Azuma M, Motegi K, Aota K, Hayashi Y, Sato M
Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, Japan.
Lab Invest. 1997 Sep;77(3):269-80.
Our aim in the present study was to understand the mechanism whereby specific destruction of acinar but not ductal structure occurs in salivary glands in Sjögren's syndrome (SS). Thus, we examined the effects of cytokines including TNF-alpha and IL-1 beta on the proteolytic activity of cultured normal human salivary gland cell clones, because degradation of the basement membrane by proteolytic enzymes leads to the disruption of acinar or ductal structure in salivary glands. Simian virus 40 (SV40)-immortalized normal human salivary gland cell clones with ductal (NS-SV-DC) or acinar (NS-SV-AC) phenotype were treated either with TNF-alpha or IL-1 beta alone or with a combination of both, and then proteolytic activity was examined. Although cytokine-treated NS-SV-AC demonstrated high matrix metalloproteinase-2 (MMP-2) activity at both protein and mRNA levels, no remarkable increase in MMP-2 activity was detected in NS-SV-DC. Expression of tissue inhibitor of metalloproteinase-2 (TIMP-2), a specific inhibitor of MMP-2, was similarly inhibited by cytokines in these cell clones. Thus, the net balance estimated by MMP-2/TIMP-2 suggested enhanced proteolysis in cytokine-treated NS-SV-AC and, to a much lesser extent, in NS-SV-DC. To examine whether enhanced expression of MMP-2 occurred in acinar cells of SS salivary glands, we carried out an immunohistochemical study using SS salivary gland tissues. This study indicated that acinar cells adjacent to the lymphocytic infiltrate exhibited enhanced expression of MMP-2 compared with those distant from infiltrated lymphocytes or with those in normal salivary glands. By Northern blot analysis, NS-SV-DC and NS-SV-AC expressed receptor mRNA for both cytokines. The signal-dependent activation of the transcription factor NF-kappa B was observed only in NS-SV-AC. I kappa B-alpha, a specific inhibitor of NF-kappa B, was down-regulated by treatment with cytokines in NS-SV-AC. However, the expression of I kappa B-alpha protein was not detected in NS-SV-DC at the basal level. Treatment of NS-SV-AC with calpain inhibitor-I restored the expression of I kappa B-alpha protein in cytokine-treated cells, thus leading to the inhibition of NF-kappa B activation. Reverse transcriptase-PCR analysis confirmed that there was a marked reduction in I kappa B-alpha mRNA expression in NS-SV-DC as compared to that in NS-SV-AC. As such, it seems likely that there is a relationship between the activation of MMP-2 and the activity of NF-kappa B, and that the NF-kappa B/I kappa B-alpha complex is not a signal mediator involved in cytokine-induced suppression of TIMP-2. These observations, therefore, indicate that the divergent response to cytokines in each constituent cell of salivary gland may result in the histopathologic manifestation of SS.
我们在本研究中的目的是了解干燥综合征（SS）患者唾液腺中腺泡结构而非导管结构发生特异性破坏的机制。因此，我们研究了包括肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）在内的细胞因子对培养的正常人唾液腺细胞克隆蛋白水解活性的影响，因为蛋白水解酶对基底膜的降解会导致唾液腺中腺泡或导管结构的破坏。用TNF-α或IL-1β单独处理或两者联合处理具有导管（NS-SV-DC）或腺泡（NS-SV-AC）表型的猿猴病毒40（SV40）永生化正常人唾液腺细胞克隆，然后检测其蛋白水解活性。虽然经细胞因子处理的NS-SV-AC在蛋白质和mRNA水平均表现出高基质金属蛋白酶-2（MMP-2）活性，但在NS-SV-DC中未检测到MMP-2活性有明显增加。基质金属蛋白酶-2的特异性抑制剂金属蛋白酶组织抑制剂-2（TIMP-2）的表达在这些细胞克隆中同样受到细胞因子的抑制。因此，通过MMP-2/TIMP-2估算的净平衡表明，经细胞因子处理的NS-SV-AC中蛋白水解增强，而在NS-SV-DC中程度要小得多。为了研究SS唾液腺腺泡细胞中MMP-2表达是否增强，我们用SS唾液腺组织进行了免疫组织化学研究。该研究表明，与远离淋巴细胞浸润的腺泡细胞或正常唾液腺中的腺泡细胞相比，与淋巴细胞浸润相邻的腺泡细胞MMP-2表达增强。通过Northern印迹分析，NS-SV-DC和NS-SV-AC均表达这两种细胞因子的受体mRNA。仅在NS-SV-AC中观察到转录因子NF-κB的信号依赖性激活。在NS-SV-AC中，用细胞因子处理可下调NF-κB的特异性抑制剂IκB-α。然而，在基础水平未在NS-SV-DC中检测到IκB-α蛋白的表达。用钙蛋白酶抑制剂-I处理NS-SV-AC可恢复细胞因子处理细胞中IκB-α蛋白的表达，从而导致NF-κB激活的抑制。逆转录酶-PCR分析证实，与NS-SV-AC相比，NS-SV-DC中IκB-αmRNA表达明显降低。因此，MMP-2的激活与NF-κB的活性之间似乎存在关联，并且NF-κB/IκB-α复合物不是参与细胞因子诱导的TIMP-2抑制的信号介质。因此，这些观察结果表明，唾液腺各组成细胞对细胞因子的不同反应可能导致SS的组织病理学表现。
