SARS的免疫致病的分子機轉---總計畫與子計畫一：干擾素-γ細胞激素風暴在SARS疾病的角色

行政院國家科學委員會專題研究計畫 成果報告

（總計畫與子計畫一）干擾素-γ細胞激素風暴在 SARS 疾病

的角色

計畫類別： 整合型計畫 計畫編號： NSC93-2751-B-006-002-Y 執行期間： 93 年 07 月 01 日至 94 年 10 月 31 日 執行單位： 國立成功大學微生物免疫學研究所 計畫主持人： 黎煥耀 報告類型： 完整報告 處理方式： 本計畫可公開查詢

中 華 民 國 94 年 11 月 22 日

行政院國家科學委員會補助專題研究計畫

█ 成 果 報 告

□期中進度報告

（總計畫與子計畫一）

干擾素-γ 細胞激素風暴在 SARS 疾病的角色

計畫類別：□ 個別型計畫

█ 整合型計畫

計畫編號：NSC93－2751－B006－002Y

執行期間：

93 年

7 月

1

日至

94 年

10 月

31 日

計畫主持人：黎煥耀

共同主持人：

計畫參與人員：

李宛霖

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█完整報告

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執行單位：

國立成功大學微生物免疫學研究所

中

華

民

國

94

年

11 月 22 日

SARS 的免疫致病的分子機轉

整合型計劃摘要

嚴重急性呼吸道微候群(SARS)是由新的冠狀病毒(SARS-CoV)感染引起的疾病，它己經 嚴重地威脅到國人的健康與生活。SARS 病人的致死率高達 10-15%，主要受影響的器官是 肺臟，在病毒急性感染上，宿主組織的傷害可以是病毒直接的傷害或免疫反應引起的。病 毒引起的細胞病變，在感染早期主要是病毒複製引起的。但在感染後期，當適應性的免疫 系統啟動後，病毒的清除通常伴随著免疫發炎反應，這些免疫反應難免會造成宿主細胞的 傷害，病毒量越多，免疫反應越強，宿主細胞的傷害越大。以 SARS-CoV 感染引起肺部的 病變為例，在感染早期肺部細胞膜呈透明，有水腫現象，之後有淋巴細胞浸潤，肺泡有肺 上皮細胞脫落，肺上皮細胞形成空泡或融合成巨大細胞，顯示肺部細胞受到刺激，有活化 及死亡的現象。我們之前的 SARS 整合型計劃研究發現有一個”干擾素-的細胞激素風暴”

存在於 SARS-CoV 感染早期，SARS 病患急性期的血清中有高量的 IFN-, IL-18, TGF-, IL-6，但沒有 TNF-, IL-2, IL-4, IL-10, IL-13 or TNFRI，另外趨化激素 IP-10, MCP-1, MIG 和

IL-8 也很高，這指出干擾素-相關的細胞激素風暴確實發生。另外發現 SARS-CoV 感染的 臨床病人血清中可偵測到抗肺上皮細胞自體抗體的存在，並證實抗體會對肺上皮細胞造成 傷害。有趣的是，自體抗體會與 SARS-CoV 棘蛋白 (Spike, S) 產生反應，其辨識位置在 S2 domain。我們提出二個新理論，分別是干擾素-的細胞激素風暴參與早期病部病變，而自 體免疫參與後期的發炎反應。我們要在此延續計劃釐清及証明這二個理論在 SARS 疾病的 角色，並解釋其分子致病機轉。子計劃一是繼續探討”干擾素-細胞激素風暴在 SARS 疾病 的角色”，瞭解干擾素-相關的細胞激素造成肺上皮細胞凋亡及纖維母細胞生長的分子機 轉，探討為何 SARS 有干擾素-相關的細胞激素的不平衡調控，以及血管緊縮素 II 及 ACE2 的角色。子計劃二是鑑定更多的自體抗原，瞭解其對細胞的生理作用，並進一步確認其抗 原決定位和 SARS-CoV 棘蛋白構造上的關連性。子計劃三是利用自體抗體辨認 SARS-CoV 的 spike 蛋白的 S2 區域上的抗原決定位，利用肺部特異性的推動子，包括 Clara cell secretory protein 和表面張力素蛋白 C，製造 S2 的基因轉殖小鼠，表現 Spike 蛋白基因的重組腺病毒， 感染基因轉殖小鼠，以期藉病毒的傷害打破免疫耐受性，引發呼吸道細胞免疫傷害，比較 轉殖小鼠呼吸道的病理變化、細胞激素、趨化素及免疫細胞種類，以驗証我們的理論。子 計劃四是製造小鼠的單株抗體，包括中和性抗體及上述致病性抗體，對於有中和作用的抗 體將利用遺傳工程改造成人類化的單株抗體，作為治療病毒感染的特異性製劑。本研究的 完成將可釐清干擾素-的細胞激素風暴和自體抗體在 SARS 中的致病角色，此免疫致病機 制的瞭解不僅對擬出更好的治療策略或研發安全有效的 SARS 疫苗都可以提供很重要的訊 息。 子計劃一的成果報告如下，另外附件一為子計劃二的成果報告，附件二為子計劃三的 成果報告。

子計劃一：干擾素-細胞激素風暴在 SARS 疾病的角色

Abstract

Recent work carried out in our laboratory showed the existence of a cytokine storm in SARS patients, dominated by Th1-type mediators. We thus hypothesized that IFN-γmay play amajor role in the pathology by triggering an immune-mediated alveolar damage. As we assessed or re-assessed some effects of IFN-γon anumberofhuman lung epithelialand fibroblastcelllines, chosen for their wide use in the literature, we found that alveolar epithelial cells were more

sensitive to IFN-in terms of proliferation inhibition and enhancement of Fas-mediated apoptosis. While similar effects were obtained on fibroblasts, concentrations of IFN-γ4-8 folds greater were required. In addition, both epithelial and fibroblastic cell lines were able to secrete large quantities of T cell-targeting chemokines, similar to the ones detected in SARS patients. Based on the clinical data collected previously, the available literature and our in vitro

experimentation, we propose that IFN-γmay beresponsibleforacutelung injury in thelatephase of the SARS pathology.

中文摘要

SARS-CoV 感染早期，SARS 病患急性期的血清中有高量的 IFN-,IL-18, TGF-,IL-6， 但沒有 TNF-, IL-2, IL-4, IL-10, IL-13 or TNFRI，另外趨化激素 IP-10, MCP-1, MIG 和 IL-8

也很高，這指出干擾素-相關的細胞激素風暴確實發生。利用人類肺上皮細胞 A549 為細胞 模式，發現干擾素-會透過抑制細胞週期、增加 Fas 表達，產生趨化激素，吸引活化 T 細 胞利用 Fas-FasL 作用造成 A549 的細胞凋亡，此種現象不見於人類肺纖維母細胞。此計劃 的特定目標是：(一)瞭解干擾素-相關的細胞激素造成肺上皮細胞凋亡的分子機轉，(二) 瞭 解干擾素-相關的細胞激素造成纖維母細胞生長的分子機轉。在 SARS 疾病中有干擾素- 細胞激素風暴產生的原因，它牽涉到 SARS-CoV 引起一些調控機制的失常，使得 T 細胞極 度的偏向 Th1，造成干擾素-過度的產生，發炎不受控制，並解釋干擾素-相關的細胞激 素造成肺上皮細胞凋亡及纖維母細胞生長的分子機轉。它和第二子計劃 SARS 因分子模擬 引起自體抗體反應，可以相互配合共同釐清 SARS 早期和後期會有嚴重發炎，引起肺部細 胞病變的致病機制。此免疫致病機制的瞭解不僅對擬出更好的治療策略或研發安全有效的 SARS 疫苗都可以提供很重要的訊息。 Introduction

In the year 2003, a new emerging infectious disease, termed severe acute respiratory disease (SARS), swept across the world, resulting in the death of 774 people [1]. With no precedent, the scientific community was able to identify the causative agent soon after its emergence [2, 3, 4]. Since then, significant advances were made in understanding this disease and less than 2 years later, vaccine candidates are already under trial [5]. However, the mechanism(s) underlying the severity of the respiratory distress remain(s) unexplained. Indeed, hypotheses are yet to be made to explain the acute lung injury (ALI) and respiratory failure observed in the severe cases, while in the presence of a declining viral load.

cases, patients also developed an atypical form of pneumonia, with acute respiratory distress (ARDS) as result of lung damage, characterized by infiltrates on chest radiography [4, 6, 7]. Amongst the changes observed in the lungs of SARS patients were epithelial cells proliferation and desquamation, hyaline membrane formation and cell infiltration (lymphocytes, neutrophils and monocytes) during the early stage of the disease, while increased fibrosis and multinucleated epithelial giant cells formation were seen at a later stage [8].

The existence of an abnormally excessive inflammatory response in the lungs has been suggested to explain the development of ALI in SARS. Indeed, patients still manifested lung injury at a time when the viral load was dropping, in support of the immune nature of the lung damage [9]. Our investigations, and the work of Wong et al., support this hypothesis as we determined the presence of several cytokines and chemokines at high concentrations in the plasma of RT-PCR-confirmed SARS patients[10, 11]. Especially, Th1-type cytokine IFN-and other related cytokine (IL-18) and chemokines (MIG, IP-10 and MCP-1) were found at unusually high levels. This seemed to place IFN-at the center of any cytokine-induced immune response.

Evidence exists to support the importance of the destruction of the alveolar epithelium in the development of conventional ARDS [12, 13]. The presence of soluble Fas ligand (FasL) was reported in ARDS patients and an agonist antibody showed to be able to induce alveolar epithelial cell injury and lung inflammation in mice [14, 15]. Although the presence of soluble FasL has not be demonstrated in SARS patients, immune cells capable of expressing membrane-form FasL were found infiltrating the lungs, and ARDS observed in late-stage SARS patients resembled other late-stage ARDS, suggesting the potential role of the Fas/FasL system in the development of ALI in SARS [7, 8, 16].

Previous work by other groups has led to conclude that human lung epithelial cells, but not lung fibroblasts were sensitive to Fas-mediated apoptosis, suggesting that phenotypic differences between these two cell types may contribute to the development of fibrosis, by rendering one type prompt to cellular damage while protecting the other [17-19]. As IFN-γseemsto play arolein SARS, we set out to compare some effects of IFN-γon anumberofhuman lung fibroblastand epithelial cell lines (chosen for their use in numerous publications as models of their primary counterparts) in order to extrapolate how these cell types co-existing in vivo may react to the presence of IFN-γ.Forthatpurpose,identicalcultureconditionswereused forallcelllines.We report here that in substance both cell types were affected similarly, i.e. susceptible to

Fas-mediated apoptosis after IFN-γstimulation,though to differentdegrees:fibroblasticlines required much larger amounts to become sensitive. These observations led us to hypothesize that specific epithelial sensitivity to IFN-γmay bethebasisunderlying the development of lung injury in the late phase of SARS.

Results

Increased Sensitivity of Epithelial Cells to Cell Proliferation Inhibition by IFN-γ

All four cell lines –A549, BEAS-2B, MRC-5 and HFL-1–were cultured in the presence of varying concentrations of IFN-γ,for24,48 or72h.Theculturein such condition resulted in an inhibition of proliferation that ranged from growth downregulation (BEAS-2B, MRC-5 and

HFL-1) to complete growth arrest (A549: p > 0.05 for 48h vs. 72h IFN-γ-treated groups), as shown in Figure 1. However, sensitivity varied among cell lines. Indeed, dose-dependent

experiments determined that epithelial cell lines were more susceptible to the effect of IFN-γthan fibroblast cell lines (data not shown). Two hundred and fifty IU.mL-1of IFN- γin A549 and BEAS-2B allowed observing very significant differences in growth, while 62.5 IU.mL-1was sufficient to detect some effects. On the contrary, MRC-5 and HFL-1 required high doses of IFN-γ:2000 IU.mL-1was necessary to observe a significant growth inhibition.

Delay and Arrest of the Cell Cycle in A549 Cells

As A549 was the only cell line whose growth was completely arrested within the

time-course of our first experiment, we decided to investigate further the effects of IFN-γon these cells. The only moderate induction of apoptosis by IFN-γ–in the range of 8-10% of the cells, as determined by annexin V/PI and active caspase-8 staining (data not shown)–was ruled out as the possible explanation. Cell cycle analysis was on the other hand able to show that, not only IFN-γwasultimately ableto dose-dependently arrest the cells at the G0/G1phase (Figures

1E and 1F), it also could first delay their progression through the S phase, as shown by the accumulation of cells in that phase. This latter effect was dose-dependent (Figure 1G). The multiple ability of IFN-γto inhibitthecellsfrom progressing through theircycleprobably explains the early growth arrest observed in A549.

IFN-γEnhanced FasExpression Indifferently in allCellLines

We next investigated the expression of Fas by epithelial and fibroblast cell lines. All cells seemed to express CD95, moderately –BEAS-2B–or more significantly –A549, HFL-1 and MRC-5–(Figure 2). Interestingly, in the presence of IFN-γ,Fasexpression wasupregulated on the surface of cells, including fibroblast cell lines. However, epithelial cells were once again more sensitive to the IFN-γstimulus,aslow concentrationsenabled Fasexpression upregulation (250 IU.mL-1), while fibroblast cell lines only responded to high concentrations (data not shown). Note also that in the particular case of MRC-5, surface Fas expression upregulation was only transient, and IFN-γceased to havean enhancing effect72h afteraddition (Figure2K).

IFN-γEnhanced Fas-Mediated Apoptosis of Epithelial Cells and Fibroblasts

Despite the existence of a constitutive expression of Fas on the surface of all 4 cell lines, triggering of apoptosis by the sole addition of a cross-linking activating anti-Fas antibody was only marginal (Figure 3). Induction of apoptosis was only significant in A549, at 36 and 60h after addition, and in MRC-5 at 12h, but concerned at any time less than 30% of the viable cell

population (Figures 3B and 3D). When IFN-γwasadded to theculturemedium,12h beforethe addition of the anti-Fas antibody, a significant enhancement of apoptosis could be observed in all cell types, although at different times and different extents. In BEAS-2B, an enhanced induction of apoptosis was only observable 72h after addition of IFN-γ(Figure3A).Moreover,this induction was to a lesser degree than in A549, and required the addition of a larger amount of anti-Fasantibody.Attheconcentration 0.1 μg.mL-1, anti-Fas antibody was unsuccessful at

inducing apoptosis, even after IFN-γpre-treatment (data not shown). In A549, IFN-γ-enhanced Fas-mediated apoptosis developed rapidly, as it was already significant by 24h (Figure 3B), and successfully, as it extended to the whole monolayer, causing all cells to be dead by 96h (data not shown). Fibroblasts were also susceptible to the enhancement of Fas-mediated apoptosis by IFN-γ.Likein BEAS-2B, apoptosis appeared enhanced only 72h after addition of IFN-γin HFL-1 (Figure 3C). However, in MRC-5 significant enhancement of Fas-mediated apoptosis was readily observed by 24h, but declined thereafter to become inexistent by 72h (Figure 3D). In this latter cell line, the enhancement of Fas-mediated apoptosis by IFN-γfollowed accurately the surface Fas expression profile of MRC-5 cells stimulated by IFN-γ.

Relevance of anti-Fas Antibody-Mediated Apoptosis to Cell-to-Cell Interaction

While the importance of the Fas/FasL system in the development of ARDS is supported by previous work [12-15], the lack of evidence of the presence of soluble FasL in SARS patients having developed ARDS required us to demonstrate the relevance for cell-to-cell interaction of work that used an anti-Fas activating antibody. A549 cells pre-treated with IFN-γwerethus co-cultured with a lymphocytic T cell line, Jurkat, which had previously been activated in order to increase the expression of surface FasL (data not shown). Stimulation by IFN-γincreased the susceptibility of A549 cells to apoptosis induced by contact with Jurkat cells (Figure 4). When the ratio of available Jurkat cells to A549 cells was increased 3-fold, however, the number of

unstimulated A549 cells undergoing apoptosis did not significantly increase. But it more than doubled when A549 cells had been stimulated by IFN-γ,clearly indicating theroleofFasin the mediation of this induction of apoptosis by cell contact, and the important role played by IFN-γin the enhancement of cellular damage.

Jurkat cells are T-lymphocyte-like cells of the kind that may be found infiltrating the lungs in SARS patients [8]. T cells are able to infiltrate tissues in response to the release of

chemoattractant messengers. It was thus interesting to notice that both A549 and MRC-5 could release, in response to IFN-γ,large amounts of the chemokines that were detected in the blood of SARS patients [8], including Mig and IP-10, some well-known chemoattractants of activated T cells (Table 1).

Discussion

A characteristic feature of patients infected with SARS-associated coronavirus is the

presence of lung consolidation, lung injury in the form of diffuse alveolar damage and fibrosis in more severe cases. In the latter cases, the development of the lung injury has led to respiratory failure and the need for respiratory assistance at an intensive care unit. Respiratory distress has appeared to be the cause of the majority of deaths related to SARS. One of the hypotheses emerging to explain the severity of the lung inflammation and damage is the development of a “cytokinestorm”, as result of the infection, leading to an unbalanced inflammatory response. Cytokines profiling of RT-PCR-confirmed SARS patients has revealed the presence of large amounts of IFN-and IFN--related cytokineand chemokinesin patients’blood,and theabsence of Th2-type cytokines [10]. IFN-is a cytokine secreted mainly, but not only, by activated and

cytotoxic T cells, as part of the immune response to eliminate virus-infected cells. Infiltration of immune cells, including lymphocytes, was observed in the lungs of SARS patients [8]. We

therefore hypothesized that IFN-γmay beamajorcontributorto theALI,foritisknown to cause cell proliferation inhibition and apoptosis. We used an array of cell lines to study how IFN-γ could be involved in selective epithelial damage and fibrosis.

The present report showed that human lung epithelial cells (A549 or BEAS-2B) and

fibroblasts (HFL-1 or MRC-5) were affected similarly, but not identically, by IFN-γin termsof Fas-mediated induction of apoptosis. This is in some ways in contradiction with previously available publications. Indeed, Tanaka et al. have reported that they found that human lung fibroblasts were resistant to Fas-mediated apoptosis, by opposition to epithelial cells that are known to be susceptible to Fas cross-linkage [17-19]. Although we do not know the actual

concentration in International Units of bioactive IFN-γused by theseauthorsto treatfibroblasts, it seems that dose of IFN-γ,duration ofpre-treatment, time-point for detection, or a combination of these is responsible for this apparent resistance to apoptosis. In our work, fibroblasts (HFL-1) required an exposure to high concentrations of IFN-γto observeany enhancement of surface Fas expression and Fas-mediated apoptosis. MRC-5, the second fibroblast cell line used here, was also sensitive to IFN-γ,although foronly alimited period.Thiscould belinked to thephenotypic differences between the two cell lines, MRC-5 possessing some characteristics of myofibroblasts [18]. The possibility that fibroblasts differentiation into myofibroblasts protects them from IFN-γ-enhanced Fas-mediated apoptosis is currently under investigation in our laboratory.

Whilst the upregulation of Fas expression on the cell surface may not be the only

mechanism underlying the enhanced induction of apoptosis in A549 cells treated with IFN-γand anti-Fas antibody, a relationship between increased expression and increased apoptosis seems to exist when timing is considered. Indeed, although these cells naturally express Fas proteins on their surface, the density seems insufficient to lead to a major induction of apoptosis (Figure 3B). However, when IFN-γincreased Fasexpression by 48 or72h(Figure 2D or 2E), more than 50% of the cells underwent apoptosis (Figure 3B). A similar, yet more convincing, observation could be made with MRC-5 cells. These cells could express Fas, but anti-Fas antibody alone was not able to provoke a clear increase in the number of cells undergoing apoptosis (Figure 3B). When Fas expression was enhanced by IFN-γ,so wasapoptosis,by 24h (Figures2Iand 3B).When the Fas expression returned to its basal level by 72h (Figure 2K), so had apoptosis (Figure 3B). The presence of a relationship in time thus seems to support the existence of a correlation between the increased density of Fas protein on the cell surface and the sensitivity of the cell to undergo apoptosis mediated by Fas.

The ability of IFN-γto enhanceFas-mediated apoptosis of lung fibroblasts is however relative, as the amount of cytokine required do not appear to correspond to the levels observed in SARS patients, even though bioactivity of the cytokines detected by ELISA and CBA kits was not determined [10]. In any way, this does not affect the fact that lung epithelial cells are clearly highly sensitive to IFN-γ,whilefibroblastsareremotely affected.IFN-γhasthustheability to prime the destruction of the lung epithelium: indeed, in addition it stimulates epithelial cells, like neighboring fibroblasts, to release significant amounts of chemokines that are known to attract

the migration of immune cells. Infiltrating lymphocytes were observed in autopsied cases of SARS [8]. It seems thus plausible that the presence of high amounts of IFN-γ,T cell chemoattractants and infiltrating lymphocytes in the lungs may have led to selective alveolar epithelial damage, as part of an innate immune response, in SARS patients.

With the exception of IL-8, which is not stimulated by IFN-γin neitherA549 norMRC-5, and MIG, IFN-γwasableto stimulatethereleaseofchemokinesfrom epithelialcells(A549)at lower concentrations than the one required by fibroblasts (MRC-5). MIG, on the other hand, was more readily produced by fibroblasts, although epithelial cells were also sensitive to the IFN-γ stimulus. This particularity could be the result of the dual effect of IFN-γon epithelialcells:while it stimulates the expression of proteins, it also inhibits the cell growth. The latter effect would undoubtedly downsize any stimulated increase in protein expression. Overall, the data point towards an increased readiness of epithelial cells to respond to IFN-γby thereleaseof chemokines, in comparison to fibroblasts that require larger concentration of the cytokine. Ultimately, the experiments investigating the production of chemokines were designed to determine if IFN-γcould furthercontributeto alung immunopathology by favouring

chemoattraction of the necessary actors (i.e. T cells) for an immune-mediated cellular damage. Whilst IFN-γisessentially regarded asan anti-fibrotic agent, evidence exists to suggest that this cytokine may play an important role in the development of the lung injury that precedes

fibrosis [21]. Indeed, the absence of IFN-γresulted in reduced inflammation in variousmodelsof lung injury using IFN-γ-knockout mice [22-24]. Several studies have also suggested that

lymphocytes may play a role in pulmonary injury in rodents. For example, T-cell depletion by anti-CD3 antibody treatment has attenuated pulmonary fibrosis and increased survival in mice treated with bleomycin [25]. A recent publication by Glass et al. indicates that, in mice, the SARS-associated coronavirus successfully replicated and induced an increase in various

proinflammatory chemokines, in the lungs, including MIG and IP-10 [26]. However, it failed to stimulate the production of cytokines or the infiltration of leukocytes. In this model, no obvious changes in pulmonary function and little signs of lung injury could be observed. Moreover, the focal necrosis and bronchiolar epithelial damage characterized in SARS-associated coronavirus infected B6 mice were not observed in RAG1-/-mice, which lack T and B lymphocytes. Such observations tend to support the fact that in SARS, cytokines and T cells may be important to the development of ALI.

Although the immune response is thought to contribute significantly to ALI and respiratory distress in SARS patients, it is not clear how a response dominated by Th1-type biological

response modifiers may lead to the development of fibrosis. Here we show lung epithelial cells to be more responsive to IFN-γ-induced damage than fibroblasts. Thus, we propose that the

Th1-dominant immune-mediated cell death may actually favor the damage of alveolar epithelial cells over the fibroblast layer, leaving the latter one relatively intact. In vivo, this would translate into the destruction of the epithelial parenchyma, a basis for the stimulation of repair mechanisms involving fibroblasts, relatively unaffected by the immune response and available for a

proliferative response (Figure 5). The association of epithelial destruction and fibrosis has been suggested in other situations, and we are currently investigating the possibility that

immune-mediated epithelial destruction may directly stimulate proliferation and/or differentiation of fibroblasts [27, 28]. To conclude, these findings are relevant to the understanding of the roles of IFN-γand theFas/FasL system in ALI,in which alveolarepithelialdamageoccursprimarily. We suggest that IFN-γmay play an important role in the development of an immune mechanistic that will lead to ALI, fibrosis and ARDS in SARS patients.

Materials and Methods Cell Lines

A549, a human lung alveolar type II epithelial carcinoma cell line, BEAS-2B, a human lung bronchiolar epithelial cell line, HFL-1 and MRC-5, human embryonic lung fibroblast cell lines, and Jurkat, a human leukemic T cell lymphoblast cell line, were all obtained from American Type Culture Collection (Rockville, MD). They were cultured under standard conditions in the

following medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine and 1% (v/v) penicillin-streptomycin solution 100X: DMEM for A549 and BEAS-2B, MEM

Eagle-Earle’sbalanced saltsolution with non-essential amino acids for MRC-5 and HFL-1, and RPMI 1640 medium for Jurkat. Adherent cells were passaged at 80-90% confluence using 0.25% (w/v) trypsin/1 mM EDTA, while Jurkat cells were subcultured every 3 days. For experimental purpose, A549, BEAS-2B, HFL-1 and MRC-5 were seeded at respectively 1x106, 1x106, 0.8x105 and 2.5x105cells per 60 mm culture dish, in 3 ml of corresponding growth medium. These seeding densities permitted to reach cell confluence within 72h for each cell type.

Cytokine, Antibodies and Flow Cytometry

Recombinant human IFN-was purchased from PreproTech EC (London, UK). Functional grade murine anti-human CD95 (Fas) monoclonal antibody (clone EOS9.1) was obtained

eBioscience (San Diego, CA). Anti-hepatitis B surface antigen culture supernatant from a murine IgM hybridoma was used as the isotype control. Goat anti-mouse IgG (H+L) conjugated to FITC served as secondary antibody (Jackson ImmunoResearch, West Grove, PA). Anti-human CD3 coupled to PerCP and its isotype control murine IgG1κwere purchased from Becton-Dickinson

(Mountain View, CA). All flow cytometric data was acquired on a FACSCalibur run with CellQuest (Becton-Dickinson), and later re-analyzed with WinMDI version 2.8 (J. Trotter).

Cell Number and Apoptosis Staining

Cells were harvested 24, 48, 72 or 96h after addition of IFN-to the culture medium, and counted with a hemacytometer, using eosin Y as an exclusion dye, before any further analysis. Apoptotic cells were stained with an annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BioVision Research, Mountain View, CA) and analyzed by flow cytometry. In some cases, the presence of active form caspase-8 was also detected using CaspGLOW Fluorescein active caspase-8 staining kit from BioVision Research, and analyzed by flow cytometry.

Cell Cycle Analysis

A549 cells were cultured in serum-free culture medium, +37 ºC, 5% CO2, for 24 or 36h, for

cell cycle synchronization. The culture supernatant was then replaced by complete medium and IFN-added to the medium. Cells were harvested 12, 18, 24, 36 or 48h later and fixed with cold 70% ethanol. The DNA content was then stained using PI and analyzed by flow cytometry with a

FACSCan (Becton-Dickinson). Cell cycle repartition was determined by analysis of the data with ModFit LT (Verity Software House, Topsham, ME).

A549/Jurkat Cells Co-culture

Resting Jurkat cells were cultured for 24 hours in 100 mm culture plates in complete

medium containing 100 ng/mL phorbol 12-myristate 13 acetate and 500 ng/mL ionomycin. At the same time, A549 cells were seeded and cultured for 24h. IFN-(250 IU/mL) was added to A549 culture 12h after seeding. After 24h of incubation, Jurkat cells were pelleted (400 g, 5 min) and resuspended with culture supernatant from individual A549 cell cultures. The Jurkat cell suspension was then transferred to A549 cell culture plates from which the culture supernatant originated. Finally whole cell cultures were harvested 24h later, stained with annexin V-FITC/PI and anti-human CD3 antibody.

Chemokines Secretion

Presence of chemokines MIG, MCP-1, IP-10, RANTES and IL-8, and their concentration, were determined using a human chemokines cytometric bead array (Becton-Dickinson). Samples analyzed were culture supernatants from cells incubated for 48h with IFN-.Each sample was centrifuged after collection (400 g, 5 min), and only the supernatant fraction was kept for assay.

Statistical Analysis

All experiments were carried out in triplicates and the data is expressed as mean ± standard error of the mean. Prism (GraphPad Software, San Diego, CA) was used to carry out

Mann-Whitney t test or one-way analysis of variance (ANOVA) for comparisons. The

Newman-Keuls multiple comparisons test was applied as a post-test in ANOVA for individual comparisons. In all analyses, statistical significance was defined as p < 0.05.

Acknowledgements

This work was supported by the SARS Research Project grant from the National Science Council, Taiwan.

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coronavirus-associated SARS pneumonia: a prospective study. Lancet 361: 1767-1772. 10. Huang KJ, Su IJ, Theron M, Wu YC, Lai SK, Liu CC, Lei HY (2004) An interferon--related

cytokine storm in SARS patients. J Med Virol 75:185-194.

11. Wong CK, Lam CWK, Wu AKL, Ip WK, Lee NLS, Chan IHS, Lit LCW, Hui DSC, Chan MHM, Chung SSC, Sung JJY (2004) Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome. Clin Exp Immunol 136: 95-103.

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Self evaluation

This paper was published in Cytokine 2005, 32:30-38. (Theron, M., K-J Huang, Y-W. Chen, C-C. Liu, and H-Y. Lei. 2005. A probable role for IFN-in the development of a lung

附件一

行政院國家科學委員會補助專題研究計畫

■ 成 果 報 告

□ 期中進度報告

SARS 的免疫致病的分子機轉-（子計畫二）SARS 自體抗體和抗原的鑑定及所

扮演的角色

計畫類別：□ 個別型計畫

■ 整合型計畫

計畫編號：

NSC 93－2751－B－006－003－Y

執行期間：

九十三年七月一日至九十四年六月三十日

計畫主持人：

林以行 教授

共同主持人：郭余民 副教授

廖寶琦 副教授

計畫參與人員：

碩士班研究生：方宜婷 (成大醫學院微生物及免疫學研究所)

江承堯 (成大醫學院微生物及免疫學研究所)

馮台雲 (成大醫學院細胞生物及解剖學研究所)

成果報告類型(依經費核定清單規定繳交)：□精簡報告

■完整報告

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、

列管計畫及下列情形者外，得立即公開查詢

□涉及專利或其他智慧財產權，□一年□二年後可公開查詢

執行單位：

國立成功大學醫學院微生物及免疫學研究所

中

華

民

國

九十四

年

九

月

二十六

日

一、中文摘要 病毒造成直接性細胞傷害以及發炎因子引發的免疫偏差反應皆可能參與 SARS-CoV 感 染症的致病機轉。本計畫的研究結果顯示在 SARS 病患血清中測得抗肺上皮細胞株 A549 的自體抗體存在，並以 IgG 為主。實驗結果顯示偵測抗上皮細胞自體抗體的存在可以在四 十一位病症晚期 (發病二十天後) 的病患血清中證實，然而在八十位病症早期(發病二十天 內)的病患血清中並無偵測抗體的存在。抗體生成的現象在非 SARS 病症之肺炎病患血清中 並無類似的結果。根據自體抗體對上皮細胞產生的病理效應，我們發現 SARS 病患血清抗 體會造成上皮細胞的細胞毒殺作用，而且血清中抗體 IgG 的效價與血清引發細胞毒殺的作 用在生物統計學上有正相關的結果。進一步利用 SARS-CoV 的棘蛋白 (spike protein) 進行 結合競爭試驗，結果顯示了病患血清中抗上皮細胞的自體抗體亦可以辨識並結合棘蛋白 domain 2 (S2)。而且利用 anti-S2 抗體以及干擾素刺激上皮細胞後會促使 PBMC 細胞吸附 黏著至上皮細胞表面的情形增加。總結本計畫的研究結果首先證實 SARS 病患血清中有抗 肺上皮細胞自體抗體的存在。而抗肺上皮細胞自體抗體的生成可能引發上皮細胞傷害以及 促進免疫細胞黏著上皮細胞的能力增加。自體免疫反應可能參與 SARS-CoV 感染引發 SARS 病症。 關鍵詞：嚴重急性呼吸道窘迫症候群、人類冠狀病毒、肺上皮細胞自體抗體、細胞毒殺、 棘蛋白 Abstract

Both viral effect and immune-mediated mechanism are involved in the pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. In this study, we showed that in SARS patient sera there were autoantibodies (autoAbs) that reacted with A549 cells, the type-2 pneumocytes, and that these autoAbs were mainly IgG. The autoAbs were detectable 20 days after fever onset. Tests of non-SARS-pneumonia patients did not show the same autoAb production as in SARS patients. After sera IgG bound to A549 cells, cytotoxicity was induced. Cell cytotoxicity and the anti-epithelial cell IgG level were positively correlated. Preabsorption and binding assays indicated the existence of cross-reactive epitopes on

SARS-CoV spike protein domain 2 (S2). Furthermore, treatment of A549 cells with anti-S2 Abs and IFN-resulted in an increase in the adherence of human peripheral blood mononuclear cells to these epithelial cells. Taken together, we have demonstrated that the anti-S2 Abs in SARS patient sera cause cytotoxic injury as well as enhance immune cell adhesion to epithelial cells. The onset of autoimmune responses in SARS-CoV infection may be implicated in SARS pathogenesis.

Keywords: severe acute respiratory syndrome; human coronavirus; anti-epithelial cell

autoantibody; cytotoxicity; spike protein

二、緣由與目的

Severe acute respiratory syndrome (SARS), an atypical pneumonia disease caused by a human coronavirus (CoV), is a new global public health problem [1-9]. Major outbreaks of SARS infection occurred in China, Hong Kong, Singapore, Vietnam, Taiwan, and Canada. Over 30 countries have reported suspected or probable cases. SARS-CoV is a mutant human CoV that may acquire new virulence factors. Although the temporal progression of the clinical,

radiological, and virological changes of SARS has been extensively studied and several

treatments have been proposed [10-13], there are as yet no effective strategies to prevent SARS, because its pathogenic mechanisms are still unresolved.

In SARS pathology, the onset of respiratory symptoms is suggested to be a result of multiple factors on respiratory epithelium disruption, including viral cytotoxicity and host factors [12]. Genetic variations of SARS-CoV and viral load seem to be responsible for the severity of SARS. In addition, abnormal immune responses to SARS-CoV infection, such as the unbalance of

immune cells and the production and dysregulation of cytokines and chemokines, may also

underlie the pathogenesis of disease [9,12,14-16]. The immune-mediated mechanisms involved in SARS pathogenesis, however, are not fully understood [9,17,18].

The onset of autoimmunity has been related to viral infections. An influenza viral

infection-induced autoimmunediseasecalled Goodpasture’ssyndromeshowstheexistenceof autoantibodies (autoAbs) against the alveolar and glomerular basement membrane [19-21], but there is no report regarding SARS-CoV-induced autoAb production. Nevertheless, murine CoV infection induces autoreactive T cells, B cell polyclonal activation, and autoAb production [22-24]. The immune-mediated pathology related to SARS-CoV infection thus merits further examination. In the present study, the generation and pathogenic role of autoAbs in SARS patients were investigated.

三、研究方法 Patients

SARS patient sera were collected by the Center for Disease Control, Department of Health, Taiwan (CDC-Taiwan), from March to June 2003. Diagnosis of SARS was based on the clinical criteria established by the WHO. Patients with SARS-CoV were confirmed by laboratory methods, including viral antigen detection, RT-PCR, and serologic methods [25]. The

epidemiological characteristics of age and gender and clinical information such as symptoms, underlying diseases, outcomes including death and hospital length-of-stay, as well as laboratory findings were obtained from CDC-Taiwan [25, 26, and unpublished data]. SARS patient sera collected from the early (80 patients, < 20 days after fever onset) and the late (41 patients,20 days) stages were included in this study. Sera of some patients were collected two or three times at the late stage; in all, 60 serum samples from 41 patients were tested. Eight serum samples from patients diagnosed with pneumonia from non-SARS etiology (obtained from Dr. T. R. Hsiue, Internal Medicine, National Cheng Kung University Hospital, Tainan) and ten serum samples from healthy individuals were used as controls.

Cell binding assay

Human lung adenocarcinoma cell line A549, hepatoma cell line Hep3B, and lung fibroblast MRC5 were grown in DMEM, and human lung epithelial cell line HL was grown in MEM, both supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamate, and 50 ng/ml gentamycin. Human microvascular endothelial cell line-1 (HMEC-1) was passed in culture plates containing endothelial cell growth medium (Clonetics, Walkersville, MD, USA) composed of 2% FCS, 1g/ml hydrocortisone, 10 ng/ml epidermal growth factor, and antibiotics. Cells were incubated in a CO2incubator at 37°C and 5% CO2in a humidified atmosphere. For

microscopic observation, monolayers of A549 cells were cultured on sterile glass slides before the experiment. For flow cytometric analysis, cell suspensions were prepared by trypsinization of cell cultures. Cells were washed briefly in phosphate-buffered saline (PBS), fixed with 1% paraformaldehyde in PBS at room temperature for 10 min, and washed again with PBS. Patient serum samples and mouse anti-SARS spike protein (anti-S), spike protein domain 1 (anti-S1), and spike protein domain 2 (anti-S2) Abs were then incubated with cells at 4°C for 1 h. After being washed three times with PBS, cells were incubated with 1lof 1 g/ml FITC-conjugated mouse anti-human IgG, IgM, or IgA and goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA), respectively, at 4°C for 1 h and washed again with PBS. The cell binding activity of sera Abs was analyzed by fluorescent microscopy and by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) with excitation set at 488 nm.

Cell cytotoxicity assay

For the cytotoxicity assay, test sera were pretreated at 56C for 30 min for complement inactivation. Monolayers of A549 cells were cultured in a 96-well plate before the experiment. Cells were washed briefly in sterile serum-free culture medium, and then treated with serum samples for 72 h. Supernatant was collected and the cytotoxicity was determined using lactate dehydrogenase (LDH) release with a kit (In Situ Cell Cytotoxicity kit; Roche Molecular

Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s instructions. The % of cytotoxicity was calculated as (ODtest –ODspontaneous release)/(ODmaximum release – ODspontaneous release) × 100%.

Preparation of SARS S2 protein

To construct the S2 expression vector, the cDNA of the SARS S2 coding region was amplified by PCR carried out using pcDNA3-Spike-EGFP (a kind gift from Dr. C. J. Huang, Academia Sinica, Taipei, Taiwan) as a template, a forward primer

(5'-GCGGAATTCGTCGACACTTCTTAT-3') containing an EcoRI restriction site (in italics), and a reverse primer (5'-GCGTCTAGATTAACTAGTCATGCAACA-3') containing a stop codon (underlined) and an XbaI restriction site (in italics). The amplified DNA fragment was cloned into the EcoRI and XbaI sites of the pMAL-c2E vector (New England Biolabs, Beverly, MA, USA) after the maltose binding protein (MBP) gene to produce pMBP-TWsp-S2. After ligation, transformation of DH5was performed using electroporation (BioRad Laboratories, Inc., Hercules, CA, USA). All constructs were verified by DNA sequencing performed using a kit (ABI-Prism dye terminator cycle sequencing kit; PerkinElmer Applied Biosystems, Foster City, CA, USA).

For expression and purification of the SARS S2 fusion proteins, the E. coli DH5bearing the construct pMBP-TWsp-S2 for expression was grown in Luria-Bertani medium with 100 g/ml ampicillin. When the cell culture reached an OD of 0.7-1.0 at 600 nm, protein expression was induced by the addition of 0.5 mM IPTG for 3 h at 37C. To verify expression, cells were collected by centrifugation and disrupted directly in SDS-PAGE sample loading buffer. For large-scale purification, cells were harvested by centrifugation and suspended in binding buffer (20 mM Tris-HCl (pH 7.4) containing 200 mM NaCl and 1 mM EDTA). Cells were lysed with sonication on ice. The cell lysate was clarified by centrifugation at 20,000g for 20 min, and then the clear supernatant was loaded onto a column containing amylose resin (New England Biolabs), equilibrated with the binding buffer. The column was washed with three volumes of binding buffer, and then the fusion proteins were eluted by the same buffer containing 10 mM maltose. Finally, the purified fusion protein MBP-S2 was concentrated using a filter unit (Centricon YM-10 Centrifugal Filter Unit; Millipore Corp., Bedford, MA, USA). Protein concentration of various fractions was determined by the Bradford spectrophotometric method (BioRad) in duplicate, and average values were calculated.

Spike peptide prediction and synthesis

Publicly available human and CoV genome sequences at the National Center for Biotechnology Information, USA, were used for in-silico prediction. Algorithms for

immunogenicity, second-structure prediction, protein topology analysis, and hydrophobicity were applied to design the tested peptides. The protein sequence of spike protein was obtained from GenBank (accession number AY274119). Immunogenic viral peptides were calculated based on the algorithm developed by Kolaskar and Tongaonkar [27]. In-silico secondary structural analyses of spike protein were performed based on two algorithms: PHD and PREDATOR. Protein topology prediction was based on the algorithm developed by TMHMM. Hydrophobic moment of the peptides was calculated based on the algorithm HMOMENT. Similarity searches were performed between spike protein and human genome databases using the NCBI Blastp program. For blastp analysis, the default database collection of all non-redundant GenBank cDNA sequence translations, PDB, SwissProt, PIR and PRF entries was used, with the species restricted to human. Finally, expert curation was applied for refinement on peptide design. Multiple antigen peptides were synthesized by CytoMol Corp. (Mountain View, CA, USA). Several synthetic peptides were used in this study: D02 (residues 658-669,

N-ASYHTVSLLRSTSQK-C), D03 (residues 733-744, N-EEGNLLLQYGSFCTQ-C), D07 (residues 927-937, N-GLGKLQDVVNQNGE-C), and D08 (residues 942-951,

N-ALNTLVKQLSSN-C). Extra amino acid residues were added at either N- or C-terminus, as indicated by italic letters, to increase the hydrophobicity.

Preabsorption of patient sera against SARS-CoV S2 and the four synthetic peptides we used was done using a solid-phase capture technique and individual peptide-coated plates. An ELISA plate was coated with or without peptides in a 10g/well and blocked by 5% bovine serum

albumin (BSA) in coating buffer (15 mM of sodium carbonate and 35 mM of sodium bicarbonate, pH 9.6). Test serum samples were 1:20 diluted and added to the plate at 4C overnight for

absorption. Supernatant was collected from each well and incubated with A549 cells for a binding assay as described above. To detect Ab titers in sera, an ELISA plate was washed with 0.05% PBS-Tween 20 (PBS-T) five times, whereafter HRP-conjugated anti-human IgG was added (0.5 mg/ml; Jackson ImmunoResearch Laboratories) in 1/5000 dilution (200l/well). After washing with PBS-T, ABTS peroxidase substrate (Trinity Biotech Plc, Bray, Co Wicklow, Ireland) was added, and the absorbance was measured using a microplate reader (Emax;

Molecular Devices Corp., Sunnyvale, CA, USA) at 405 nm.

Adhesion assay

A549 cells (5 × 104cells/well) were plated into 8-well glass chamber slides (Nalge Nunc International, Naperville, IL, USA). When monolayers were confluent, the cells were treated with anti-S2 hyperimmune sera in serum-free culture medium. After 1 h of incubation, the cells were washed once with medium and incubated for 1 h at 37C with isolated healthy human peripheral blood mononuclear cells (PBMC) (1 × 105cells/well) in a total volume of 250l/well. At the end of the incubation period, the nonadherent cells were removed by washing twice with 0.1% BSA in PBS. Adherent cells were stained with Liu’sstain (TONYAR Biotech,Taipei,Taiwan)and viewed with light microscopy. The adherent cells were counted on three consecutive microscopic fields [28,29].

Statistical analysis

Comparisons between various treatments were performed using Student’s t test with SigmaPlot version 8.0 for Windows (Cytel Software Corp., Cambridge, MD, USA). Non-paired Student’s t tests were used for the data analyses in Table 1 and Fig. 2, and paired Student’s t tests were used for the data analyses in Figs. 3 and 5. Values were considered statistically significant at P < 0.05.

四、結果

SARS patients produced Abs cross-reactive with A549 cells

Pulmonary defects are clinical features of SARS-CoV infection. In an attempt to investigate the role played by SARS patient sera, the binding activity of patient sera with human A549 epithelial cells was determined. Fluorescent microscopic observation showed that Abs present in SARS patient sera reacted with uninfected A549 epithelial cells, while sera from healthy controls did not (Fig. 1a). By flow cytometric analysis, the average IgG level was significantly higher at the late stage (20 days after fever onset) compared to the early stage (< 20 days after fever onset) and the healthy controls (Fig. 1b and Table 1). Although some of the patient sera IgM and IgA at the late stage exhibited high A549 cell binding activity (Fig. 1b), the average levels were not significantly different from those of the early stage and the healthy controls (Table 1). The epithelial cell binding activity of Abs was not significantly elevated in non-SARS-pneumonia patient sera (Fig. 1b and Table 1). The autoAb levels started to increase by day 20, reached the highest levels around day 40, and declined gradually thereafter (60 samples in 41 patients; Fig. 1b and Table 1).

Cytotoxic effect of cross-reactive Abs on A549 cells

The consequences of serum binding to A549 cells were next assessed. A549 cell cytotoxicity induced by patient sera was measured using an LDH activity assay. Sera from the late phase were used and results showed that SARS patient sera induced A549 cytotoxicity (average OD = 0.43 0.15, n = 60) compared to sera from healthy controls (average OD = 0.190.04, n = 10) and non-SARS-pneumonia patient sera (average OD = 0.290.03, n = 8) (Fig. 2a, upper panel). The

% of cytotoxicity showed a pattern similar to that of OD values (Fig. 2a, lower panel). Preabsorption of the IgG fraction by protein G resulted in a reduction of A549 cytotoxicity to levels similar to those of healthy controls, indicating that the cytotoxic effect was caused by the IgG present in the SARS patient sera (data not shown). Complement inactivation of patient sera was performed before experiments. Therefore, epithelial cell injury must have been mediated by patient IgG via a complement-independent pathway. However, the possibility that

complement-mediated cytotoxicity may play a role cannot be excluded. Further analysis showed a statistically significant correlation between the levels of anti-A549 IgG, but not IgM or IgA, and the magnitudes of epithelial cell cytotoxicity (Fig. 2b).

In addition to A549 cells, we examined whether autoAbs present in SARS patient sera also reacted with other cell types. Some of the SARS patient sera detected bound to the human

endothelial cell line HMEC-1 and to the human hepatoma cell line Hep3B, and, to a lesser extent, to the lung fibroblast MRC5 cells, as demonstrated by flow cytometric analysis (Fig. 3).

Cross-reactive epitopes on SARS-CoV spike protein

Several synthetic peptides were designed from the viral epitopes sharing sequence homology with human proteins (Fig. 4a). After a panel of screening proceeded, two spike-protein peptides, designated D07 and D08, appeared to be bound by SARS patient sera (Fig. 4b). These two peptides are located at the S2 domain of the spike protein. Binding of patient sera to S2 was also confirmed (Fig. 4b). To further validate the epitopes shared between viral- and self-antigens, SARS patient sera were preabsorbed with various peptides, and A549 binding activity was determined. Results indicated that A549 cell binding activity of patient sera was reduced by preabsorption with S2, D07, and D08 (Fig. 4c). The D02 and D03 peptides were not bound by patient sera (Fig. 4b) and, accordingly, did not cause an inhibition (Fig. 4c) The existence of cross-reactive epitopes on SARS-CoV S2 shared homology with host cell proteins was therefore demonstrated.

To further characterize the epithelial cell cross-reactivity of anti-S Abs, mouse Abs directed against S, S1, and S2 were tested for their binding activities with A549 cells. Using flow

cytometry analysis, there were higher levels of cell binding ability by anti-S and anti-S2, but not anti-S1, Abs (Fig. 5a). The binding ability of anti-S and anti-S2 Abs on the surface of epithelial cells was also confirmed by confocal microscopy (Fig. 5b). Further study using human lung epithelial cell line HL for the binding activity indicated that anti-S and anti-S2 Abs could also bind to these lung epithelial cells (Fig. 5c).

Effect of anti-S2 Abs on immune cell adhesion to A549 cells

To further explore the effects of anti-S2 Abs binding on epithelial cells, the adherence of PBMC to A549 cells was investigated. Because a cytokine storm such as the increased production of IFN-was previously shown [26], the adhesion of PBMC to IFN--treated A549 cells in the presence or absence of anti-S2 Abs was also assessed. Results showed that higher levels of PBMC adherence could be observed in cells treated with IFN-for 24 h compared to the untreated group (Fig. 6a). Interestingly, anti-S2 promoted the adhesion of PBMC to A549 cells and this was greatly enhanced in combination with IFN-treatment (Fig. 6a). Therefore, in addition to cell injury after cross-linking, anti-S2 Abs can also upregulate the immune cell

adhesion to epithelial cells. Study using immunostaining indicated that monocytes in PBMC were the major cell population with preferential binding to the anti-S2-treated A549 cells (data not shown). This is in accordance with the finding that macrophages are the prominent leukocyte in the alveoli of SARS patients (12). In a competition binding assay, synthetic peptides (D07 and D08) and S2 protein inhibited the anti-S2-mediated adhesion of PBMCs to A549 cells (Fig. 6b).

五、討論

A novel human CoV may cause SARS, which is characterized by fever, myalgia, dry cough, and lymphopenia. SARS patients develop an atypical form of pneumonia. Among the changes

observed in the lungs of SARS patients are epithelial cell proliferation and desquamation, hyaline membrane formation along alveolar walls, and immune cell infiltration during the early stage of the disease, and increased fibrosis and multinucleated epithelial giant cell formation during a later stage. The increasing viral load suggests an effect caused by viral replication in the early phase [11]. In addition, proinflammatory cytokine production and dysregulation may be involved in the pathogenesis of SARS [12,15,16,26]. One study documented IgG seroconversion in 93% of the patients at a mean of 20 days. The timing of IgG seroconversion, which started on day 10, correlated with falls in viral load, which occurred between days 10-15, when several clinical worsenings, which cannot be explained by uncontrolled viral replication, occurred. The lung damage at this phase is therefore related to immune-mediated pathological effects [11]. In the present study, we showed that autoAb production is also involved in SARS-CoV infection. The autoAbs, mainly IgG, started to appear on day 20.

Viral infections have been associated with the development of autoimmune diseases [30]. Most autoimmune disorders are chronic diseases, but there are several acute autoimmune responses that are initiated shortly after infection. Structural similarities between viral proteins and self-antigens have long been proposed as targets for immune cross-reactivity associated with the initiation of autoimmunity. We have reported this phenomenon in dengue virus infection and hypothesized its role in the immunopathogenesis of dengue virus-induced dengue hemorrhagic fever and dengue shock syndrome [31-34]. Our finding that anti-epithelial cell autoAbs are produced after SARS-CoV infection is another example of acute viral infection-induced autoimmunity. Both viruses have some characteristics in common: both are lymphotrophic; the infections cause high fever, lymphopenia, thrombocytopenia, hemorrhage, and mild hepatitis; cytokine storm occurs in the acute phase; and molecular mimicry exists between virus proteins and self-antigens. Mouse CoV infection induces B cell polyclonal activation and the generation of autoAbs [23,24]. Although autoAb production in SARS patients causes cytotoxicity to A549 cells (type-2 pneumocytes), whether the autoimmune response may act as a pathological factor in SARS disease needs further investigation. Interestingly, the elevated Ab titer was associated with the aggravation of respiratory failure [35]. In addition to Ab-mediated cytotoxicity, the release of chemokines and cytokines by A549 cells has been detected. Our preliminary results indicated an increase in the production of IL-6, MCP-1, MIP-1and MIP-1after anti-S2 stimulation (unpublished data).

Recent reports [36,37] have shown that the SARS-CoV spike protein might elicit the protective Ab responses in mice. The neutralizing effect of anti-SARS-CoV spike Abs was demonstrated. Based on our results by sequence comparison from the NCBI protein database, however, several regions of SARS-CoV spike protein show sequence homology to self-antigens expressed in human cells. We have identified several candidate proteins recognized by SARS patient sera and anti-SARS-CoV spike Abs, including annexin II, glyceraldehyde-3-phosphate dehydrogenase, albumin,1-antitrypsin, aldo-keto reductase, and transferrin (Fig. 7). The local alignment by Java-European Molecular Biology Open Software Suite (JEMBOSS)-Water analysis showed that these proteins have a high degree of homology with the spike peptide sequences of D07 and D08 located in S2 portion. Recombinant S2 protein or the spike peptides D07 and D08 blocked the binding of SARS patient sera to A549 cells. Furthermore, murine anti-S2, but not S1, antisera can bind A549 cells. These results suggest that anti-SARS-CoV Abs can recognize the cross-reactive epitope on human lung epithelial A549-cell proteins by

molecular mimicry between spike proteins and self-antigens. The epitope for neutralizing Abs is in the S1 domain [38,39], while the cross-reactive Abs recognize the epitope on the S2 domain of spike protein. Analysis of Ab expression profile from SARS patient sera used in this study indicated that anti-S2 IgG started to appear around day 20, while anti-nucleocapsid (N) IgG could be detected around day 10 [40]. This is consistent with the previous report that IgG

seroconversion started on day 10 [11] and with our findings that the autoAbs, which are mainly anti-S2 IgG, appeared on day 20.

The onset of autoimmune responses in SARS-CoV infection may have important

implications. It is still not clear why intense lung inflammation develops even after the viral load has dropped two weeks after the onset of fever. The virus itself and virus-induced cytokine

production might be responsible for the early stage of lung epithelial cell damage, while the late autoAb-mediated or infiltrating cell-mediated inflammation causes continued and sustained alveolar damage and fibrosis. In addition to A549 cells, other cell types, including endothelial cells, hepatocytes, and fibroblasts, were also bound by autoAbs in SARS patients. The autoAb production caused by the molecular mimicry between self-antigens and the spike protein of SARS-CoV might relate to the systemic effects and the sequelae of SARS disease [41].

Furthermore, the binding of immune cells with lung epithelial cells, which is accelerated by the combination of IFN-and anti-S2, may contribute to the inflammatory responses associated with SARS pathogenesis. This hypothesis needs to be further tested. Studies on the role of autoAbs will be crucial to gain an insight into SARS immunopathologic mechanism.

六、計畫成果自評 雖然目前 SARS 感染症的致病機制仍無完整的定論，一般認為這是病毒與宿主交互反 應後的病理結果。感染 SARS-CoV 病毒會造成嚴重急性呼吸道窘迫症候群，此新型傳染性 的爆發流行顯示出病毒的傳播是極具生命威脅的。臨床觀察顯示，SARS 患者易出現嚴重 的肺部呼吸功能障礙，我們認為來自病毒的直接感染和抗體的合併效應，亦或是抗體直接 的影響都有可能是造成病症的發生。自體免疫反應的發生可能參與病毒感染的致病機轉 上，過去的研究發現在某些小鼠冠狀病毒的感染症上證實包括有 B 淋巴細胞的多株落活化 作用及自體抗體的生成進而可能參與細胞傷害。自體免疫疾病”Goodpasture症候群”被證實 在某些類似感冒症狀的病例上出現，此自體免疫病症伴隨自體抗體的生成並造成宿主體內 肺上皮細胞的傷害而往往引發宿主猝死現象。根據本計畫的研究結果，我們證實 SARS 病 患對於 SARS-CoV 的感染會產生自體抗體。而生成的自體抗體進一步會造成細胞病理效應 例如細胞的直接毒殺作用。初步結果雖然排除自體抗體參與疾病早期的病症發生，然而在 晚期病患持續性的發炎反應與自體抗體的生成是否具關聯性是值得繼續研究的。另外，抗 肺上皮細胞自體抗體所辨識並結合的肺上皮細胞表面分子目前也已有初步的結果。而這些 自體抗原極可能在細胞激素的發炎性作用下被刺激增加其表現。本計畫的實驗內容依據原 訂計畫目標執行，證實 SARS 病人血清中自體免疫抗體生成的研究結果已於今年九月刊登 於臨床實驗免疫學雜誌 (Clinical and Experimental Immunology, 141 (3), 500-508)。自體抗原 鑑定部份也已開始彙整並將投稿於國際期刊發表。根據本計畫的發現，將可釐清自體免疫 是否參與 SARS-CoV 感染引發 SARS 病症，並且提供未來 SARS 病症的治療與疫苗研發之 考量。根據本計劃的研究結果顯示抗病毒棘蛋白抗體似乎與肺上皮細胞自體抗體的生成極 為相關。基於這些結果的發現，警示我們在研發疫苗之前，針對抗體所可能參與的自體免 疫反應，有必要去釐清它所隱藏的潛在性致病角色。

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