Immunization with an immunodominant self-peptide derived from glucose-6-phosphate isomerase induces arthritis in DBA/1 mice

Introduction  

T-helper (Th) lymphocytes are critically required for the pathogenesis of glucose-6-phosphate isomerase (G6PI)-induced arthritis, but neither the G6PI epitopes recognized by arthritogenic T cells nor their pathogenic effector functions have been fully elucidated to date. We aimed at identifying arthritogenic G6PI peptides.     

Induction of a B-cell-dependent chronic arthritis with glucose-6-phosphate isomerase

Antibodies specific for glucose-6-phosphate isomerase (G6PI) from T-cell receptor transgenic K/BxN mice are known to induce arthritis in mice, and immunization of DBA/1 mice with G6PI led to acute arthritis without permanent deformation of their joints. Because rheumatoid arthritis is a chronic disease, we set out to identify the capacity of G6PI to induce chronic arthritis in mice. Immunization with recombinant human G6PI induced a chronically active arthritis in mice with a C3H genomic background, whereas the DBA/1 background allowed only acute arthritis and the C57BL/10 background permitted no or very mild arthritis. The disease was associated with the major histocompatibility region sharing an allelic association similar to that of collagen-induced arthritis (i.e. q > p > r). All strains developed a strong antibody response to G6PI that correlated only in the C3H.NB strain with arthritis severity. Similarly, a weak response to type II collagen in a few mice was observed, which was associated with arthritis in C3H.NB mice. Mice on the C3H background also developed ankylosing spondylitis in the vertebrae of the tail. Both C3H.Q and B10.Q mice deficient for B cells were resistant to arthritis. We conclude that G6PI has the ability to induce a chronic arthritis, which is MHC associated and B-cell dependent. Thus, there are striking similarities between this and the collagen-induced arthritis model.     

Specific activities of 6-phosphogluconate dehydrogenase,glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase during Bufo bufo development

It has been suggested by some authors that during amphibian development, due to the higher glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity compared to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.43), 6-phosphogluconate could accumulate in the embryo tissues and regulate the channelling of glucose-6-phosphate into glycolysis. Here, on the base of the specific activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glucose-6-phosphate isomerase (EC 5.3.1.9) found in the embryos of Bufo bufo during development, it is discussed whether 6-phosphogluconate can accumulate and play a regulative role on glucose-6-phosphate metabolism in the anuran embryo.     

Immunization of mice with T cell-dependent antigens promotes IL-6 and TNF-alpha production in muscle cells

IL-6 and TNF-alpha are proinflammatory cytokines involved in various inflammatory or non-inflammatory disorders characterized by muscle wasting. While infiltrating leukocytes are known to be the major source of these cytokines, it is unclear whether muscle cells also contribute to local inflammation. In this study, we first showed that cultured muscle cells and naive mouse muscle tissue were capable of producing IL-6 and TNF-alpha. We demonstrated an increased expression of IL-6 and TNF-alpha on muscle sections of C57BL/6J mice immunized with acetylcholine receptor (AChR) in the complete Freund's adjuvant (CFA) or with CFA only. In the presence of IL-6 or TNF-alpha, cultured AChR-expressing mouse (G-8) and human (TE671) skeletal muscle cells showed significantly decreased alpha-bungarotoxin-binding sites as measured by cellular ELISA. Moreover, IL-6- or TNF-alpha-treated muscle cells displayed a considerable increase in apoptotic cell ratios. Collectively, this study suggests a direct role for these two cytokines in muscle cell destruction and a possibility of muscle cell damage via an autocrine fashion.     

葡萄糖-6-磷酸异构酶抗原对类风湿关节炎的诊断价值

目的:通过比较类风湿关节炎(Rheumatoid arthritis,RA)患者、非RA风湿病患者及健康对照者血清中葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase,GPI)抗原的阳性率来探讨GPI对RA的诊断意义,并探讨GPI,抗CCP抗体和RF联合应用对RA的诊断价值.方法:对110例RA患者、223例非RA风湿病患者和55例健康对照者共388份血清标本进行了检测.GPI抗原和抗CCP抗体采用ELISA方法,RF采用免疫比浊法定量检测.结果:RA组、非RA风湿病组和健康对照组血清中的GPI抗原阳性率分别为83.64%,30.04%和20.00%,RA组阳性率显著高于其它组(P＜0.01).GPI抗原对RAt诊断的敏感性和特异性分别为83.64%和71.58%.GPI和抗CCP抗体联合检测的敏感性和特异性分别为90.91%和71.22%,GPI和RF联合检测的敏感性和特异性分别为92.73%和 61.87%,如果三者同时检测,其敏感性和特异性分别为94.55%和60.43%.结论:RA患者的血清GPI阳性率明显高于其它自身免疫性疾病患者和健康对照者.GPI抗原时RA具有诊断价值,联合检测GPI、抗CCP抗体和RF可以提高RA诊断的敏感性.     

Molecular basis for the isozymes of bovine glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase exists as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis, isoelectric focusing, and ion-exchange chromatography. GPI from bovine heart was purified to homogeneity and each of the isozymes resolved. Four of the five isozymes were characterized with regard to their physical, chemical, and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118,000) and subunit (59,000) molecular weights but had different apparent pI values of 7.2, 7.0, 6.8, and 6.6. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes in either reaction direction. Structural analyses showed that the amino termini were blocked and the carboxyl terminal sequences were -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes at pH extremes, at high ionic strength, in the presence of denaturants, or upon exposure to proteases. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release of ammonia along with the spontaneous shift to more acidic isozymes indicates deamidation as the molecular basis for the formation of the acidic isozymes both in vivo and in vitro. The change in the peptide fragmentation patterns following cleavage by hydroxylamine further suggests that deamidation of specific Asn-Gly bonds accounts for the structural basis of the isozymes.     

Cell age dependent decay of human erythrocytes glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase shows a biphasic decay pattern during red blood cell aging, which is very fast during the first part of cell's life span in circulation. This decay is not due to accumulation of inactive enzyme molecules, as shown by immunological studies, but is accompanied by the formation of secondary isozymes (i.e., chemically modified forms). Electrophoretic and ion-exchange chromatographic experiments show that glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9) consists of only one isozymic form in young erythrocytes but is present in two components, with different electric charge, in mature and old cells. This secondary isozyme is more stable to heat treatment and is inactivated by IgG anti-glucose-6-phosphate isomerase with a lower affinity than the native isozyme. In vitro incubation of homogeneous human glucose-6-phosphate isomerase under conditions known to produce enzyme deamination does not reproduce the isozymic pattern found in erythrocytes, suggesting that one or more mechanisms other than those previously described to explain glucose-6-phosphate isomerase microheterogeneity occur in the human erythrocyte.     

Molecular basis of the isozymes of bovine glucose-6-phosphate isomerase

Glucose-6-phosphate isomerase occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). Most differentiated tissues have five distinct forms with apparent pI values of 7.2, 7.0, 6.8, 6.6, and 6.4. Young, mitotically active, cells of the intestinal mucosa and the epithelium of the eye lens show only the two more basic isozymes, while old cells in the cortex and nucleus of the eye lens accumulate the more acidic isozymes. All of the isozymes exhibit equal separation based on charge-to-mass ratio (PAGE) and charge (IEF), thus indicating only charge changes. The isozyme patterns are unchanged in the presence of reducing agents or protease inhibitors. Each isozyme was purified to homogeneity and shown to exhibit identical subunit molecular weights (59,000) on SDS-gel electrophoresis. Each of the isolated isozymes, when subjected to PAGE or IEF, exhibited a single band, indicating that the isozymes are not generated as a result of electrophoresis. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with properties identical to those found in vivo. The isozymes, thus, appear to be the result of spontaneous, postsynthetic modifications involving the addition of equal numbers of negative charges and are consistent with the deamidation of specific asparagine and/or glutamine residues.     

Altered peptide ligands inhibit arthritis induced by glucose-6-phosphate isomerase peptide

Introduction

Immunosuppressants, including anti-TNFα antibodies, have remarkable effects in rheumatoid arthritis; however, they increase infectious events. The present study was designed to examine the effects and immunological change of action of altered peptide ligands (APLs) on glucose-6-phosphate isomerase (GPI) peptide-induced arthritis.

Methods

DBA/1 mice were immunized with hGPI_325-339, and cells of draining lymph node (DLN) were stimulated with hGPI_325-339 to investigate the T-cell receptor (TCR) repertoire of antigen-specific CD4⁺ T cells by flow cytometry. Twenty types of APLs with one amino acid substitution at a TCR contact site of hGPI_325-339 were synthesized. CD4⁺ T cells primed with human GPI and antigen-presenting cells were co-cultured with each APL and cytokine production was measured by ELISA to identify antagonistic APLs. Antagonistic APLs were co-immunized with hGPI_325-339 to investigate whether arthritis could be antigen-specifically inhibited by APL. After co-immunization, DLN cells were stimulated with hGPI_325-339 or APL to investigate Th17 and regulatory T-cell population by flow cytometry, and anti-mouse GPI antibodies were measured by ELISA.

Results

Human GPI_325-339-specific Th17 cells showed predominant usage of TCRVβ8.1 8.2. Among the 20 synthesized APLs, four (APL 6; N329S, APL 7; N329T, APL 12; G332A, APL 13; G332V) significantly reduced IL-17 production by CD4⁺ T cells in the presence of hGPI_325-339. Co-immunization with each antagonistic APL markedly prevented the development of arthritis, especially APL 13 (G332V). Although co-immunization with APL did not affect the population of Th17 and regulatory T cells, the titers of anti-mouse GPI antibodies in mice co-immunized with APL were significantly lower than in those without APL.

Conclusions

We prepared antagonistic APLs that antigen-specifically inhibited the development of experimental arthritis. Understanding the inhibitory mechanisms of APLs may pave the way for the development of novel therapies for arthritis induced by autoimmune responses to ubiquitous antigens.     

Application of a rapid glucose-6-phosphate isomerase assay in white cabbage breeding

In the studied conditions of cellulose acetate electrophoresis only glucose-6-phosphate isomerase (PGI, EC 5.3.1.9) appeared to be a polymorphic isozyme. The PGI analysis of 23 cabbage (Brassica oleracea var. capitata) inbred lines, carried out in two years, allowed detecting off-type individuals in four lines. Seed contamination caused by sib-pollination was detected in 10 out of 19 F₁ hybrids. In five of them the contamination did not exceed 5 %, and in the remaining five, ranged from 27–52 %. Also homozygosity of 67 plants obtained through anther culture in vitro was confirmed. The results indicate that PGI electrophoresis in cellulose acetate matrix is very fast and can be useful in the assessment of genetic purity in cabbage breeding materials.     

Arthritogenic T cell epitope in glucose-6-phosphate isomerase-induced arthritis

Introduction  

Arthritis induced by immunisation with glucose-6-phosphate isomerase (GPI) in DBA/1 mice was proven to be T helper (Th) 17 dependent. We undertook this study to identify GPI-specific T cell epitopes in DBA/1 mice (H-2q) and investigate the mechanisms of arthritis generation.     

Inhibition of hexose transport by glucose in a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae

The rate of hexose transport was approximately 60% lower for both the high- and the low-affinity components of hexose uptake when a glucose-6-phosphate isomerase mutant ofSaccharomyces cerevisiae was preincubated with glucose, as compared with preincubation with water. Similarly theJ _max value of the high-affinity system of the mutant was 25–35 % of the correspondingJ _max value for normal cells incubated with glucose. Accumulation of glucose 6-phosphate or of some other metabolite, such as fructose 6-phosphate or trehalose, may be responsible for this striking inhibition.     

葡萄糖-6-磷酸-异构酶抗原对老年人类风湿性关节炎的诊断价值

目的:探讨葡萄糖-6-磷酸-异构酶抗原(GPI抗原)及类风湿因子(PF)的检测对老年人类风湿性关节炎(EORA)的诊断价值.方法:分别用ELISA法、速率散射比浊法检测49例RA患者和49例非RA的老年健康对照者血清GPI抗原和RF.运用四格表和受试者工作特征曲线评估两者对EORA诊断效能.结果:GPI抗原对EORA的敏感性、特异性和准确性分别为81.63%、93.87%及87.75%,阳性和阴性拟然比分别为13.3和0.19.RF诊断EORA的敏感性、特异性和准确性分别为73.46%、79.59%、76.53%,阳性和阴性拟然比分别为3.16和0.35.GPI抗原诊断准确性高于RF(P=0.013).GPI抗原诊断EORA的ROC曲线下面积(AUC)为0.913(95%CI,0.852-0.975),优于RF(P<0.001)结论:GPI抗原对EORA具有较好的敏感性和很高的特异性,在诊断EORA时较RF有更高的价值.     

Red blood cell dysfunction in septic glucose-6-phosphate dehydrogenase-deficient mice

Glucose-6-phosphate dehydrogenase (G-6-PDH) deficiency is the most common known human genetic polymorphism. This study tested the hypothesis that G-6-PDH deficiency worsens sepsis-induced erythrocyte dysfunction. Sepsis (24 h) was induced by cecal ligation and puncture in wild-type (WT) and G-6-PDH-deficient (G-6-PDH activity 15% of WT) mice. Erythrocyte responses were tested in whole blood as well as in subpopulations of circulating erythrocytes. Whereas erythrocyte deformability was similar in unchallenged deficient and WT animals, sepsis decreased erythrocyte deformability that was more pronounced in deficient than WT animals. Sepsis also resulted in anemia and hemolysis in deficient compared with WT animals. Mean corpuscular hemoglobin content and erythrocyte deformability decreased in younger erythrocyte subpopulations from septic deficient compared with WT animals. Sepsis decreased the reduced-to-oxidized glutathione ratio in erythrocytes from both deficient and WT animals; however, plasma glutathione increased more in deficient than in WT animals. Erythrocyte content of band 3 associated with the cytoskeleton was elevated in deficient compared with WT erythrocytes. The antioxidant N-acetyl-l-cysteine in vivo alleviated the sepsis-induced decrease in erythrocyte deformability in deficient animals compared with sham-operated control animals. This study demonstrates that a mild degree of G-6-PDH deficiency (comparable to the human class III G-6-PDH deficiencies) worsens erythrocyte dysfunction during sepsis. Increased erythrocyte rigidity and tendency for hemolysis together with alterations in band 3-spectrin interactions may contribute to the immunomodulatory effects of G-6-PDH deficiency observed after major trauma and infections in humans.