行政院國家科學委員會專題研究計畫 成果報告
（子計畫四）SARS 病人抗肺部組織及細胞自體抗體之偵測
計畫類別： 整合型計畫 計畫編號： NSC92-2751-B-002-011-Y 執行期間： 92 年 07 月 01 日至 93 年 06 月 30 日 執行單位： 國立臺灣大學醫學院小兒科 計畫主持人： 楊曜旭 計畫參與人員： 黃鈺惠 報告類型： 完整報告 處理方式： 本計畫可公開查詢中 華 民 國 93 年 9 月 14 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
（計畫名稱）
嚴重急性呼吸道症候群的免疫致病機轉-（子計畫四）SARS 病人抗肺
部組織及細胞自體抗體之偵測
計畫類別： □ 個別型計畫 ■ 整合型計畫
計畫編號：NSC 92－2751－B－002－011－Y
執行期間：2003 年 07 月 01 日至 2004 年 06 月 30 日
計畫主持人：楊曜旭
共同主持人：江伯倫
計畫參與人員：
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：國立台灣大學醫學院小兒科
中 華 民 國 93 年 09 月 14 日
目錄
中文摘要………(I)
英文摘要………(II)
前言及研究目的………(1)
研究方法………(3)
研究結果………(9)
討論………(12)
文獻探討………(17)
附圖說明………(24)
附圖………(26)
中文摘要
關鍵字：嚴重急性呼吸道症候群，自體抗體，內皮細胞，上皮細胞，細胞毒殺
嚴重急性呼吸道症候群 (SARS)是一種由 SARS-associated 冠狀病毒感染而引起 肺部嚴重發炎，甚至纖維化的非典型性肺炎。在這個研究中，我們將探討感染此 冠狀病毒後身體是否會產生抗血管內皮細胞及肺部上皮細胞之自體抗體。血清來 自病人疾病不同時期(phase I, phase II/III)及健康對照組。目標細胞我們選用 1) A549 human pulmonary epithelial cell-line，2) human umbilical venous endothelial
cells (HUVEC)，3) primary human pulmonary endothelial cells (HPEC)。自體抗體 的偵測是採用 cell-based ELISA，indirect immunofluorescence staining，及 flow cytometry 三種方法。結果顯示不管用那一種方法，病人於 phase II/III 的血清中 存 IgG anti-A549 cells antibodies，IgG anti-HUVEC antibodies，及 IgM anti-HPEC antibodies 三種自體抗體。進一步的研究顯示 IgG anti-A549 cells 自體抗體具有
complement-dependent 細胞毒殺作用。本實驗結論：感染 SARS-associated 冠狀病 毒後期，病人血清會產生抗內皮細胞及上皮細胞之自體抗體，而這些感染後產生 的自體抗體所扮演的角色則須要更多的研究來確定。
Key words: SARS, autoantibodies, epithelial cell, endothelial cell, cytotoxicity
Abstract
The severe acute respiratory syndrome (SARS), an atypical pneumonia emerged in
21st century, is caused by the invasion of the SARS-associated coronavirus
(SARS-CoV) and characterized by severe pulmonary inflammation and fibrosis. In
this study, we investigate the possibilities of the development of autoantibodies
against human epithelial cells and endothelial cells in patients with SARS at different
time periods (the first week: phase I, 1-2 months after the disease onset: phase
II/phase III). Antibodies in sera of patients and normal healthy controls against 1)
A549 human pulmonary epithelial cell-line, 2) human umbilical venous endothelial
cells (HUVEC), 3) primary human pulmonary endothelial cells (HPEC) were detected
by the methods of cell-based ELISA, indirect immunofluorescence staining, and flow
cytometry. The results of ELISA revealed that serum levels of IgG anti-A549 cells
antibodies, IgG anti-HUVEC antibodies, and IgM anti-HPEC antibodies were
significantly higher in SARS patients at phase II/phase III than those in healthy
controls. Results of the other two tests, indirect immunofluorescence staining and
flow cytometry, were consistent with the previous one. Sera from SARS patients at
phase II/phase III could mediate complement dependent cytotoxicity against A549
endothelial cells would be developed after SARS-CoV infection and this phenomenon
may indicate the post-infectious cellular injury and also provide the possibility of
報告內容
Introduction
In early 2003, a new infectious disease-severe acute respiratory syndrome (SARS)
swept the world including Taiwan [1-2]. The pathogen was later identified as
SARS-associated coronavirus (SARS-CoV) and spread soon among human beings
through the close contact of droplets [3-4]. Those who infected by this virus present
with persistent high fever, cough, dyspnea, and the disease may eventually progress to
respiratory and/or multiple organs failure [1,5]. The autopsies of patients died from
SARS have revealed extensive pulmonary consolidation, localized hemorrhage and
necrosis, proliferation and desquamation of alveolar epithelial cells, monocytes,
lymphocytes and plasma cells infiltration in alveoli, and hyaline membrane formation
[6-7]. Besides, systemic vasculitis has also been found and characterized by edema,
thrombosis, localized fibrinoid necrosis, and infiltration of monocytes, lymphocytes,
and plasma cells into vessel walls in many organs including heart, lung, liver, kidney,
and the stroma of striated muscles [7]. All these pathological changes are now thought
to be mediated by direct viral destruction and followed by immune-mediated
processes [8].
Epithelial cells and endothelial cells, according to the pathological findings, may
be the two major target cells that are damaged in the inflammatory process of SARS.
ulcerative colitis and prostate cancer [9-11], and anti-endothelial cell antibodies
(AECA) have also been found in many disorders such as systemic lupus
eyrthematosus (SLE), Kawasaki disease, Henoch-Schönlein purpura (HSP), Behcet's
disease, and some post-infectious immune-mediated diseases [12-17]. Although some
of them appear as a result of inflammatory tissue injury, others are proved to have a
pathogenic potential to induce further damage. The aims of this study was to
investigate the development of autoantiboies against human epithelial cells and
endothelial cells after the SARS-coronavirus infection by using the methods of
cell-based ELISA, indirect immunofluorescence staining, and flow cytometry, and to
Materials and Methods
Patients and controls
Twenty-two previously healthy Chinese adults suffering from SARS in early 2003
were included in this study. The diagnosis was confirmed according to the typical
clinical presentations with fever, cough, and dyspnea, and positive viral PCR.
Informed consents and institutional approval were obtained for this study. Blood was
sampled during the first one-week (phase I) and one-two months after the disease
onset (phase II or phase III) [8]. Twenty normal healthy adults were enrolled as
controls. In the study of indirect immunofluorescence staining, patients with
streptococcal necrotizing pneumonia were also recruited as controls. For safety, serum
samples of patients derived from the acute stage were inactivated at 56℃ for 30 min
before testing.
Antibodies against SARS-CoV nucleocapsid protein
To detect the presence of anti-SARS-CoV nucleocapsid (N) antibodies in SARS
patients, the 96-well microplate was coated with purified His-N protein at a
concentration of 5 µg/ml. Each well was then blocked by PBS containing 0.05﹪
Tween-20 (PBS/Tween 20) (Sigma) and 5﹪BSA at 37℃ for 2 hours. Diluted serum
400 with 1﹪BSA) were added to the wells at room temperature for 2 hours and then
removed. Following the washing procedure, peroxidase-conjugated mouse
anti-human IgG, IgA and IgM (1: 5000 in 1﹪BSA) was added to each well and
incubated at room temperature for 1 hour. The plates were washed by PBS/Tween 20
before adding teramethylbezidine (TMB) (KPL, USA) substrate and the reactions
were stopped by the addition of 2 N H2SO4. The optical density (OD) of each well
was read at a wavelength of 450nm minus 540 nm by an ELISA reader. The serum
levels of antibodies between patients and controls were expressed as OD values.
Cells culture
A549 cells, a human pulmonary epithelial cell-line, were cultured with DMEM
supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamin,
150 mM HEPES, and 100 µg/ml penicillin/streptomycin. Primary human pulmonary
endothelial cells (HPEC) were cultured with EGM-2 MV (SingleQuots, USA)
supplemented with EBM (Cambrex Bio Science Walkersville, Inc. USA). Human
umbilical venous endothelial cells (HUVEC) were obtained from human umbilical
vein by collagenase (GIBCO BRL Life Technologies) digestion as described
previously [18]. The separated cells were seeded in 75 ml flasks precoated with 1%
supplemented with 15% heat inactivated FCS, heparin sulfate, L-glutamine,
endothelial cell growth factor (BM) (final concentration, 20 µg/mL), and 100 µg/ml
penicillin/streptomycin. All cultures were incubated at 37℃ in 5% CO2, and the cells
were used between the 2nd and the 6th passage.
Cell-based ELISA
A549, HUVEC, and HPEC were prepared to detect autoantibodies in sera of SARS
patients. Cells were seeded on gelatin-coated 96-well microtitre plates (NuncTM,
Denmark) at a concentration of 1×105 cells/well. When the cellular growth became
confluent 3-4 days later, cells were fixed with 0.2% glutaraldehyde in PBS for 10 min
at room temperature and incubated with blocking buffer (1% bovine serum albumin
(BSA)/0.05% azide/0.1 M Tris in ddH2O) for 60 min at 37℃ to prevent non-specific
binding. After washing with PBS/Tween 20, the serum samples, diluted in blocking
buffer at 1:200 for IgG/IgM detection; 1:25 for IgA detection, were incubated for 2 h
at 37℃. The sera were then removed and the plates were washed, 100 µl of
peroxidase-conjugated rabbit antihuman IgG, IgM and IgA immunoglobulins were
added to each well for a further 2 h at 37℃. After washing, TMB solution was added
for 15 min, and stop solution (1M hydrochloric acid) for 5 min. The optical density of
patients by immunofluorescence staining and ELISA had identified the patients with
high antibody binding activity to three cell types, and who were adopted as the
positive control (SLE serum for IgG and IgM detection; HSP serum for IgA detection).
A normal control serum with relative low binding activity was used as the negative
control. The results were expressed as ELISA ratio (ER) = 100×(S-A)/(B-A), where S
is absorbance of sample, A is absorbance of negative control and B is positive control.
Indirect immunofluorescence staining
A549, HPEC and HUVEC were prepared on 12-well Teflon-printed slides, fixed in
4% paraformaldehyde overnight at 4℃, and incubated with blocking buffer (5% fetal
calf serum in phosphate buffered saline (PBS)) for 30 min at 37℃. Cells on slides
were then incubated with sera of SARS patients, patients with necrotizing pneumonia,
and healthy controls for 1 h at 37℃. The slides were washed three times by PBS and
FITC-conjugated antihuman immunoglobulins (CHEMICON, Australia), diluted in
blocking buffer at 1:100, were added to each well for a further 1 h at 37℃. The
interactions of cells and PBS only (without adding any serum) were as negative
controls to establish backgrounds of various immunofluorescence staining. The
specimens were then washed three times, mounted in glycerol and examined using a
Flow cytometry analysis
A549, HUVEC and HPEC cells were detached from the culture plates and suspended
at 2×105 cells for flow cytometry. Cells were washed once with FACS buffer (2%
FCS/0.05% azide/0.5M EDTA in PBS) and incubated with 50 µl sera of SARS
patients or healthy controls for one hour at 4℃. After washing with FACS buffer once,
cells were incubated with 50 µl of FITC-conjugated antihuman immunoglobulins
(1:100 in FACS buffer) for another one hour at 4℃, washed once again, and then
fixed by 2% paraformaldehyde in FACS buffer. Isotype matched control mouse IgG
was used to eliminate non-specific bindings. The binding activities of antibodies to
cells were finally analyzed using FACSort (Becton Dickinson, USA).
Complement dependent cytotoxicity assay
Cells were seeded in 48-well culture plates at 1 × 104
cells/well overnight for cell
lysis assay. The culture medium was replaced by test medium (RPMI-1640
supplemented with 2 mM L-glutamine but without phenol red) before the addition of
patient sera. Patient and normal control sera were preheated at 56℃ for 30 min to
inactivate complement, diluted (1:25 dilution) and incubated with Low-Tox-M rabbit
complement (1:20 dilution; Celardane Laboratories Ltd., Hornby, Ontario, Canada) at
levels of lactate dehydrogenase activity in the culture supernatant were determined
using a Cytotoxicity Detection Kit (Boehringer Mannheim GmbH, Germany). The
absorbance of the sample was measured at 490 nm and the reference wavelength was
620 nm. To determine the cytotoxicity index, the absorbance values are substituted in
the following equation: cytotoxicity index (%) = (sample value-low control)/(high control-low control) × 100%. Low control is the absorbance from the supernatant of the cells cultured with test medium, and high control is the absorbance from the
supernatant of the cells cultured with 1% Triton X-100 in test medium.
Statistical analysis
The values of OD, ELISA ratio, and cytotoxicity index were expressed as mean ± SEM. Each two-group comparison was conducted using the Mann-Whitney U test. A
Results
Anti-N protein antibodies in SARS patients
Serum levels of antibodies against SARS-CoV nucleocapsid protein in SARS patients
at phase II/phase III and those in healthy controls were analysed and compared. The
results showed that IgG and IgM anti-N protein antibodies elevated significantly in SARS patients (IgG: 1.16 ± 0.10 vs 0.23 ± 0.04 p < 0.001, IgM: 0.84 ± 0.13 vs 0.46 ± 0.07 p = 0.03), and there was no statistical difference of IgA isotype between SARS patients and healthy controls (0.69 ± 0.12 vs 0.42 ± 0.07 p = 0.08) (Fig 1).
AEpCA and AECA detection by cell-based ELISA
Figure 2 summarized the ELISA ratios of serum antibodies (IgG, IgA, IgM) against
A549 cells, HPEC, and HUVEC in healthy controls and SARS patients at different
time periods, phase I and phase II/phase III. During the first week (phase I), patients
presented with high fever, general malaise, myalgia and cough. The levels of these all
antibodies in this period were not different between patients and healthy controls.
When the disease progressed, patients received combined therapy of ribavirin,
intravenous immunoglobulin, and steroid. Although serum samples in this period were
collected during or after treatment, the serum levels of IgG anti-A549 cells antibodies,
increased when compared with healthy controls (IgG anti-A549 cells: 45.44 ± 6.26 vs 22.63 ± 4.57, p = 0.009; IgM anti-HPEC antibodies: 65.45 ± 7.38 vs 42.94 ± 6.67, p = 0.036; IgG anti-HUVEC antibodies: 27.12 ± 4.28 vs 13.47 ± 2.92, p = 0.025). The levels of IgA anti-HPEC antibodies and IgA anti-HUVEC antibodies in patients, no
matter at phase I or phase II/phase III, were not statistically different from healthy
controls; however, these two antibodies were decreased significantly after the acute phase (comparisons between different phases, IgA anti-HPEC antibodies: 25.04 ± 9.17 vs 7.94 ± 3.26, p = 0.018; IgA anti-HUVEC antibodies 25.13 ± 7.52 vs 11.75 ± 5.51, p = 0.018) (Fig 2(B), (C)).
Anti-epithelial cell antibodies (AEpCA) and AECA detection by indirect
immunofluorescence staining and flow cytometry
In the study of autoantibodies against A549 cells, Fig 3(A) showed that IgG anti-A549
cells antibodies existed in SARS patients during phase II/phase III, but not in healthy
controls and patients with necrotizing pneumonia that also had severe pulmonary
inflammation and damage. In AECA detection, although some binding activities of
IgM anti-HPEC and IgG anti-HUVEC antibodies were detected in patients with
necrotizing pneumonia, the intensity of immunofluorescence of these antibodies in
healthy controls (Fig 3(B), (C)). By using another method of flow cytometry to
re-check AEpCA and AECA, serum was interacted directly with the suspended target
cells. The results also showed that the binding activities of IgG antiA549 cells
antibodies, IgM anti-HPEC antibodies, and IgG anti-HUVEC antibodies in patients at
phase II/phase III were higher than those in healthy controls (Fig 4).
SARS patient sera induce A549 cell lysis
In the present experiment, purified IgG and IgM immunoglobulins could not be
available due to the limitations regarding blood sampling from SARS patients.
Therefore, in order to investigate if these autoantibodies have the pathogenic effects,
sera from SARS patients with high-level autoantibodies were used for the cytotoxic
assay. The results showed that in the presence of complement, sera from patients at
phase II/phase III induced greater A549 cell lysis than sera from healthy controls (cytotoxicity index: 45.43 ± 5.21 vs 29.74 ± 2.86 p = 0.007) (Fig 5).
Discussion
SARS is a new emerging infectious disease with global impact. The diagnosis could
be confirmed by the positive viral PCR, and patients were also found to have positive
serologic results with elevated IgG and IgM antibodies against SARS-CoV
nucleocapsid protein at later phases. Although the pathogen has been identified, the
real pathogenesis is to be determined. A prospective study from Peiris JSM, et al
concluded that the clinical progression of SARS had a tri-phasic pattern according to
the clinical presentations and pathological changes. Phase I (the first week),
characterized by fever, myalgia and other systemic symptoms was supposed to be the
effect of viral rapid replication and cytolysis. As the disease progressed into phase II
and phase III, the rates of viral shedding from nasopharynx, stood, and urine
decreased gradually, however, severe clinical worsening often occurred at this time
[8,19]. In addition to pulmonary damage, some autopsies also revealed an impressive
finding of systemic vasculitis [7,20]. Taken together with some therapeutic effects of
immunoglobulin and steroid to block the disease progression [21-22], all these
findings suggested that the later phases of SARS are related to immunopathological
damage.
Focusing on the immune-mediated pathogenesis, after SARS-CoV infection, we
beyond the onset of the disease; including IgG anti-A549 cell antibodies, IgM
anti-HPEC antibodies, and IgG anti-HUVEC antibodies. There are many
well-established methods to detect antibodies against whole cells. The use of
fluorescein-conjugated antisera to human immunoglobulins (indirect
immunofluorescence staining) is the standard method with high specificity; however,
this method is limited by low sensitivity [23-25]. Cell-based ELISA is now the
method most widely used. In this assay, whole cells from different sources are used as
the substrate and fixed by glutaraldehyde treatment. The procedure of cell fixation
may lead to false-positive results, probably because that autoantibodies reacting to
intracellular antigens are also detected as well [25-26]. Another method is
demonstrated by immunoglobulin binding to suspension of cells in a
fluorescence-activated cell sorter (flow cytometry) analysis. This method needs a
number of cells in suspension. Besides, the cell surface antigens are at high risk to be
contaminated with nuclear and cytoplasmic components through the procedures of
detachment of cells, grown in vitro, and enzymatic treatment [25,27]. Each test
described above has its own advantages and limitations, in order to confirm the results
of our study; we used three methods to re-check the laboratory data and gained
consistent results.
different tissue such as mucosal epithelial cells in pemphigus and recurrent oral ulcer,
and intestinal epithelial cells in ulcerative colitis [9,10,28]. There is no literature
concerning about AEpCA detection in those disorders with pulmonary involvement
using respiratory tract epithelial cells as target cells. In our study, we use A549 cells,
an easily available and commonly used human respiratory epithelial cell-line, as the
substrate. Our results showed the development of IgG anti-A549 cell antibodies in
SARS patients but not in patients with streptococcal necrotizing pneumonia, and this
is the first report in the study of the association between autoantibodies development
and infectious pulmonary disorders. In contrast to AEpCA, AECA are extensively
studied. AECA have been found in a wide range of diseases, especially in systemic
autoimmune diseases and primary autoimmune vasculitis [12-17,25]. Vasculopathy or
vasculitis would develop after some viral infection including hepatitis C virus (HCV)
[29], cytomegalovirus (CMV) [16], and dengue virus [17]. In these conditions, AECA
could also be detectable. SARS primarily affects lung, but vasculopathy/vasculitis of
other organs can also be found as the disease progresses [7,20]. This phenomenon
indicates that SARS-CoV like HCV, CMV, or dengue virus has the ability to damage
vessels directly or indirectly, and this may be the reason why we could detect IgM
anti-HPEC antibodies, and IgG anti-HUVEC antibodies in SARS patients. Another
antibodies, although were not statistically increased at phase I, decreased significantly
when the disease progressed to phase II/phase III. This phenomenon may be
explained by the invasion of SARS-CoV that activates mucosa-associated immune
system, a specialized system for IgA globulins production, and induces the formation
of IgA AECA that decline gradually when the viral load is decreased.
The mechanisms of these autoantibodies development in SARS patients were
speculated: after the contact of SARS-CoV, possibly through the epithelial cell surface
receptor recently identified as angiotensin-converting enzyme 2 [30], the virus
invades into the epithelial cells, and that can be directly observed by the electro
microscope [31]. During the first week (phase I), SARS-CoV replicates rapidly and
induces cytolysis. At the same time, macrophages accumulate around local
inflammatory site; and these activated macrophages and other cells may release tumor necrosis factor α, interleukin-1, and other proinflammatory cytokines that have been found increased after SARS-CoV infection [32-33]. The damaged cells and the
stimulation by proinflammatory cytokines may reveal some cryptic autoantigens.
Macrophages infiltrated around the lesion may play another role as the antigen
presenting cells, initiate the process of adaptive immunity, and lead to the formation
of autoantibodies. However, patients with necrotizing pneumonia that also
cytokines did not have the same phenomenon. Another possibility may be that
SARS-CoV shares some specific antigenic determinants with epithelial cells and
endothelial cells individually. The antibodies primarily against virus then cross-react
with these cells due to the mimic molecules. Autoantibodies against cells like AECA
are functionally heterogeneous, most probably depending on their specificity [34].
They may only be epiphenomenon of pulmonary epithelial and vascular injury, but
they could also have pathogenic roles to cause further cellular damage by apoptosis,
complement or antibody-dependent cytotoxic pathway [17,34-35]. In SARS patients,
we found that autoantibodies binding to epithelial cells could activate the complement
system and induce cells lysis.
In summary, although more studies should be designed and performed to identify
the disease-specific autoantigens, the presence of AEpCA and AECA after
SARS-CoV infection may represent the severe pulmonary injury and vascular damage
in these SARS patients. Among these autoantibodies, AEpCA have a pathogenic role
to damage the epithelial cells, and this reaction provides another immunological clue
for a better understanding of the pathogenesis of SARS. Because of the possible
pathogenic potential of these autoantibodies, it is suggested to exclude the possibility
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Figure 1. The comparisons of serum IgG, IgA, and IgM antibodies against SRAS-CoV
nucleocapsid protein between SARS patients at phase II/phase III and healthy controls.
The relative serum levels of these immunoglobulins were expressed as optical density
(OD) values. (* P < 0.05, ** P < 0.01)
Figure 2. Quantitative analysis of serum levels of IgG, IgA, and IgM autoantibodies
against (A) A549 cells, (B) human pulmonary endothelial cells (HPEC), and (C) human umbilical venous endothelial cells (HUVEC) in healthy controls ( ) and SARS patients at phase I (hatched bars) and phase II/phase III (). The levels of antibodies (ELISA ratios) are expressed as mean ± SEM. (* P < 0.05, ** P < 0.01)
Figure 3. Immunofluorescence analysis for the binding activities of (A) IgG
antibodies against A549 cells, (B) IgM antibodies against HPEC, and (C) IgG
antibodies against HUVEC in patients with streptococcal necrotizing pneumonia (2nd
row), healthy controls (3rd row), and SARS patients at phase II/phase III (4th row).
PBS was used in this test as a control to eliminate non-specific bindings (1st row).
Figure 4. Cytofluorographic analysis for serum IgG anti-A549 cells antibodies (1st
in healthy controls and SARS patients at phase II/phase III by using of liquid-phase
sandwich assay. Isotype matched control mouse IgG was used to eliminate
non-specific bindings.
Figure 5. Complement-dependent cytotoxicity assay. Sera from SARS patients (n = 10)
at phase II/phase III with autoantibodies and from healthy controls (n = 10) were used
for this assay. Low-Tox-M rabbit complement (1:20 dilution) was incubated with
pre-inactived sera, and the cytotoxic effects were evaluated by the supernatant levels
of lactate dehydrogenase. Data were calculated and expressed as cytotoxicity index
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