GAPDH measured as optical density between the groups.
Neutralizing antibodies against CVB4.
There were no differences in the titer of neutralizing antibodies against CVB4 between the three groups.
Correlation between A1C values and immune response parameters.
There were no correlations between the A1C values in the children with type 1 diabetes and the expression of CCR2, CXCR6, IL-18R, IL-12R2-chain, or Tbet or the secretion of IFN- (Table 1).
DISCUSSION
In children with type 1 diabetes, a decreased number of CD4 and CD8 T-cells expressing CCR2, CXCR6, and IL-18R and a lower intensity of these receptors on CD4 and CD8 T-cells after stimulation of PBMCs with CVB4 was found in comparison with healthy children with or without HLA risk haplotype. Furthermore, also the number of CD8 T-cells expressing IL-12R2-chain and the intensity of this receptor on CD8 T-cells tended to be lower in children with type 1 diabetes compared with both groups of healthy children after CVB4 stimulation. Type 1 immune response is associated with expression of the receptors CCR2 (23), CXCR6 (25), IL-18R, and IL-12R2-chain (24); the cytokine IFN- (31); and the transcription factor Tbet (24). Thus, our results suggest that the induction of type 1 immune response against CVB4 is impaired in children with type 1 diabetes. This interpretation was further supported by the findings of decreased secretion of IFN- and decreased mRNA levels of the transcription factor Tbet in CVB4-stimulated PBMCs in children with type 1 diabetes. A similar trend was also seen in the protein levels of Tbet. Impaired development of type 1 immune response to CVB4 may cause a delay in the elimination of CVB4 followed by systemic spread of the virus and infection of the -cells. Enteroviruses are able to infect human -cells in vitro (32). Also, enterovirus-infected -cells have been reported in vivo in human pancreata (33). The appearance of -cell autoimmunity has been reported to be associated with enterovirus infections diagnosed by antibody increases and RNA in the serum (2,34).
In our study, we have used inactivated CVB4, but we still observed activation of both CD4 and CD8 cells. CD8 cell activation in PBMCs was thus not dependent on the presentation of intracellular viral proteins produced in infected cells. It has been previously shown that inactivated virus can be cross-presented by dendritic cells and induce a cytotoxic T-cell response in in vitro cell cultures (35).
For an efficient elimination of viruses, the induction of Th1 cell responses and effector Tc1 cells play a major role. Interaction between chemokines and their receptors allows migration of effector Tc1 cells to inflammation sites, leading to an efficient elimination of the virus.
In our study, we found that the percentage of CD4 and CD8 T-cells expressing CCR2 in response to CVB4 is decreased in children with type 1 diabetes. Previous reports have reported that viruses induce CCR2 expression on CD8 T-cells in vivo (36). Several studies have shown that CCR2-deficient mice have a severe Th1 defect seen as a reduced IFN- production in response to purified protein derivative immunization and defects in elimination of intracellular pathogens (37,38).
We found impaired response to CVB4 stimulation in several of the parameters, which characterize type 1 immune response. Discrepant results showing increased IFN- production in response to CVB4 antigens in young adults with newly diagnosed type 1 diabetes have been reported (19). However, the choice of CVB4 antigens and the age of the subjects differ significantly between the studies. Our results are limited to peripheral blood, and this response may not mirror the local response in the pancreas.
Our measures of type 1 response included IFN- secretion, which is known to induce IL-12R and is secreted by CXCR6-expressing type 1 cells and by NK cells, macrophages, and dendritic cells. We also studied the expression of Tbet, a transcription factor that activates the IFN- gene (39), leading to increased IFN- secretion. Tbet further induces IL-12R2-chain (40), supporting via IL-12&/STAT-4 signaling IFN- production. IFN- production is induced synergistically by IL-12 and IL-18 through the IL-12R and IL-18R, respectively (41). In CD8 T-cells, Tbet has been reported to control both cytokine production and effector function (42). In T-beteC/eC OT-1 TCR+ mice, a reduced cytotoxicity was observed, and T-beteC/eC mice were also reported to be more susceptible to lymphocytic choriomeningitis virus, which implies an impaired elimination of viruses. CVB4 has been shown to induce production of IFN-, IL-1, TNF-, IL-2, and IL-10 in PBMCs from healthy donors (43), and proper signaling of IFNs is important in protecting the -cell from destruction by the CVB4 infection (44).
Because we measured IFN- in PBMC population we are not able to identify the origin of IFN- responses. The blood volume taken from the children is limited and did not allow us to study the response in isolated cell populations. A reduced secretion of IFN- of CVB4-stimulated NK cells, macrophages, and dendritic cells in the PBMC cultures may contribute to decreased T-cell responses detected as lower amounts of Tbet on both mRNA and protein levels. Reduced levels of Tbet may further be responsible for the reduced number of T-cells expressing IL-12R2-chain and IL-18R. On the other hand, reduced IFN- secretion in the PBMC cultures may also reflect poor IFN- production from the T-cells.
We and others have previously reported that children with type 1 diabetes show a generally impaired type 1 immune response in vitro (30,45eC47). A previous study in patients with newly diagnosed and longstanding type 1 diabetes reported reduced levels of intracellular IFN- in CD4 T-cells from PMA+I-stimulated PBMCs (45). Another study reported lower secretion of IFN- and IL-10 from phytohemagglutinin-stimulated PBMCs at diagnosis of type 1 diabetes in children and young adults (46). Lohmann et al. (47) have reported a reduced secretion of IFN- and TNF- from fresh PBMCs in children with newly diagnosed type 1 diabetes, but this was normalized 6 months after diagnosis.
Immune responses against CVB4 or CVB4 proteins in individuals with type 1 diabetes have also been investigated. Patients with type 1 diabetes tended to respond less frequently with IFN- production and proliferation of PBMCs stimulated with a fusion protein between maltose binding protein and the nonstructural CVB4 protein P2C (MBP-P2C) compared with healthy controls (18). It has also been reported that the T-cell proliferation response to purified CVB4 (20) or different CVB4 proteins (19) is lower at onset of type 1 diabetes and increases with duration of type 1 diabetes. In contrast, we did not see any relation between the IFN- secretion or expression of cytokine and chemokine receptors on T-cells and the duration of type 1 diabetes in the present study (data not shown). In addition, there were no correlations between the actual A1C values of the children with type 1 diabetes and the immune response parameters. This suggests that the immune response against CVB4 is not affected by the metabolic balance in individuals with type 1 diabetes. We also studied the effect of in vitro glucose concentration on the upregulation of CCR2, CXCR6, and IL-12R after CVB4 stimulation in PBMCs from four healthy individuals. Preincubation of the PBMCs at hyperglycaemic conditions before CVB4 stimulation did not result in decreased immune response (data not shown). Although we did not see a direct effect of high glucose concentrations in vitro or in vivo (A1C) on immune response to CVB4, we are not able to exclude the possibility that hyperglycemia may modulate the cell-mediated response to CVB4. If this is the case, decreased elimination of viruses may then be an accelerating contributor to -cell destruction in slightly hyperglycemic pre-diabetic individuals. We demonstrate here a poor induction of type 1 immune response against CVB4, and suggest that this could explain the more frequent occurrence of enterovirus infections in children who develop type 1 diabetes.
In our study, the impaired immune response against CVB4 could not be explained by HLA risk haplotypes of type 1 diabetes. Juhela et al. (20) have previously shown that the T-cell proliferation against purified CVB4 was lower in children with type 1 diabetes compared with HLA-matched controls, which is in accordance with our findings. It has also been reported that the proliferation and cytokine response against MBP-P2C in individuals with HLA risk haplotypes is not different from individuals without HLA risk haplotypes (18). However, antibody response to CVB4 is modulated by HLA genotype (21).
In conclusion, children with type 1 diabetes seem to have an impaired type 1 immune response against CVB4, which was not explained by disease-associated HLA genotypes. The impaired type 1 immune response in children with type 1 diabetes may explain the more frequent CVB4 infections and systemic spread of the infection, which could then cause -cell damage ultimately leading to the development of type 1 diabetes.
ACKNOWLEDGMENTS
This study was supported by grants from Swedish Child Diabetes Foundation (Barndiabetesfonden), Swedish Research Council (K2002-72X-11242-08A and K2003-72X-14690-01A), Swedish Diabetes Association, Medical Research Fund of the County of stergtland, Samaritens’ Foundation, The Board of the Research Fellowship Award, The Strategic Area "Prevention of Diabetes and its Complications," the Juvenile Diabetes Research Foundation, the Academy of Finland, and the Sigrid Juselius Foundation.
We thank Gunilla Hallstrm, Eva Isacsson, and Ann-Marie Sandstrm for recruitment of healthy children and Cecilia Runnqvist for technical assistance.
FOOTNOTES
CVB4, Coxsackie virus B4; IFN-, -interferon; IL, interleukin; PBMC, peripheral blood mononuclear cell.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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2 Laboratory for Immunobiology, Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland
3 Enterovirus Laboratory, Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland
4 Turku Immunology Centre and Department of Virology, University of Turku, Turku, Finland
ABSTRACT
Enteroviruses, particularly Coxsackie virus B4 (CVB4), are considered to be involved in the pathogenesis of type 1 diabetes. We wanted to compare the characteristics of T-cell immune response to CVB4 in children with type 1 diabetes and healthy children with and without HLA risk-associated haplotypes (HLA-DR3-DQ2 or HLA-DR4-DQ8) for type 1 diabetes. Peripheral blood mononuclear cells (PBMCs) were isolated and cultured with CVB4 and analyzed for cytokine and chemokine receptors by flow cytometry and for expression of transcription factors Tbet and GATA-3 by RT-PCR and Western blot. Culture supernatants were analyzed for secretion of -interferon (IFN-). In children with type 1 diabetes, a decreased percentage of T-cells expressed CCR2, CXCR6, interleukin (IL)-18R, and IL-12R2-chain after in vitro stimulation with CVB4 in comparison with healthy children with or without HLA risk genotype. Moreover, we found that children with type 1 diabetes had decreased IFN- secretion and expression of Tbet, both on mRNA and protein level, in CVB4-stimulated PBMCs. Accordingly, children with type 1 diabetes show an impaired type 1 immune response against CVB4 compared with healthy children. This may lead to a delayed clearance of the virus and, at least partly, explain why children with type 1 diabetes may be more prone to CVB4 infections and related complications, such as -cell damage.
Type 1 diabetes is an autoimmune disease caused by destruction of the insulin producing -cells in pancreas, which is probably mediated by autoreactive T-cells. The risk of developing type 1 diabetes is to some extent genetically determined, but environmental factors are also involved in the autoimmune process. This is demonstrated, e.g., by the rapid increase of the disease incidence during the last decades (1).
Enteroviruses, in particular Coxsackie virus B4 (CVB4), have been proposed to be an environmental trigger of -cell autoimmunity and type 1 diabetes. An increased number of enterovirus infections have been shown in pre-diabetic individuals in a number of prospective studies using serological tests and enterovirus RNA detection (2eC5). Enterovirus infections during pregnancy have also been suggested to be associated with an increased risk of developing type 1 diabetes in the offspring (5eC7); however, the results are contradictory (8,9).
Several case-control studies have reported serological evidence for recent enterovirus infection (10eC13) and enterovirus RNA (14eC17) in the peripheral circulation in patients with type 1 diabetes. Moreover, cellular and humoral immune responses to enteroviruses have been reported to be aberrant in children with type 1 diabetes carrying risk genotype for type 1 diabetes (18eC21). Despite these findings, more studies and especially prospective follow-up studies are still needed to determine whether there is a causal association of CVB4 infection and type 1 diabetes (22).
Several mechanisms for the induction of -cell destruction by viruses have been suggested. Viral antigens may show molecular mimicry with -cell antigens and induce autoreactivity, viruses may also cause a direct cytolysis of infected -cells or induce bystander activation of autoreactive T-cells due to the inflammatory mediators released in infected islets.
The balance of type 1 and type 2 T-cells plays a major role in the regulation of cellular and humoral immune responses to give rise to an appropriate immune response against foreign antigens, e.g., viruses. Nave T-cells express the chemokine receptors CXCR4 and CCR7 (23). Type 1 T-cells are characterized by expression of the cytokine receptors interleukin (IL)-12R2-chain and IL-18R (24) and the chemokine receptors CXCR3, CCR5, CCR2 (23), and CXCR6 (25). The transcription factor Tbet is associated with type 1 response, such as IFN- activation (24). The chemokine receptors CCR3 and CCR4 (23) and the transcription factors GATA-3 and c-maf are associated with type 2 T-cells (24).
To study whether the immune response to CVB4 differs between children with type 1 diabetes and healthy children, we stimulated peripheral blood mononuclear cells (PBMCs) with inactivated CVB4 and studied expression of intracellular transcription factors, secretion of IFN-, and the expression of cytokine and chemokine receptors on T-cells.
RESEARCH DESIGN AND METHODS
Cell culture.
After informed consent from the participants, venous blood was collected into Cell Preparation Tubes (Becton Dickinson, Stockholm, Sweden) from children with type 1 diabetes (mean for duration of type 1 diabetes was 20.2 months, range 1eC48 months; mean HbA1c [A1C] 6.0%) (n = 15, mean age 11 years; range 6eC17 years) and healthy children with (n = 13, mean age 11 years; range 10eC18 years) or without (n = 14, mean age 12 years; range 10eC16 years) HLA risk genes. The month for collection of blood samples did not differ between the groups. Selected HLA-DQB1 and -DQA1 alleles were typed as described previously (26,27) to detect the presence of type 1 diabetes risk-associated DR3-DQ2 (DQA105-DQB102) and DR4-DQ8 (DQAB10302) haplotypes. The healthy children without HLA risk-associated haplotypes were negative for autoantibodies against GAD, tyrosine phosphatase, and insulin. The healthy children with HLA risk genotype were also negative for autoantibodies, except for two individuals, one with tyrosine phosphatase autoantibodies and the other with insulin autoantibodies.
PBMCs were isolated from the blood samples by centrifugation at 1,500 x g for 20 min, and then the cells were washed three times with RPMI 1640 (Gibco&/Life Technologies, Paisley, U.K.) supplemented with 5% inactivated human AB+ serum (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). The cells were then diluted to 1 x 106 cells&/ml with RPMI 1640 supplemented with inactivated human AB+ serum (5%), L-glutamine (2 mmol&/l; Invitrogen, Stockholm, Sweden), and gentamicin (25 e蘥&/ml; Sigma-Aldrich, Stockholm, Sweden). The PBMCs were cultured in wells with 2 x 106 cells per well.
The PBMCs were stimulated on day 0 with heat-inactivated (+56°C for 30 min) CVB4 strain J.V.B. (1 e蘥&/ml; obtained from the American Type Culture Collection, Manassas, VA). On day 3, IL-2 was added (32 pg&/ml; BD PharMingen, Stockholm, Sweden). Supernatants from the cultures were collected on day 7 and stored at eC70°C for further analysis of cytokine profiles. On day 7, the cells were analyzed using flow cytometry. Lysed cells for mRNA and cell pellets for Western blot analysis were collected and saved at eC70°C and in liquid nitrogen, respectively, for further analysis.
Flow cytometry.
The cells were stained with 5 e蘬&/300,000 cells allophycocyanin-anti-CD4, peridinin chlorophyll protein-anti-CD8, phycoerythrin-anti-IL-12R-2-chain, phycoerythrin-anti-CCR4, fluorescein isothiocyanate (FITC)-anti-CCR5 as well as isotype-matched controls (BD PharMingen, San Josee, CA), FITC-anti-CXCR4, FITC-anti-CXCR3, phycoerythrin-anti-IL-18R, FITC-anti-CCR7, phycoerythrin-anti-CCR2, FITC-anti-CCR3, and phycoerythrin-anti-CXCR6 (R&D Systems, Minneapolis, MN) for 30 min. After being washed with PBS (Medicago, Uppsala, Sweden) supplemented with 0.5% BSA (Difco Laboratories, Detroit, MI), the cells were resuspended in 160 e蘬 PBS supplemented with 0.5% BSA. The labeled cells were analyzed with four-color flow cytometry using a FACSCalibur and CellQuest software (Becton Dickinson, San Josee, CA).
The lymphocytes were gated on forward and side scatter. Cells (15,000) were acquired, and two parameter dot plots were created. For isotype controls, 5,000 cells were acquired. Compensation was performed to adjust for spectrally adjacent dye pairs. The quadrants in the dot plots were placed according to the staining of the unstained cells and isotype controls.
Autoantibodies.
Levels of autoantibodies against GAD and thyrosine phosphatase were analyzed in plasma by immunoprecipitation described previously (28). Positivity for GAD and thyrosine phosphatase autoantibodies was determined as antibody levels above the 98th percentile.
Diabetes Autoantibody Standardization Program 2003 was used to set the specificity and sensitivity levels. For GAD autoantibody assay, the specificity was 98% and sensitivity was 78%, and for thyrosine phosphatase autoantibody assay, the specificity was 100% and the sensitivity was 48%.
Autoantibodies against insulin were analyzed by a radio binding assay according to Williams et al. (29). The cutoff for positivity was determined as antibody levels above the 98th percentile. In the Diabetes Autoantibody Standardization Program 2003, we had specificity for autoantibodies against insulin of 100%, whereas the sensitivity was 24%.
Standard curves and interpolated values of samples were performed using GraphPad Prism 4 (GraphPad Software, San Diego, CA).
Enzyme-linked immunosorbent assay for IFN-.
The concentration of IFN- in supernatant samples was studied according to the method described previously (30).
Real-time RT-PCR.
For the mRNA expression measurements, the total RNA were isolated from the samples by using Genelute total RNA isolation kit (Sigma-Aldrich, St. Louis, MO). cDNA was prepared using Applied Biosystems TaqMan cDNA transcription kit (Applied Biosystems, Foster City, CA) according to manufacturer’s protocol. Elimination of the genomic DNA was done by additional DNAse I (Roche Diagnostics, Mannheim, Germany) treatment before the cDNA synthesis. Random Hexamers (Applied Biosystems) was used to prime the first-strand synthesis, and the reaction was carried out in a total volume of 20 e蘬 with Multiscribe Reverse transcriptase enzyme according to manufacturer’s protocol (Applied Biosystems). A total of 1.8 e蘬 cDNA was used for each TaqMan measurement triplicate.
6-carboxyfluoresceineClabeled TaqMan Gene Expression assays were used to measure transcription levels of the selected genes (T-bet, Hs00203436_m1; GATA-3, Hs00231122_m1; c-maf, Hs00193519_m1; IL-12R, Hs00234651_m1; IL-4R, Hs00166237_m1; and ribosomal 18s, Hs99999901_s1).
Real-time PCR was carried out according to manufacturer’s protocols using TaqMan Universal PCR master mix (Applied Biosystems). ABI Prism 7700 sequence detector instrumentation was used for signal detection. Sequence detector was programmed to an initial step of 2 min at 50°C and 10 min at 95°C, followed by 50 thermal cycles of 15 s at 95°C and 1 min at 60°C.
The quantitative value obtained from the TaqMan run is a threshold cycle (Ct), which indicates the number of PCR cycles at which the amount of amplified target molecule exceeds a predefined threshold value. The difference value (Ct) is the normalized quantitative value of the expression level of the target gene achieved by subtracting the Ct value of the housekeeping gene (18s) from the Ct value of the target gene.
In vitro-transcribed cDNA pool denoted as a calibrator was considered as an interassay standard and to which all the other samples were compared. Calculations are expressed as follows: Ct(x1) = Ct(x1) eC Ct (calibrator). The difference in expression level is given by 2eCCt.
Western blot analysis for Tbet and GATA-3.
Total protein (5 e蘥) isolated from CVB4-stimulated PBMCs was run on a 10% SDS-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA) and transferred onto polyvinylidene difluoride nitrocellulose blotting membrane (Amersham Biosciences UK, Buckinghamshire, U.K.). The membrane was blocked for 1 h in PBS-Tween (Medicago) supplemented with 5% nonfat dried milk (Bio-Rad Laboratories) and 0.2% sodium fluoride (Merck, Damstadt, Germany). The membrane was incubated with anti-T-bet (final dilution 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GATA-3 (final dilution 1:1,000; Santa Cruz Biotechnology) followed by washes and incubation with horseradish peroxidase-conjugated rabbit anti-mouse IgG antibody (final dilution 1:15,000; Bio-Rad Laboratories). The membrane was blocked again as previously described. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (final dilution 1:1,000; Santa Cruz Biotechnology) was then incubated with the membrane followed by washes and incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (final dilution 1:15,000; Bio-Rad Laboratories). The proteins were detected with ECL Plus kit (Amersham Biosciences) and chemiluminiscence films (Amersham Biosciences). The immunoblots were semiquantified by densiometric scanning (Gel-PRO analyzer; Media Cybernetics, Silver Spring, MD).
Micorneutralization test for CVB4 antibodies.
CVB4 antibodies were determined by a microneutralization assay in green monkey kidney cells, a continuous cell line from green monkey kidneys. Pretitrated virus (75 e蘬) corresponding to 30eC300 TCID_50 units was mixed with 75 e蘬 fourfold dilutions of plasma specimens on microtiter plates (96-well Nunclon Microtest plates). The mixture was incubated for 1 h at 36°C and then left overnight at 4°C. Then, 30,000 cells were added to each well, and the plates were incubated at 36°C for 6 days before staining with crystal violet. The highest dilution with complete inhibition of virus-induced cytopathic effect was considered the end point titer.
Statistical analysis.
For comparison among three groups, Kruskal-Wallis was used as pretest and Mann-Whitney U test was used for comparison of two unrelated groups. For correlation analysis of variables, Spearman’s rank order correlation coefficient test was used. P values below 0.05 were considered significant. All statistical analyses were made using GraphPad Prism 4.
Ethical considerations.
The study was approved by the Regional Ethics Committee for Human Research at the Faculty of Health Sciences, Linkping University.
RESULTS
Flow cytometry analysis of cytokine- and chemokine-receptor expression on T-cells after CVB4 stimulation of PBMCs.
Children with type 1 diabetes showed decreased response to CVB4 when compared with healthy children with or without HLA risk-associated haplotypes as seen in the percentage of CD4 and CD8 T-cells expressing CCR2 (medians for CD4 T-cells 1.50, 5.98, and 7.36, respectively, and for CD8 T-cells 3.56, 7.50, and 13.97, respectively) and CXCR6 (medians for CD4 T-cells 49.53, 67.01, and 67.11, respectively, and medians for CD8 T-cells 49.68, 79.58, and 70.26, respectively) (Figs. 1A and B). This was also seen in the intensity of CCR2 (medians for CD4 T-cells 3.02, 4.10, and 4.13, respectively, and medians for CD8 T-cells 3.22, 4.22, and 4.55, respectively) and CXCR6 (medians for CD4 T-cells 10.00, 13.70, and 13.78, respectively, and medians for CD8 T-cells 10.00, 20.35, and 16.58, respectively) on CD4 and CD8 T-cells (Figs. 2A and B).
The percentage of CD4 and CD8 T-cells expressing IL-18R (medians for CD4 T-cells 24.30, 29.31, and 27.29, respectively, and medians for CD8 T-cells 18.18, 24.71, and 27.45, respectively, Figs. 3A and B) and the intensity of IL-18R (medians for CD4 T-cells 5.42, 6.32, and 6.16, respectively, and medians for CD8 T-cells 4.61, 6.15, and 6.44, respectively; Figs. 4A and B) on CD4 and CD8 T-cells were also lower in children with type 1 diabetes in comparison with healthy children with or without risk-associated HLA haplotypes. The percentage of CD8 T-cells expressing IL-12R2-chain and the intensity of IL-12R2-chain on CD8 T-cells tended to be lower in children with type 1 diabetes in comparison with healthy children with or without HLA risk haplotypes (median percentage 1.07, 2.61, and 2.60, respectively, and median intensity 2.55, 3.16, and 2.84, respectively) (Figs. 3B and 4B). There were no differences in expression of CXCR3, CXCR4, CCR3, CCR4, CCR5, or CCR7 between the groups.
Cytokine secretion of PBMCs stimulated with CVB4.
The secretion of IFN- by PBMCs after 7 days of CVB4 stimulation was significantly lower in children with type 1 diabetes in comparison with healthy children with or without HLA risk haplotypes (medians 10,441, 36,680, and 79,080 pg&/ml, respectively) (Fig. 5).
Real-time RT-PCR analysis of Tbet, GATA-3, c-maf, IL-12R, and IL-4R in CVB4-stimulated PBMCs.
The CBV4-induced mRNA levels of Tbet were significantly lower in children with type 1 diabetes in comparison with both healthy children with and without type 1 diabeteseCassociated risk haplotypes (medians 16.89, 36.52, and 36.39, respectively) (Fig. 6A). There were no differences in mRNA levels of GATA-3, c-maf, IL-12R, and IL-4R between the three groups.
Western blot analysis of Tbet and GATA-3 in CVB4-stimulated PBMCs.
A tendency of lower ratio of Tbet to GAPDH measured as optical density was observed in children with type 1 diabetes in comparison with healthy children without HLA-associated risk genotype (medians 0.83 and 1.12, respectively) (Fig. 6B). There were no differences in the ratio of GATA-3 to GAPDH measured as optical density between the groups.
Neutralizing antibodies against CVB4.
There were no differences in the titer of neutralizing antibodies against CVB4 between the three groups.
Correlation between A1C values and immune response parameters.
There were no correlations between the A1C values in the children with type 1 diabetes and the expression of CCR2, CXCR6, IL-18R, IL-12R2-chain, or Tbet or the secretion of IFN- (Table 1).
DISCUSSION
In children with type 1 diabetes, a decreased number of CD4 and CD8 T-cells expressing CCR2, CXCR6, and IL-18R and a lower intensity of these receptors on CD4 and CD8 T-cells after stimulation of PBMCs with CVB4 was found in comparison with healthy children with or without HLA risk haplotype. Furthermore, also the number of CD8 T-cells expressing IL-12R2-chain and the intensity of this receptor on CD8 T-cells tended to be lower in children with type 1 diabetes compared with both groups of healthy children after CVB4 stimulation. Type 1 immune response is associated with expression of the receptors CCR2 (23), CXCR6 (25), IL-18R, and IL-12R2-chain (24); the cytokine IFN- (31); and the transcription factor Tbet (24). Thus, our results suggest that the induction of type 1 immune response against CVB4 is impaired in children with type 1 diabetes. This interpretation was further supported by the findings of decreased secretion of IFN- and decreased mRNA levels of the transcription factor Tbet in CVB4-stimulated PBMCs in children with type 1 diabetes. A similar trend was also seen in the protein levels of Tbet. Impaired development of type 1 immune response to CVB4 may cause a delay in the elimination of CVB4 followed by systemic spread of the virus and infection of the -cells. Enteroviruses are able to infect human -cells in vitro (32). Also, enterovirus-infected -cells have been reported in vivo in human pancreata (33). The appearance of -cell autoimmunity has been reported to be associated with enterovirus infections diagnosed by antibody increases and RNA in the serum (2,34).
In our study, we have used inactivated CVB4, but we still observed activation of both CD4 and CD8 cells. CD8 cell activation in PBMCs was thus not dependent on the presentation of intracellular viral proteins produced in infected cells. It has been previously shown that inactivated virus can be cross-presented by dendritic cells and induce a cytotoxic T-cell response in in vitro cell cultures (35).
For an efficient elimination of viruses, the induction of Th1 cell responses and effector Tc1 cells play a major role. Interaction between chemokines and their receptors allows migration of effector Tc1 cells to inflammation sites, leading to an efficient elimination of the virus.
In our study, we found that the percentage of CD4 and CD8 T-cells expressing CCR2 in response to CVB4 is decreased in children with type 1 diabetes. Previous reports have reported that viruses induce CCR2 expression on CD8 T-cells in vivo (36). Several studies have shown that CCR2-deficient mice have a severe Th1 defect seen as a reduced IFN- production in response to purified protein derivative immunization and defects in elimination of intracellular pathogens (37,38).
We found impaired response to CVB4 stimulation in several of the parameters, which characterize type 1 immune response. Discrepant results showing increased IFN- production in response to CVB4 antigens in young adults with newly diagnosed type 1 diabetes have been reported (19). However, the choice of CVB4 antigens and the age of the subjects differ significantly between the studies. Our results are limited to peripheral blood, and this response may not mirror the local response in the pancreas.
Our measures of type 1 response included IFN- secretion, which is known to induce IL-12R and is secreted by CXCR6-expressing type 1 cells and by NK cells, macrophages, and dendritic cells. We also studied the expression of Tbet, a transcription factor that activates the IFN- gene (39), leading to increased IFN- secretion. Tbet further induces IL-12R2-chain (40), supporting via IL-12&/STAT-4 signaling IFN- production. IFN- production is induced synergistically by IL-12 and IL-18 through the IL-12R and IL-18R, respectively (41). In CD8 T-cells, Tbet has been reported to control both cytokine production and effector function (42). In T-beteC/eC OT-1 TCR+ mice, a reduced cytotoxicity was observed, and T-beteC/eC mice were also reported to be more susceptible to lymphocytic choriomeningitis virus, which implies an impaired elimination of viruses. CVB4 has been shown to induce production of IFN-, IL-1, TNF-, IL-2, and IL-10 in PBMCs from healthy donors (43), and proper signaling of IFNs is important in protecting the -cell from destruction by the CVB4 infection (44).
Because we measured IFN- in PBMC population we are not able to identify the origin of IFN- responses. The blood volume taken from the children is limited and did not allow us to study the response in isolated cell populations. A reduced secretion of IFN- of CVB4-stimulated NK cells, macrophages, and dendritic cells in the PBMC cultures may contribute to decreased T-cell responses detected as lower amounts of Tbet on both mRNA and protein levels. Reduced levels of Tbet may further be responsible for the reduced number of T-cells expressing IL-12R2-chain and IL-18R. On the other hand, reduced IFN- secretion in the PBMC cultures may also reflect poor IFN- production from the T-cells.
We and others have previously reported that children with type 1 diabetes show a generally impaired type 1 immune response in vitro (30,45eC47). A previous study in patients with newly diagnosed and longstanding type 1 diabetes reported reduced levels of intracellular IFN- in CD4 T-cells from PMA+I-stimulated PBMCs (45). Another study reported lower secretion of IFN- and IL-10 from phytohemagglutinin-stimulated PBMCs at diagnosis of type 1 diabetes in children and young adults (46). Lohmann et al. (47) have reported a reduced secretion of IFN- and TNF- from fresh PBMCs in children with newly diagnosed type 1 diabetes, but this was normalized 6 months after diagnosis.
Immune responses against CVB4 or CVB4 proteins in individuals with type 1 diabetes have also been investigated. Patients with type 1 diabetes tended to respond less frequently with IFN- production and proliferation of PBMCs stimulated with a fusion protein between maltose binding protein and the nonstructural CVB4 protein P2C (MBP-P2C) compared with healthy controls (18). It has also been reported that the T-cell proliferation response to purified CVB4 (20) or different CVB4 proteins (19) is lower at onset of type 1 diabetes and increases with duration of type 1 diabetes. In contrast, we did not see any relation between the IFN- secretion or expression of cytokine and chemokine receptors on T-cells and the duration of type 1 diabetes in the present study (data not shown). In addition, there were no correlations between the actual A1C values of the children with type 1 diabetes and the immune response parameters. This suggests that the immune response against CVB4 is not affected by the metabolic balance in individuals with type 1 diabetes. We also studied the effect of in vitro glucose concentration on the upregulation of CCR2, CXCR6, and IL-12R after CVB4 stimulation in PBMCs from four healthy individuals. Preincubation of the PBMCs at hyperglycaemic conditions before CVB4 stimulation did not result in decreased immune response (data not shown). Although we did not see a direct effect of high glucose concentrations in vitro or in vivo (A1C) on immune response to CVB4, we are not able to exclude the possibility that hyperglycemia may modulate the cell-mediated response to CVB4. If this is the case, decreased elimination of viruses may then be an accelerating contributor to -cell destruction in slightly hyperglycemic pre-diabetic individuals. We demonstrate here a poor induction of type 1 immune response against CVB4, and suggest that this could explain the more frequent occurrence of enterovirus infections in children who develop type 1 diabetes.
In our study, the impaired immune response against CVB4 could not be explained by HLA risk haplotypes of type 1 diabetes. Juhela et al. (20) have previously shown that the T-cell proliferation against purified CVB4 was lower in children with type 1 diabetes compared with HLA-matched controls, which is in accordance with our findings. It has also been reported that the proliferation and cytokine response against MBP-P2C in individuals with HLA risk haplotypes is not different from individuals without HLA risk haplotypes (18). However, antibody response to CVB4 is modulated by HLA genotype (21).
In conclusion, children with type 1 diabetes seem to have an impaired type 1 immune response against CVB4, which was not explained by disease-associated HLA genotypes. The impaired type 1 immune response in children with type 1 diabetes may explain the more frequent CVB4 infections and systemic spread of the infection, which could then cause -cell damage ultimately leading to the development of type 1 diabetes.
ACKNOWLEDGMENTS
This study was supported by grants from Swedish Child Diabetes Foundation (Barndiabetesfonden), Swedish Research Council (K2002-72X-11242-08A and K2003-72X-14690-01A), Swedish Diabetes Association, Medical Research Fund of the County of stergtland, Samaritens’ Foundation, The Board of the Research Fellowship Award, The Strategic Area "Prevention of Diabetes and its Complications," the Juvenile Diabetes Research Foundation, the Academy of Finland, and the Sigrid Juselius Foundation.
We thank Gunilla Hallstrm, Eva Isacsson, and Ann-Marie Sandstrm for recruitment of healthy children and Cecilia Runnqvist for technical assistance.
FOOTNOTES
CVB4, Coxsackie virus B4; IFN-, -interferon; IL, interleukin; PBMC, peripheral blood mononuclear cell.
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