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,1
* Department of Pathology, School of Basic Medical Sciences, and
Infectious Disease Center, Peking University, Beijing, China; and
Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA
1Correspondence: Department of Pathology, School of Basic Medical Sciences, Peking (Beijing) University, 38 Xueyuan Rd., 100083 Beijing, China. E-mail: jianggu{at}bjmu.edu.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-2,3-linked and -2,6-linked sialic acids, respectively. Until today, the distributions of these two receptor types had never been investigated in H5N1-infected human tissue samples. Here, the expression of avian (AIV-Rs) and human influenza receptors (HuIV-Rs) is studied in various organs (upper and lower respiratory tracts, brain, placenta, liver, kidney, heart, intestines, and spleen) of two H5N1 cases and 14 control cases. Histochemical stains using biotinylated Maackia amurensis lectin II and Sambucus nigra agglutinin were performed to localize AIV-Rs and HuIV-Rs, respectively. Immunohistochemical stainings were performed to identify the receptor-bearing cells. AIV-Rs were detected on type II pneumocytes; a limited number of epithelial cells of the upper respiratory tract; and the bronchi, bronchioli, and trachea; as well as on Kupffer cells, glomerular cells, splenic T cells, and neurons in the brain and intestines. HuIV-Rs were abundantly present in the respiratory tract and lungs. They were also detected on Hofbauer cells, glomerular cells, splenic B cells, and in the liver. Moreover, endothelial cells of all organs examined expressed both receptor types. In conclusion, the distribution pattern of AIV-Rs is partially inconsistent with the pattern of infected cells as detected in previous studies, which suggests there may be other receptors or mechanisms involved in H5N1 infection. In addition, the diffuse presence of receptors on endothelial cells may account for the multiple organ involvement in H5N1 influenza. Finally, the relative lack of AIV-Rs in the upper airway may be a one of the factors preventing efficient human-to-human transmission of H5N1 influenza. Yao, L., Korteweg, C., Hsueh, W., Gu, J. Avian influenza receptor expression in H5N1-infected and noninfected human tissues.
Key Words: sialic acid • human influenza virus receptors • lectin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The fact that a massive outbreak has not occurred may be due, at least in part, to differences between avian and human influenza viruses in their ability to bind to cell surface receptors. Avian and human influenza viruses bind to sialyloligosaccharides with different binding molecules. The hemagglutinin (HA) of avian influenza viruses favors sialic acids linked to galactose by -2,3 linkage (SA-2,3 Gal); the HA of human influenza viruses binds to sialic acids with -2,6 linkage (SA-2,6 Gal) (2
3
4
5)
. The distribution of these two forms of sialic acid compounds is known to be cell and species specific. It is widely believed that the low prevalence of SA-2,3 Gal molecules, the so-called avian influenza virus receptors (AIV-Rs), on the epithelial cells of the human upper respiratory tract decreases the susceptibility of humans to infection by avian influenza virus (6)
. However, genomic mutations of the HA of avian influenza virus have been known to change binding preference from the -2,3 linked to the -2,6 linked SA receptor (7
8
9
10)
. Potentially such an occurrence could unleash extremely serious, widespread epidemics. Despite this, efforts to localize AIV-Rs in human tissues have been few, and those available are mostly limited to the respiratory tract (11
12
13
14
15
16
17)
. Furthermore, these previous studies used either cultured airway epithelial cells or tissue sections of lungs from patients who did not die of infectious diseases. Although the major target for the virus appears to be the lungs, avian influenza is a systemic disease that affects multiple organs (18)
. We have demonstrated in a previous study that except the lungs, other organs may also be infected by the H5N1 virus, including the trachea, intestines, brain, and placenta (19)
. Hence the importance of recognizing patterns of avian influenza virus receptor expression in specific types of human cell populations is apparent. The present study aims to 1) detect the expression of AIV-Rs in a variety of human tissues; 2) compare them with the expression of human influenza virus receptors (HuIV-Rs); and 3) compare the patterns of receptor expression between H5N1 cases and noninfected control cases. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The clinical data of this case have been reported in detail (20)
. The other victim (case 2) was a previously healthy 35-year-old man, who presented with pneumonia and high fever. A diagnosis of avian influenza was made by the Chinese Center for Disease Control and Prevention in Beijing. Despite treatment with Oseltamivir (Tamiflu®) and supportive care, he died 3 wk after admission of multiorgan failure, including impairment of cardiac and renal functions. We have used lectin histochemistry to study the distribution of avian and human influenza virus receptors in the tissues collected at the autopsy of these two patients. In addition, human tissues of 14 autopsy cases were selected as controls from the tissue archives of the Department of Pathology at Peking University Health Science Center. The control group consisted of Chinese adult patients of both genders. None of the control patients had infectious pulmonary diseases. The following are the organs that were studied: upper respiratory tract (nasal mucosa and pharynx), lower respiratory tract (trachea, bronchi, bronchioli, and lungs), brain, placenta, liver, kidneys, heart, small and large intestines, and spleen. The brain of case 1 was not available for investigation. In addition, neither the pharyngeal nor the nasal tissues of the H5N1 cases were available for investigation. Also the thymus, thyroid gland, and pancreas could not be investigated. We compared the receptor distribution of H5N1-infected cases with that of control cases to the extent the relevant tissues were available.
As H5N1 viral sequences and viral proteins have previously been detected in the lower respiratory tract, the brain, the intestines, placental cytotrophoblasts, and immune cells, including Hofbauer cells (placental macrophages) and circulating mononuclear cells (19)
, the present study also mainly focuses on these organs and cells in an attempt to better understand the virus-receptor-cell interaction.
Lectin histochemistry
Paraffin-embedded consecutive sections of 4-µm thickness from all of these cases were stained with biotinylated, sialic acid-specific lectins. Biotinylated Maackia amurensis lectin II (MAA), which is specific for -2,3-linked sialic acid (a marker for AIV-R), and biotinylated Sambucus nigra agglutinin (SNA), specific for binding the -2,6 linked sialic acid (a marker for HuIV-R), were purchased from Vector Laboratories (Burlingame, CA, USA). Lectin histochemistry was performed according to the procedures described by Cerna et al. (21)
. Briefly, the formalin-fixed, paraffin-embedded tissue sections were deparaffinized and immersed in 3% hydrogen peroxide to eliminate endogenous peroxidase activity. 5% bovine serum albumin was used to block nonspecific staining. The tissue sections were incubated with SNA (0.625 µg/ml) and MAA (1.25 µg/ml) in buffer at 4°C overnight. Optimal contrast between the specific labeling and the background was obtained with the use of a SABC kit (Dako, Carpinteria, CA, USA). Biotinylated lectin binding was visualized using a DAB (3,3'-diaminobenzidine-tetrahydrochloride) substrate-chromogene kit (Zymed Labs, South San Francisco, CA, USA), which gives a brown color, and slides were counterstained with hematoxylin. Negative controls were done by using slides incubated with phosphate-buffered saline (PBS) instead of the lectin.
Neuraminidase pretreatment
To confirm the specificity of the lectin stains, neuraminidase pretreatment was performed. Because neuraminidase digests both -2,3-linked and -2,6-linked sialic acid residues, negative lectin staining after neuramidase pretreatment would indicate that the lectin stainings used are specific for detection of -2,3-linked and -2,6-linked sialic acids. The paraffin-embedded tissue sections were deparaffinized and immersed in 3% hydrogen peroxide to eliminate endogenous peroxidase activity. Slides were covered with 12.5 U/µl neuraminidase (NEB, Ipswich, MA, USA) for 24 h incubation at 37°C. The slides were then washed three times with PBS. 5% BSA was used to block nonspecific staining. Lectin staining was subsequently performed as described above. Additional negative controls were performed by using slides incubated with PBS instead of neuraminidase.
Immunohistochemistry
To identify the specific cell types, immunohistochemical stains were performed using monoclonal antibodies to CD3 (T lymphocytes) (Zymed Labs), CD20 (B lymphocytes) (Zymed Labs), CD68 (macrophages) (Zymed Labs), tubulin β (ciliated epithelial cells) (Zymed Labs), microtubule-associated protein 2 (MAP2) (neurons) (Zymed Labs), and surfactant protein A (type II pneumocytes) (Dako Cytomation, Glostrup, Denmark). Briefly, deparaffinized tissue sections were incubated with primary antibodies after being treated by 3% hydrogen peroxide and processed for antigen retrieval (by heating in a microwave oven at 96°C, in 0.01 M citrate buffer, at pH 6, for 15 min.). The reaction products were colorized with PV9000 immunohistochemistry (IHC) kit (Zymed Labs) and DAB substrate-chromogen kit, resulting in a brown signal. Negative controls were carried out with PBS instead of the primary antibody.
Double labeling and serial sections
To identify the specific cell types expressing AIV-Rs or HuIV-Rs, both lectin and IHC stains were performed on serial sections. Double labeling with MAA or SNA staining for receptor detection and IHC with antibodies to tubulin β for ciliated epithelial cells was also carried out. Briefly, deparaffinized sections were treated as described above. After incubation with lectin overnight, alkaline phosphatase-labeled strepatividin (1:400) (Zymed Labs) was added to the slides at room temperature for 30 min of incubation. Nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Promega Corp., Madison, WI, USA), which gives a purplish-blue color, was subsequently used to visualize the signals. Following the lectin colorization reaction, sections were incubated with 3% hydrogen peroxide to quench endogenous peroxidase activity, and then they were incubated with monoclonal antibodies to tubulin β with incubation overnight at 4°C. After washing in PBS, sections were incubated with goat anti-mouse IgG labeled with horseradish peroxidase (HRP) at room temperature for 30 min. Tubulin β antibodies were detected with 3-amino-9-ethylcarbazole (AEC) (Sigma, St. Louis, MO, USA), which gives a brownish-red reaction color.
Because lymphocytes are too small to show up in sufficient numbers in consecutive sections of splenic tissue, we have instead compared the receptor distribution patterns of HuIV-Rs and AIV-Rs with the cell distribution patterns of both splenic B and T cells in B-cell and T-cell-rich regions in the spleen.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. This table lists the expression of receptors for AIV (-2,3-linked sialic acid) and for human influenza virus (HuIV) (-2,6-linked sialic acid), respectively, in each organ investigated. Furthermore, it indicates per organ whether a comparison could be made between the receptor distribution in H5N1 cases and that in control cases, and if so, the outcome of such a comparison.
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For each organ studied, the receptor distribution was consistent throughout all cases within the relevant group. Table 1
, however, shows that the detection of AIV-R and HuIV-R differed markedly for each organ.
Figures 1
, 2,
and 3
further illustrate our results. To avoid redundancy only images from control cases are shown, in case of similar results in H5N1-infected and control subjects. With respect to the lungs, representative tissues from both H5N1 and control cases are shown, as in the H5N1 cases the structure of the lower airway was distorted to such an extent that it was impossible to discern the types of cells expressing the receptors. Only images of the spleen in control cases are presented, due to the severe lymphocyte depletion in H5N1 cases.
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In the upper airway (Fig. 1A-F
), only a small proportion of nonciliated epithelial cells and a few ciliated cells stained positive for AIV-R in the nasal mucosa (Fig. 1A, B
). In contrast, the HuIV-R was expressed abundantly on both ciliated and nonciliated cells of the nasal mucosa (Fig. 1C
). Only nonciliated epithelial cells, putative basal cells stained focally positive for AIV-R in the pharynx (Fig. 1D, E
), whereas HuIV-R staining was diffusely expressed on both ciliated and nonciliated epithelial cells (Fig. 1E, F
). No comparison in the receptor distribution of the upper respiratory tract could be made between the H5N1 and control cases, due to the fact that the nasal mucosal and pharyngeal tissues of the H5N1 cases were not available for investigation.
In the lower respiratory tract, the trachea showed a receptor distribution similar to that of the pharynx, with AIV-Rs only sparsely present on nonciliated cells (Fig. 1G, H
) and HuIV-Rs diffusely detectable on both ciliated and nonciliated epithelial cells (Fig. 1H, I
). This receptor expression pattern differed from the one seen in bronchi and bronchioles, where the AIV-R type was expressed focally on both ciliated and nonciliated cells (Fig. 1J, K
). The HuIV-R type, in contrast, was diffusely expressed on ciliated and nonciliated cells (Fig. 1L
). In the pulmonary parenchyma, a relative restriction of AIV-R distribution was again noted: AIV-R expression seemed limited to type II pneumocytes (Fig. 1M, N
). Alveolar macrophages (Fig. 1P, Q
) and type I pneumocytes were negative for AIV-R staining (Fig. 1M
). In contrast, SNA staining was diffusely present on both type I and type II pneumocytes, but not on alveolar macrophages (Fig. 1N, O, Q, R
). The distribution of AIV-Rs and HuIV-Rs in the trachea did not notably differ in H5N1 and control cases. As the structures of the bronchi and lungs of the H5N1 cases were distorted due to avian influenza infection, it was not possible to accurately compare the receptor distribution in the H5N1-infected and noninfected subjects. To the extent visible, however, the overall distribution and receptor density in the bronchi and lungs appeared to be broadly similar for both groups.
In the brain AIV-R staining was detected on neurons (Fig. 2A
), although the positive signals were weak compared to the high background staining. Following neuraminidase pretreatment the background staining was still visible, whereas the positive staining of the neurons had disappeared (Fig. 2B
). HuIV-R staining was focally present only on endothelial cells of blood vessels (Fig. 2C
). In the placenta, endothelial cells expressed both HuIV-R and AIV-R, whereas Hofbauer cells stained only positive for HuIV-R (Fig. 2D-F
). Both cytotrophoblasts and syncytiotrophoblasts were negative for both receptor types. In the liver, Kupffer cells were focally and weakly positive for both receptors, but only HuIV-R was expressed on the bile duct epithelium. Hepatocytes were only positive for HuIV-R (Fig. 2G-I
). In the kidney, the endothelial cells of the glomerulus stained diffusely for both HuIV-R and AIV-R. The distal convoluted tubular cells showed positive staining for HuIV-R, but not for AIV-R. Other tubular cells were negative for both receptors (Fig. 2J, K
). The heart was negative for both receptors, except for the endothelial cells of the blood vessels (Fig. 2L, M
). In the small and large intestines, both receptor types were seen on the endothelial cells of blood vessels and inflammatory cells but were absent in the epithelial cells (Fig. 2N, O
). Neurons of both small and large intestines were positive for AIV-R staining but not for HuIV-R staining (Fig. 2P-R
). As to the brain, placenta, liver, kidney, heart, and intestines, there were no differences in AIV-R and HuIV-R expression among the H5N1 cases and the control cases.
Because of the severe lymphocyte depletion in the spleens of H5N1 cases, the splenic receptor distribution could not be examined in these cases in the manner described in the section Double Labeling and Serial Sections. Therefore, only the results of receptor staining in the control group spleens are shown (Fig. 3A-D
). AIV-R was expressed mainly on T cells (identified by CD3 staining), whereas HuIV-R was expressed primarily on B cells (identified by CD20 staining). Accordingly, no comparison in receptor distribution between H5N1-infected cases and control cases could be made.
In addition, it is noteworthy that vascular endothelial cells expressed both receptor types. As all organs examined possessed blood vessels, both receptor types were detected in all organs investigated. Many blood cells (including erythrocytes and leukocytes) that were present in the blood vessels or sinuses of these organs also stained positive for both AIV and HuIV receptors.
The specificity of IHC staining was confirmed by the negative results in the negative controls. Treatment with neuraminidase prior to lectin staining resulted in absence of staining and thus confirmed the specificity for both SNA and MAA (Fig. 1U, V
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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11
12
13
14
15
16
, 22)
. In the current study, only a small proportion of nonciliated cells of the nasal mucosa and the pharynx expressed AIV-Rs. Although viral recognition of cell surface sialic acid linkages is not the only determinant of infectivity, this finding allowed us to hypothesize that the lack of AIV-Rs on the superficial epithelium of the upper airway may account for the low contagiousness of AIV in humans—until now no evidence of efficient human-to-human transmission has been provided (22)
. However, the current findings together with several observations made in previous studies (19
, 23)
appear to contradict this hypothesis. First, productive viral replication has been demonstrated in ex vivo nasopharyngeal tissues despite the lack of AIV-R expression on these cells (23)
. Second, H5N1 infection has been detected in a significant number of epithelial cells in the trachea (19)
, contrasting with the relative lack of AIV-Rs on these cells as seen in the current study. Therefore, it seems unlikely that the relative lack of AIV-Rs in the upper respiratory tract would be the only factor accounting for the low contagiousness of AIV in humans.
In contrast to the upper respiratory tract, AIV-Rs are abundantly present in the lower respiratory tract, especially in type II pneumocytes. The presence of AIV-Rs on type II pneumocytes is supportive of previous studies that have detected H5N1 viral antigens (19
, 24)
and genomic sequences (19)
in type II pneumocytes of the lungs of H5N1 autopsy cases and H5N1 virus attachment to pneumocytes (25)
. It is remarkable, however, that despite the widespread and abundant expression of AIV-Rs in the lungs, only a limited number of pneumocytes have been found to be infected in previous studies (19
, 24)
, implying that the presence of AIV-Rs is not the only factor affecting the capability of AIV to infect cells.
It is noteworthy that several cell types in the respiratory tract, including type II pneumocytes and nonciliated epithelial cells of the respiratory tract, express both receptor types. The presence of both receptors in the respiratory tract not only implies that humans are susceptible to both forms of influenza, albeit not to the same extent, but also that they could become the repository where a potential genomic reassortment may be realized. When two strains of viruses exist in the same host, the possibility exists of genomic interchange (reassortment). In fact, the deadly 1957 Asian and 1968 Hong Kong epidemics of influenza A virus are known to have been caused by reassortment of genomic subunits between human and avian influenza viruses (9
, 26)
. From our study and previous studies, it appears that the human respiratory tract, with its high avidity for influenza virus, could be a site for emergence of reassortants possessing genes that confer to them wider receptor-binding range.
Conflicting results regarding the cell types expressing AIV-Rs in the trachea and bronchi have been reported. Some previous studies have detected AIV-Rs in human airway tissue sections primarily on either ciliated cells (11
, 12
, 15
16
17)
or goblet cells (13
, 14)
, whereas others have failed to detect AIVRs in the human trachea or bronchi (10)
. In our study AIV-Rs were primarily found on nonciliated cells of the trachea and bronchi. These differences may be due to restrictions of lectin staining techniques (15)
. Theoretically, other factors could also be involved, although there is no evidence supporting this. We have detected HuIV-Rs on both ciliated and nonciliated cells of the trachea and bronchi, which is consistent with the findings of others (12
, 15
, 16)
.
AIV-Rs are expressed on neurons in the brain which is consistent with the detection of H5N1 genomic sequences and antigens in the brain of a H5N1 case (19)
and with H5N1 virus isolation from cerebrospinal fluid of a boy who suffered from acute encephalitis (27)
.
Neither HuIV-Rs nor AIV-Rs are detectable in epithelial cells of the intestines. The lack of HuIV-Rs is in accord with previous studies (28)
. However, the absence of AIV-Rs does not agree with the findings of Sata et al. (28)
, who did detect -2,3-linked sialic acid residues in the intestines. It also conflicts with the infection of epithelial cells as suggested in a previous study on tissue tropism (19)
. The presence of AIV-Rs on neurons in the intestines may be of interest, as infection of such cells could explain the gastrointestinal symptoms often observed in H5N1 cases (18
, 27
, 29)
.
Similar to our findings in the intestines, the absence of AIV-Rs on ciliated epithelial cells of the trachea is remarkable as it contrasts with the detection of H5N1 viral genomic sequences and proteins in a large number of such cells (19)
. In addition, the absence of AIV-R expression on alveolar macrophages, bronchiolar epithelial cells, cytotrophoblasts, and Hofbauer cells is not in line with the detected infection of these cells (19
, 23)
. It is conceivable that other yet unidentified receptors or mechanisms may be involved in the interaction between the H5N1 virus and the target cells. Nicholls et al. (23)
have put forward a similar suggestion based on their findings of productive viral replication in ex vivo nasopharyngeal tissues despite the lack of AIV-R expression on these cells.
Hofbauer cells have been found to express HuIV-Rs but not AIV-Rs. The implication of this finding remains to be explored.
We also detected AIV receptors and HuIV receptors on both red and white blood cells. MAA has previously been reported to bind to eosinophils, neutrophils, lymphocytes, and erythrocytes (30)
. It is not difficult to imagine that attachment of AIV to blood cells facilitates its spread to all regions of the body. In line with previous studies (31)
, we found that AIV-Rs are primarily expressed on T lymphocytes, whereas HuIV-Rs are mainly expressed on B lymphocytes. This might be related to the frequent occurrence of lymphopenia in H5N1-infected patients (18
, 29
, 32)
.
For a number of organs, a comparison could be made between the receptor distribution of H5N1-infected vs. noninfected subjects. In these organs, no differences in pattern or intensity in the staining of AIV-R were detected. This seems to suggest that, at least in these organs, the receptors for the virus are neither up- nor down-regulated after the individual is infected by the virus. However, there were also a number of organs and tissues where no comparison could be made because of either tissue unavailability (nasal mucosa and pharyngeal tissues) or severe distortion of the tissue architecture (lungs and spleen). Therefore, no definite conclusions can be drawn in this respect, and further investigation will be necessary.
The widespread distribution of AIV-Rs, as observed in this study, is remarkable. The evidence of strong expression in endothelial cells throughout the body seems especially significant, particularly in view of the necrotizing, fulminant nature of the pneumonia caused by AIV subtype H5N1 (33
34
35)
. Diffuse endothelial vascular expression of AIV-Rs may also provide an anatomical correlate for the presence of viral RNA in multiple organs as detected by RT-PCR (19)
and for the diffuse intravascular coagulation, which is observed in patients who succumb to this disease (36)
. In this context, animal experiments have amply demonstrated a coagulopathy in chickens with AIV infection (37)
.
In conclusion, the diffuse presence of the receptors on endothelial cells may account for the multiple organ involvement in avian influenza. Infection with avian influenza virus may not change its receptor expression in tissues. The absence of AIV-Rs in several cell types, including ciliated epithelial cells of the trachea, intestinal epithelial cells, cytotrophoblasts, and Hofbauer cells appears to be in contrast with the detected H5N1-infection of these cells. This suggests that other receptors or coreceptors might be involved in virus-target-cell interaction in H5N1 avian influenza.
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ACKNOWLEDGMENTS |
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Received for publication July 17, 2007. Accepted for publication September 6, 2007.
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REFERENCES |
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