Gebhard J R, Perry C M, Harkins S, Lane T, Mena I, Asensio V C, Campbell I L, Whitton J L
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA.
Am J Pathol. 1998 Aug;153(2):417-28. doi: 10.1016/S0002-9440(10)65585-X.
Viral myocarditis is remarkably common, being detected in approximately 1% of unselected asymptomatic individuals. Many cases are attributable to enteroviral infection, and in particular to coxsackievirus B3. The underlying pathogenesis is controversial, but most studies admit the important immunopathological role of infiltrating CD8+ (cytotoxic) T lymphocytes (CTLs). We have previously shown that CTLs play conflicting roles in coxsackievirus B (CVB) myocarditis; they assist in controlling virus replication, but also are instrumental in causing the extensive inflammatory disease, which often results in severe myocardial scarring. A role for perforin, the major CTL cytolytic protein, in CVB myocarditis has been suggested, but never proven. In the present study we use perforin knockout (PKO) mice to show that perforin plays a major role in CVB infection; in broad terms, perforin is important in immunopathology, but not in CVB clearance. For example, PKO mice are better able to withstand a normally lethal dose of CVB (100% survival of PKO mice compared with 90% death in +/+ littermates). In addition, PKO mice given a nonlethal dose of CVB develop only a mild myocarditis, whereas their perforin+ littermates have extensive myocardial lesions. The myocarditis in PKO mice resolves more quickly, and these mice show minimal histological sequelae; in contrast, late in disease the perforin+ mice develop severe myocardial fibrosis. PKO mice, despite lacking this major CTL effector function, can control the infection and eradicate the virus; growth kinetics and peak CVB titers are indistinguishable in PKO and perforin+ mice. Therefore, the immunopathological and antiviral effects of CTLs can be uncoupled by ablation of perforin; this offers a promising target for therapy of myocarditis. Furthermore, we evaluate the possible roles of apoptosis, and of chemokine expression, in CVB infection. In perforin+ mice, apoptotic cells are detected within the inflammatory infiltrate, whereas in their PKO counterparts, apoptotic myocyte nuclei are seen. Chemokine expression in both PKO and perforin+ mice precedes and parallels the course of myocarditis. Several chemokines are detectable earlier in PKO mice than in perforin+ mice, but PKO mice show reduced peak levels, and chemokine expression decays sooner. In particular, MIP-1alpha expression is barely detectable at any time point in PKO mice, but it is readily identified in perforin+ animals, peaking just before the time of maximal myocarditis; this is particularly interesting, given that MIP-1alpha knockout mice are resistant to CVB myocarditis, but remain able to control viral infection. Thus, the chemokine pathway offers a second route of intervention to diminish myocarditis and its sequelae, while permitting the host to eradicate the virus.
病毒性心肌炎非常常见，在约1%未经挑选的无症状个体中可检测到。许多病例归因于肠道病毒感染，尤其是柯萨奇病毒B3。其潜在发病机制存在争议，但大多数研究承认浸润的CD8 +（细胞毒性）T淋巴细胞（CTLs）的重要免疫病理作用。我们之前已经表明CTLs在柯萨奇病毒B（CVB）心肌炎中发挥着相互矛盾的作用；它们有助于控制病毒复制，但也在引发广泛的炎症性疾病中起作用，这种疾病常导致严重的心肌瘢痕形成。有人提出穿孔素（主要的CTL溶细胞蛋白）在CVB心肌炎中起作用，但从未得到证实。在本研究中，我们使用穿孔素基因敲除（PKO）小鼠来表明穿孔素在CVB感染中起主要作用；总体而言，穿孔素在免疫病理学中很重要，但在CVB清除中并非如此。例如，PKO小鼠更能耐受通常致死剂量的CVB（PKO小鼠100%存活，而其同窝野生型小鼠90%死亡）。此外，给予非致死剂量CVB的PKO小鼠仅发展为轻度心肌炎，而其穿孔素阳性的同窝小鼠有广泛的心肌损伤。PKO小鼠的心肌炎消退更快，并且这些小鼠显示出最小的组织学后遗症；相反，在疾病后期穿孔素阳性小鼠发展为严重的心肌纤维化。PKO小鼠尽管缺乏这种主要的CTL效应功能，但能够控制感染并清除病毒；PKO小鼠和穿孔素阳性小鼠的病毒生长动力学和CVB滴度峰值没有区别。因此，通过去除穿孔素可以使CTLs的免疫病理作用和抗病毒作用解偶联；这为心肌炎的治疗提供了一个有前景的靶点。此外，我们评估了凋亡以及趋化因子表达在CVB感染中的可能作用。在穿孔素阳性小鼠中，在炎性浸润内可检测到凋亡细胞，而在其PKO对应小鼠中，可见凋亡的心肌细胞核。PKO小鼠和穿孔素阳性小鼠中的趋化因子表达在心肌炎病程之前出现并与之平行。几种趋化因子在PKO小鼠中比在穿孔素阳性小鼠中更早可检测到，但PKO小鼠显示出峰值水平降低，并且趋化因子表达衰减更快。特别是，在PKO小鼠的任何时间点几乎都检测不到MIP - 1α表达，但在穿孔素阳性动物中很容易识别，在心肌炎最严重之前达到峰值；鉴于MIP - 1α基因敲除小鼠对CVB心肌炎有抗性，但仍能控制病毒感染，这一点特别有趣。因此，趋化因子途径提供了第二条干预途径，以减轻心肌炎及其后遗症，同时允许宿主清除病毒。
