Martignat L, Elmansour A, Audrain M, Julien J F, Charbonnel B, Saï P
Laboratory of Cellular and Molecular Immuno-Endocrinology, University School of Medicine, Nantes, France.
J Autoimmun. 1995 Aug;8(4):465-82. doi: 10.1016/0896-8411(95)90002-0.
Diabetes-prone NOD mice of both sexes and at different ages were compared to control mice with regard to the level of pancreatic expression of certain autoantigens: antigens for islet cell antibodies (ICA antigens) and glutamic acid decarboxylase (GAD) 67 kDa. ICA antigens were compared by immunofluorescence using serial dilutions of ICA positive human sera so that differences of fluorescence intensity were due only to differences in amounts of antigen. Pancreatic GAD67 mRNAs were compared by polymerase chain reaction followed by Southern hybridization with 32P-probes and densitometry of autoradiographic bands. GAD67 product and gamma-aminobutyric acid (GABA) were compared by immunoperoxidase staining. As compared to BALB/c, C57BL6, Swiss, or F1 mice, NOD mice displayed higher ICA antigen levels (P < 0.01) both before and after insulitis onset (at 7 days, 15 days, 1 month, 2 months). ICA antigens were scarcely detectable by the first day of life, and increased with age from 7 days to 2 months (P < 0.01; n = 10 for each strain and at each age). Both before and after insulitis onset (4 days, 7 days, 15 days, 1 month, 2 months), amounts of GAD67 mRNAs were higher (P < 0.01) in NOD mice than in BALB/c mice (n = 8 for each age in each strain). This was already noted in foetuses on Day 18 of gestation (n = 8). After birth, amounts of GAD67 mRNAs increased up to 1 month (P < 0.04) and then decreased in older mice. The staining intensity of pancreatic sections with antisera against either GAD67 or GABA was higher (P < 0.04) in islets from NOD mice than in those from control mice. Whatever the age, no significant difference was noted between female and male NOD mice with regard to ICA antigens or GAD67. The expression of ICA antigens and GAD67 was intermediate in NOD x BALB/c F1 mice when compared to parental strains. We conclude that whatever the age, NOD mice strongly express ICA antigens and GAD67. This peculiarity was detectable very early, in embryos for GAD67 but after birth for ICA antigens. The timing of antigen expression may underlie the development of diabetes. The antigen overexpression might affect early completion of self-tolerance and, during later life, might also contribute to amplification of the anti-beta cell autoimmune response due to the existence of more targets for effector mechanisms.
将不同年龄的糖尿病易感NOD雌雄小鼠与对照小鼠在某些自身抗原的胰腺表达水平方面进行比较：胰岛细胞抗体（ICA抗原）和67 kDa谷氨酸脱羧酶（GAD）的抗原。通过使用ICA阳性人血清的系列稀释液进行免疫荧光来比较ICA抗原，以使荧光强度的差异仅归因于抗原量的差异。通过聚合酶链反应，随后用32P探针进行Southern杂交以及对放射自显影片条带进行光密度测定来比较胰腺GAD67 mRNA。通过免疫过氧化物酶染色比较GAD67产物和γ-氨基丁酸（GABA）。与BALB/c、C57BL6、瑞士或F1小鼠相比，NOD小鼠在胰岛炎发作之前和之后（7天、15天、1个月、2个月）均显示出更高的ICA抗原水平（P < 0.01）。在出生第一天几乎检测不到ICA抗原，并且从7天到2个月随年龄增加（P < 0.01；每个品系和每个年龄n = 10）。在胰岛炎发作之前和之后（4天、7天、15天、1个月、2个月），NOD小鼠中GAD67 mRNA的量均高于BALB/c小鼠（每个品系每个年龄n = 8）。在妊娠第18天的胎儿中就已观察到这种情况（n = 8）。出生后，GAD67 mRNA的量增加至1个月（P < 0.04），然后在老年小鼠中减少。用抗GAD67或抗GABA抗血清对胰腺切片进行染色，NOD小鼠胰岛中的染色强度高于对照小鼠（P < 0.04）。无论年龄如何，雌性和雄性NOD小鼠在ICA抗原或GAD67方面均未观察到显著差异。与亲本品系相比，NOD×BALB/c F1小鼠中ICA抗原和GAD67的表达处于中间水平。我们得出结论，无论年龄如何，NOD小鼠均强烈表达ICA抗原和GAD67。这种特性在很早的时候就可以检测到，对于GAD67在胚胎期，但对于ICA抗原在出生后。抗原的表达时间可能是糖尿病发生的基础。抗原的过度表达可能会影响自身耐受性的早期完成，并且在晚年，由于存在更多的效应机制靶点，也可能有助于抗β细胞自身免疫反应的放大。
