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Inge Hamming
Wim Timens
Marian Bulthuis
Titia Lely
Gerjan Navis
Harry van Goor
Journal of Pathology 2004; 203(2):631-637
CHAPTER
CHAPTER CHAPTER
CHAPTER
5
55
5
Tissue distribution of ACE2 protein, the functional receptor
Tissue distribution of ACE2 protein, the functional receptor Tissue distribution of ACE2 protein, the functional receptor
Tissue distribution of ACE2 protein, the functional receptor
for SARS Coronavirus
for SARS Coronavirusfor SARS Coronavirus
for SARS Coronavirus
Chapter 5
90
Abstract
AbstractAbstract
Abstract
Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via
the respiratory route. A distinct coronavirus (SARS-CoV) has been identified as the etiological
agent of SARS. Recently, a metallopeptidase named angiotensin converting enzyme 2 (ACE2) has
been identified as the functional receptor for SARS-CoV. Although ACE2 mRNA is known to be
present in virtually all organs, its protein expression is largely unknown. Since identifying the
possible route of infection has major implications for understanding the pathogenesis and future
treatment strategies for SARS, we investigated the localization of ACE2 protein in various human
organs (oral and nasal mucosa, nasopharynx, lung, stomach, small intestine, colon, skin, lymph
nodes, thymus, bone marrow, spleen, liver, kidney and brain).
The most remarkable finding is the surface expression of ACE2 protein on lung alveolar epithelial
cells and enterocytes of the small intestine. Furthermore, ACE2 is present in arterial and venous
endothelial cells and arterial smooth muscle cells in all studied organs.
In conclusion, ACE2 is abundantly present in epithelia of the lung and small intestine in humans,
which might provide possible routes of entry for the SARS-CoV. This epithelial expression,
together with the presence of ACE2 in vascular endothelium, also provides a first step in
understanding the pathogenesis of the main SARS disease manifestations.
Tissue distribution of ACE2
91
Introduction
IntroductionIntroduction
Introduction
Severe acute respiratory syndrome (SARS) is an acute infectious disease that spreads mainly via
the respiratory route. Recently, a distinct coronavirus (SARS-CoV) has been identified as the
etiological agent of SARS
1-4
. The spike proteins of this RNA virus associate with cellular receptors
of sensitive cells, to mediate infection of their target cells after which it starts replicating in the
cytoplasm. The main targets of SARS-CoV are the lungs, immune organs and systemic small
vessels, resulting in systemic vasculitis, decreased immune function and respiratory distress
caused by extensive pulmonary consolidation and diffuse alveolar damage with hyaline
membrane formation
5
, which causes death in 10% of infected individuals
6
.
Recently, Li et al identified a metallopeptidase named angiotensin converting enzyme 2 (ACE2),
isolated from SARS-CoV – permissive Vero-E6 cells, that effectively binds to the S1 domain of the
SARS-CoV protein. ACE2 transfected 293T cells formed multinucleated syncytia with cells
expressing S proteins. The virus was shown to replicate effectively in ACE2-transfected, but not in
mock-transfected 293T cells. ACE2 antibodies, but not ACE1 antibodies, blocked the viral
replication on Vero E6 cells
7
. These data indicated convincingly that ACE2 is a functional receptor
for SARS-CoV.
Although real-time PCR revealed that ACE2 messenger RNA expression is present in 72 human
tissues
8
, ACE2 protein expression has thus far been identified only in heart, kidney and testis
9-12
.
Since identifying the possible route of infection has major implications for understanding the
pathogenesis and future treatment options for SARS, we investigated the immunolocalization of
ACE2 protein in various human organs.
Methods
MethodsMethods
Methods
Human tissue specimen
Human tissue specimenHuman tissue specimen
Human tissue specimen
Human tissues from 15 different organs were obtained from patients undergoing biopsy
procedures for diagnostic purposes or surgery for various reasons, predominantly cancer.
Additional tissue was obtained from unused donor organs (because of technical reasons; often in
case unilateral transplantation with lack of an adequate acceptor for the other lung). Extensive
specification of the diagnosis is given for the lung and small intestine only (see below). Brain
tissue was obtained from autopsies. Tissues were chosen to represent organ systems were the
SARS virus has been detected in human
13
and in experimentally infected macaques
3
. Routine
morphology was evaluated by hematoxylin and eosin by a qualified pathologist. Tissues were only
used if characterized as non-diseased. Tissues were investigated from 93 different subjects: lung
(cancer n=4, unused donorlung n=5, alpha 1 antitrypsin deficiency n=1); skin (n=6); oral mucosa
(n=4); nasal mucosa (n=5); nasopharynx (n=6), gastric cardia and corpus (n=9); different parts of
the small intestine: duodenum (cancer n=2, ulcus n=2), jejunum (chronic inflammation n=1,
atresia n=1, cancer n=1, resection ileostoma n=1) and ileum (resection ileostoma n=1, chronic
inflammation n=1, metastatic cancer n=3, primary cancer n=1, M.Hirschsprung n=1,
Chapter 5
92
angiodysplasia n=1); colon (n=5); spleen (n=4); thymus (n=4); lymph nodes (n=6); bone marrow
(n=5); liver (n=6); kidney (n=4); and brain (n=3). All procedures and use of (anonymized) tissue
were performed according to recent national guidelines.
The lung type II alveolar epithelial cell line A 549 and fibrotic lung tissue from patients (n=4) with
usual interstitial pneumonia were used to confirm the findings on type II pneumocytes.
Immunohistochemistry and ACE2 loca
Immunohistochemistry and ACE2 locaImmunohistochemistry and ACE2 loca
Immunohistochemistry and ACE2 localization
lizationlization
lization
Tissues were deparaffinized, rehydrated and subjected to heat induced antigen retrieval by
overnight incubation in 0.1 M Tris/HCl buffer pH 9 at 80°C. Endogenous peroxidase was blocked
with 0.075% H
2
O
2
in phosphate-buffered saline (PBS, pH 7.4) for 30 minutes. Cytospin
prepreations from A549 cells were fixed in PBS buffered paraformaldehyde (2%) at 4ºC for 10
minutes. Subsequently, they were dried and stained for ACE2. A polyclonal rabbit anti-ACE2
antiserum (Millenium Pharmaceuticals, Inc, Cambridge, MA)
10
diluted in PBS and supplemented
with 1% bovine serum albumin was used in a concentration of 1:1000 for 1 hr at room
temperature. Antibody binding was detected using sequential incubations with peroxidase-
labeled goat anti-rabbit and peroxidase-labeled rabbit anti-goat antibodies (GARPO/RAGPO Dako,
Glostrup, Denmark). Human AB serum (1%) was added to the secondary antibodies. Peroxidase
activity was developed by using 3,3-diaminobenzidine tetrachloride (DAB) for 10 minutes.
Counterstaining was performed using Mayer’s hematoxylin. Three types of control tests were
performed to determine the specificity of the antibody. First, control sections were incubated with
anti-ACE2 antibody solutions, which were pre-incubated with the synthetic peptide to which the
antibody was raised (peptide sequence: NTNITEENVQNMNNAGDKW aa51-69, Pepscan Systems
BV, Lelystad, The Netherlands). Second, sections were incubated with unrelated rabbit polyclonal
antibodies (anti-alpha 1 Inhibitor 3 or anti-nitrotyrosine) and third, sections were incubated with
PBS in the absence of the primary antibodies. These control sections did not reveal any staining
(Figure 1F and 2F). A qualified pathologist analyzed the staining for structures positive for ACE2.
Tissue distribution of ACE2
93
Figure 1.
Figure 1.Figure 1.
Figure 1. Normal lung tissue on overview (A)
(A)(A)
(A) and larger magnification (B)
(B)(B)
(B). Positive sta ining f or ACE2 is clearly present on
alveolar epithelial cell s (arrow) and capillairy endothelium (arrow-head). Fibrotic lung tissue (C)
(C)(C)
(C) a nd a larger magnification
(D)
(D)(D)
(D). Positive staining for ACE2 i s clearly present on type II cells (arrow). Cultured lung type II alveolar epithelial cells (A549)
stain strongly positive for ACE2 (E)
(E)(E)
(E). Control section stained with anti-ACE2 in the presence of the synthetic ACE2 peptide
shows negative staining in lung tis sue (F)
(F)(F)
(F).
Chapter 5
94
Figure 2.
Figure 2.Figure 2.
Figure 2. Oral mucosa on overview (A)
(A)(A)
(A). Strong staining is observed in vascular endothelium (arrow) and vascular smooth
muscle cells (arrow-head). Granular ACE2 staining is present in the basal layer of the epithelium. In the small intestine
(ileum) (B)
(B)(B)
(B) strong staining can be seen i n t he villous brush border (arrow), the muscularis mucosae (arrow-head) and the
muscularis propria (star). In a larger magnification of the submucosa (C)
(C)(C)
(C), strong staining is present in vascular endothelium
(arrow) and vascular smooth muscle cells (arrow-head). In a larger magnification of the villi (D)
(D)(D)
(D), abundant staining is seen on
the brush border of the enterocytes (arrow). In the colon (E)
(E)(E)
(E), ACE2 staining is present in endothelium and vascular smooth
muscle cells from the blood vessels (arrow-head) and in the muscular layers. Control section stained w ith anti-ACE2 in the
presence of the synthetic ACE2 pep tide shows no staining in the small intestine (ileum) (F)
(F)(F)
(F).
Tissue distribution of ACE2
95
Results
ResultsResults
Results
The mean age of patients (n=93) was 52 ± 22 year and the male-to-female ratio was 50/43.
The ACE2 staining pattern was consistent in the same type of tissues regardless of the
pathological condition of the organ and disease status of the patient.
The first remarkable finding was that ACE2 is present in endothelial cells from small and large
arteries and veins in all studied tissues. Moreover, arterial smooth muscle cells were consistently
positive for ACE2. Positive staining for ACE2 was also noted in myofibroblasts and the membrane
of fat cells in various organs. Furthermore, ACE2 was found at specific sites in each organ as
described below.
Marked ACE2 immunostaining was found in type I and type II alveolar epithelial cells in normal
lungs (Figure 1A and B). This finding was confirmed by ACE2 expression in the lung type II
alveolar epithelial cell-line A549 (Figure 1E) and by lungs with fibrotic changes which revealed
abundant staining of type II epithelial cells (Figure 1C and D). Cytoplasm of bronchial epithelial
cells showed also weak positive ACE2 staining.
In nasal and oral mucosa and the nasopharynx, we found ACE2 expression in the basal layer of
the non-keratinizing squamous epithelium (Figure 2A ).
Beside ACE2 localization in the smooth muscle cells and endothelium of the vessels from
stomach, small intestine, and colon we found ACE2 in smooth muscle cells of the muscularis
mucosae and the muscularis propria (Figure 2B, C, E). Remarkably, ACE2 was abundantly
present in the enterocytes of all parts of the small intestine including duodenum, jejunum and
ileum, but not in enterocytes of the colon. The staining in enterocytes was confined to the brush
border (Figure 2B and D).
In the skin, ACE2 is present in the basal cell layer of the epidermis extending to the basal cell
layer of hair follicles (Figure 3A, C and D). Smooth muscle cells surrounding the sebaceous
glands were also positive for ACE2. Weak cytoplasmic staining was observed in sebaceous gland
cells. Strong granular staining pattern for ACE2 was seen in cells of the eccrine glands (Figure
3B).
Consistent with findings in other organs, the brain only revealed endothelial and smooth muscle
cell staining (Figure 4A). Despite the clear endothelial staining of many small vessels, the
endothelial lining of the sinusoids in the liver was negative for ACE2. Surface staining in bile
ducts was occasionally observed. Kupffer cells and hepatocytes were negative (Figure 4B).
In the spleen, thymus, lymph nodes, and bone marrow, cells of the immune system such as B
and T lymphocytes, and macrophages were consistently negative for ACE2 (Figure 4C). In some
lymph nodes, we noted positive staining in sinus endothelial cells in a granular staining pattern.
In the kidney, weak glomerular visceral ACE2 staining was observed, whereas the parietal
epithelial cells were moderately positive. Despite the clear endothelial staining of vessels, the
mesangium and glomerular endothelium were negative for ACE2. Abundant staining was seen in
the brush border of the proximal tubular cells, whereas the cytoplasm of these cells was weakly
Chapter 5
96
positive. Epithelial cells from the distal tubules and collecting ducts showed weak cytoplasmic
staining (Figure 4D).
Figure 3.
Figure 3.Figure 3.
Figure 3. Skin tissue (A)
(A)(A)
(A) with larger magnification (C and D)
(C and D)(C and D)
(C and D). Staining is abundantly present in blood vessels/ capillaries and
in the basal layer of epidermis of the skin (arrow) and hair follicles (arrow-head ). Eccrine gl ands are also positive for ACE2
(B)
(B)(B)
(B).
Discussion
DiscussionDiscussion
Discussion
In the present paper we report the immunolocalization of Angiotensin-converting enzyme 2
(ACE2), the functional receptor for SARS-CoV, in human tissues. The most remarkable finding is
the surface expression of ACE2 protein on lung alveolar epithelial cells and enterocytes of the
small intestine, i.e. cells in contact with the external environment. Furthermore, ACE2 is present
in arterial and venous endothelial cells and arterial smooth muscle cells in all studied organs.
These data are consistent with previous findings that low levels of ACE2 mRNA are found in many
tissues and that ACE2 mRNA is highly expressed in renal, cardiovascular and gastrointestinal
tissues
8,10,12
.
The physiological role of ACE2 in most tissues has not been elucidated, although ACE2 is thought
Tissue distribution of ACE2
97
Figure 4.
Figure 4.Figure 4.
Figure 4. In the brain (A)
(A)(A)
(A), ACE2 is expressed only in endothelium (arrow) and smooth muscle cells of the vessels. In the liver
(B)
(B)(B)
(B) Kupffer cells, hepatocytes and the endothelium of sinusoids were negative. Surfac e staining in bile ducts was
occasionally ob served (arrow-head). Vascular endothelium (arrow) and smooth muscle cells were positive. In the spleen (C)
(C)(C)
(C)
ACE2 was not expressed in cells of the immune s ystem. Vascular- a nd red pulp sinus endothelium was positive. In the
kidney (D)
(D)(D)
(D) ACE2 is present in visceral (arrow) and parietal (arrow-head) epithelium, in the brush border (short arrow) and
cytoplasm of proximal tubular cells a nd in cytoplasm of distal tubules and collecting ducts.
to be an essential regulator of cardiac function and blood pressure control
9
, possibly by acting as
a natural counterpart of ACE1
14
. ACE2 has recently been identified as the functional receptor for
SARS-CoV
7
. Li et al showed that ACE2 can be immunoprecipitated by the S1 domain of the SARS-
CoV virus and that ACE2 can promote viral replication. The demonstration of ACE2 expression in
human organs can potentially identify the possible routes of infection for SARS-CoV, and possible
routes of spread and replication throughout the body.
SARS is mainly a lower respiratory tract disease, causing pulmonary lesions and respiratory
distress
5
. Furthermore, SARS-CoV is spread via the respiratory tract. Recent studies in autopsy
series using viral isolation, culture techniques and in-situ hybridization showed that SARS-CoV is
present in pneumocytes
13
. Transmission electron microscopy revealed presence of coronavirus-
like particles and viral inclusion bodies in pneumocytes. We found that type I and type II
Chapter 5
98
pneumocytes are markedly positive for ACE2 and that bronchial epithelial cells only show weak
staining. The type II alveolar epithelial cell line A549 confirmed the presence of ACE2 protein in
type II pneumocytes. This data, combined with the fact that ACE2 is the functional receptor for
SARS-CoV, indicates that alveolar pneumocytes in the lung are a possible site of entrance for
SARS-CoV. Furthermore, this expression pattern provides a possible explanation for the
pathologic lung manifestations and its rapid progression. Initial viral entrance may cause
cytopathological changes at the epithelial alveolo-capillary interface, initially resulting in
induction of type II alveolar cells as a first attempt to repair. In case of SARS, the abundant
expression of ACE2 in type II alveolar cells may cause a base for rapid viral expansion and a
vicious circle of local alveolar wall destruction, resulting in rapidly progressive severe diffuse
alveolar damage.
Upper respiratory tract symptoms occur in the minority of SARS patients and SARS-CoV RNA can
be detected in nasopharyngeal aspirates
15
. However, tissues of the upper respiratory tract, like
oral and nasal mucosa and nasopharynx did not show ACE2 expression on the surface of
epithelial suggesting that these tissues are not the primary site of entrance for SARS-CoV. The
upper respiratory tract symptoms cannot be explained by our findings, but patients with SARS
might be susceptible for secondary infections
16
. Moreover, SARS-CoV RNA detected in
nasopharyngeal aspirates might be derived from infected lower respiratory tract.
Extrapulmonary manifestations of SARS-CoV infection like gastrointestinal symptoms have been
reported and include watery diarrhoea
15,17,18,18,19
. Using in-situ hybridization, To et al. found
SARS-CoV in the surface of small intestine enterocytes
13
. Active viral replication in the
enterocytes of the small intestine has been reported by Leung et al
19
and SARS-CoV RNA can be
detected in stool of patients
15,17,19
. We showed that ACE2 protein is abundantly expressed in the
brush border of enterocytes of all parts of the small intestine, including duodenum, jejenum and
ileum. Surprisingly, other organs of the digestive tract as stomach and colon did not show this
brush border staining. The presence of ACE2 as a functional receptor for SARS-CoV and the
presence of SARS-CoV in enterocytes of the small intestine, combined with the fact that virus is
present in stool of patients is consistent with the possibility of oral-faecal transmission.
In addition to pulmonary and gastrointestinal problems, SARS-CoV infection also causes massive
necrosis of the spleen and lymph nodes. Furthermore, most patients develop lymphopenia
20
which, in analogy with respiratory syncytial virus disease, measles and sepsis has been ascribed
to increased apoptosis of lymphocytes
21
. The consistent absence of ACE2 in immune cells in all
haemato-lymphoid organs suggests that direct viral infection is unlikely to be the cause of these
manifestations and that the pathological changes seen in these organs are probably related to
the systemic effects of the abnormal immune reactions towards the virus.
Other SARS-CoV related manifestations include systemic vasculitis, apoptosis and swelling of
endothelial cells and inflammation in various organs like heart, kidney, liver and adrenal glands
5
.
The abundant expression of ACE2 on endothelia and smooth muscle cells in virtually all organs
Tissue distribution of ACE2
99
suggests that the SARS-CoV, once present in the circulation, can spread easily through the body.
The absence, however, of SARS-CoV in these organs as shown by in situ hybridization studies
13
is
at variance with this assumption. The vascular abnormalities and inflammatory changes in
various organs might therefore be related to systemic toxic effects of the immune reactions
elicited by SARS-CoV infection.
It is remarkable that despite the presence of ACE2 in endothelia of all organs and SARS-CoV in
blood plasma of infected individuals, so few organs become virus positive. This may imply that, in
analogy with HIV infection, where the current general model of viral entry requires not only
binding of the viral envelope to a cell surface receptor (CD4), but also to a chemokine co-receptor
[CXCR4 or CCR5(BBA)]
22
, SARS-CoV also needs the presence of a co-receptor for cellular entry.
Future studies have to elucidate whether SARS-CoV binding to a co-receptor in addition to ACE2
might be involved in the specific infection of lung and small intestine .
In conclusion, ACE2 is abundantly present in epithelia of the lung and small intestine in humans,
which might provide possible routes of entry for the SARS-CoV. This epithelial expression,
together with the presence in vascular endothelium, also provides a first step in understanding
the pathogenesis of the main SARS disease manifestation, in particular in the lung. Whether the
abundant expression in the vascular systems may also serve as a route of spread and
replication, should be further investigated in functional studies applying blockade of the ACE2
protein.
Acknowledgements
AcknowledgementsAcknowledgements
Acknowledgements
We thank M. Donoghue and S. Acton (Millennium Pharmaceuticals, Inc, 75 Sidney St, Cambridge,
MA 02139) for their kind gift of the ACE2 antibody. The authors thank Iris van Sen for skilled
photographical work.
Chapter 5
100
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