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Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses.

Nicholls JM, Bourne AJ, Chen H, Guan Y, Peiris JS - Respir. Res. (2007)

Bottom Line: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract.In addition we found that there were differences in tissue distribution of the alpha2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used.This finding is important if conclusions about the potential binding sites of SAalpha2,3 binding viruses, such as influenza or human parainfluenza are to be made.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pathology Department, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong SAR. nicholls@pathology.hku.hk

ABSTRACT

Background: Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of alpha2,3-linked and alpha2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAalpha2,6Gal and Maackia amurensis agglutinin (MAA) for SAalpha2,3Gal in the respiratory tract of normal adults and children.

Methods: We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers.

Results: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the alpha2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs.

Conclusion: The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAalpha2,3 binding viruses, such as influenza or human parainfluenza are to be made.

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Lectin binding to upper and lower respiratory tract. Tissue distribution of Sambucus nigra agglutinin (SNA) for SAα2,6, Maackia amurensis agglutinin 1 (MAA1), and Maackia amurensis agglutinin 2 (MAA2) for SAα2,3 binding in the adult and paediatric respiratory tract. Serial sections of nasopharynx (A-C), adult bronchus (D-F), adult lung (G-I), paediatric bronchus (J-L), and paediatric lung (M-O) are shown and stained with SNA (A,D,G,J,M), MAA1 (B,E,H,K,N) and MAA2 (C,F,I,L,O). The adult nasopharynx shows SNA and MAA1 binding in the epithelium but no MAA2 binding. A similar pattern is also present in the adult bronchus and in addition the pneumocytes show MAA1 and MAA2 binding (E,F). Alveolar macrophages (G-I) demonstrate minimal SNA and no MAA2 binding but are positive for MAA1. The paediatric bronchus shows a greater binding of the epithelium with MAA1 (K) than the adult (E). The pneumocytes (M) also show more SNA binding than the adult (G). Staining using HRP conjugated SNA and biotin conjugated MAA1 and MAA2. (A-F) and (J-L) at 200 × magnification and (G-I) and (M-O) at 400 × magnification.
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Figure 2: Lectin binding to upper and lower respiratory tract. Tissue distribution of Sambucus nigra agglutinin (SNA) for SAα2,6, Maackia amurensis agglutinin 1 (MAA1), and Maackia amurensis agglutinin 2 (MAA2) for SAα2,3 binding in the adult and paediatric respiratory tract. Serial sections of nasopharynx (A-C), adult bronchus (D-F), adult lung (G-I), paediatric bronchus (J-L), and paediatric lung (M-O) are shown and stained with SNA (A,D,G,J,M), MAA1 (B,E,H,K,N) and MAA2 (C,F,I,L,O). The adult nasopharynx shows SNA and MAA1 binding in the epithelium but no MAA2 binding. A similar pattern is also present in the adult bronchus and in addition the pneumocytes show MAA1 and MAA2 binding (E,F). Alveolar macrophages (G-I) demonstrate minimal SNA and no MAA2 binding but are positive for MAA1. The paediatric bronchus shows a greater binding of the epithelium with MAA1 (K) than the adult (E). The pneumocytes (M) also show more SNA binding than the adult (G). Staining using HRP conjugated SNA and biotin conjugated MAA1 and MAA2. (A-F) and (J-L) at 200 × magnification and (G-I) and (M-O) at 400 × magnification.

Mentions: The 8 samples of fetal tissue at 20 weeks gestation showed strong binding (++) of MAA to the bronchial epithelium and to the developing pneumocytes with absent SNA detected. With advancing development the SNA binding increased and there was a switch in the MAA1 and MAA2 binding between the alveoli and bronchi (Table 1). The paediatric tissues from children with CCAM showed strong (++) binding in all cases of MAA to the bronchial epithelium but not to the alveoli. SNA was also strongly (++) bound to the bronchial epithelium but there was weaker (+) binding to the alveoli. Because we found greater binding of MAA in the respiratory tract than previously reported using the MAA from one supplier (EY Laboratories) we tested the MAA from another supplier (Vector) to verify the results. The MAA from EY labs has been identified as a combination of 2 isoforms of MAA – MAA1 and 2 which though both identifying SAα2,3Gal have different recognition patterns for the inner fragments. While MAA2 is specific towards SAα2,3Galβ1,3GalNAc and has been used to detect the "traditional" avian influenza receptor, MAA1 is more specific towards SAα2,3Galβ1,4GlcNAc [13]. When an analysis of sequential sections from the upper and lower respiratory tract was performed a number of consistent findings were observed. Firstly, SNA binding was more widespread in the upper than the lower respiratory tract and this was more pronounced in the adult tissues (Fig 2A,D,G,J,M). It was also present in the mucus secreting cells as well as the ciliated cells with a strong intensity (++). In the adult lung the pneumocytes showed only weak binding (+) of SNA (Fig 2G) but there appeared to be stronger binding (++) to the epithelium of the lungs of children (Fig 2M). Secondly, MAA1 was widely bound throughout the all the respiratory tract of children and adults and did not vary with age after delivery (Fig 2B,E,H,K,N). It bound to ciliated cells as well as the mucus secreting cells. In the paediatric bronchus it also bound to the basal cells (Fig 2K). Thirdly, MAA 2 binding appeared to be limited only to pneumocytes (+ – ++) and did not bind to cells in the nasopharynx or the bronchial epithelium (Fig 2F,O).


Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses.

Nicholls JM, Bourne AJ, Chen H, Guan Y, Peiris JS - Respir. Res. (2007)

Lectin binding to upper and lower respiratory tract. Tissue distribution of Sambucus nigra agglutinin (SNA) for SAα2,6, Maackia amurensis agglutinin 1 (MAA1), and Maackia amurensis agglutinin 2 (MAA2) for SAα2,3 binding in the adult and paediatric respiratory tract. Serial sections of nasopharynx (A-C), adult bronchus (D-F), adult lung (G-I), paediatric bronchus (J-L), and paediatric lung (M-O) are shown and stained with SNA (A,D,G,J,M), MAA1 (B,E,H,K,N) and MAA2 (C,F,I,L,O). The adult nasopharynx shows SNA and MAA1 binding in the epithelium but no MAA2 binding. A similar pattern is also present in the adult bronchus and in addition the pneumocytes show MAA1 and MAA2 binding (E,F). Alveolar macrophages (G-I) demonstrate minimal SNA and no MAA2 binding but are positive for MAA1. The paediatric bronchus shows a greater binding of the epithelium with MAA1 (K) than the adult (E). The pneumocytes (M) also show more SNA binding than the adult (G). Staining using HRP conjugated SNA and biotin conjugated MAA1 and MAA2. (A-F) and (J-L) at 200 × magnification and (G-I) and (M-O) at 400 × magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: Lectin binding to upper and lower respiratory tract. Tissue distribution of Sambucus nigra agglutinin (SNA) for SAα2,6, Maackia amurensis agglutinin 1 (MAA1), and Maackia amurensis agglutinin 2 (MAA2) for SAα2,3 binding in the adult and paediatric respiratory tract. Serial sections of nasopharynx (A-C), adult bronchus (D-F), adult lung (G-I), paediatric bronchus (J-L), and paediatric lung (M-O) are shown and stained with SNA (A,D,G,J,M), MAA1 (B,E,H,K,N) and MAA2 (C,F,I,L,O). The adult nasopharynx shows SNA and MAA1 binding in the epithelium but no MAA2 binding. A similar pattern is also present in the adult bronchus and in addition the pneumocytes show MAA1 and MAA2 binding (E,F). Alveolar macrophages (G-I) demonstrate minimal SNA and no MAA2 binding but are positive for MAA1. The paediatric bronchus shows a greater binding of the epithelium with MAA1 (K) than the adult (E). The pneumocytes (M) also show more SNA binding than the adult (G). Staining using HRP conjugated SNA and biotin conjugated MAA1 and MAA2. (A-F) and (J-L) at 200 × magnification and (G-I) and (M-O) at 400 × magnification.
Mentions: The 8 samples of fetal tissue at 20 weeks gestation showed strong binding (++) of MAA to the bronchial epithelium and to the developing pneumocytes with absent SNA detected. With advancing development the SNA binding increased and there was a switch in the MAA1 and MAA2 binding between the alveoli and bronchi (Table 1). The paediatric tissues from children with CCAM showed strong (++) binding in all cases of MAA to the bronchial epithelium but not to the alveoli. SNA was also strongly (++) bound to the bronchial epithelium but there was weaker (+) binding to the alveoli. Because we found greater binding of MAA in the respiratory tract than previously reported using the MAA from one supplier (EY Laboratories) we tested the MAA from another supplier (Vector) to verify the results. The MAA from EY labs has been identified as a combination of 2 isoforms of MAA – MAA1 and 2 which though both identifying SAα2,3Gal have different recognition patterns for the inner fragments. While MAA2 is specific towards SAα2,3Galβ1,3GalNAc and has been used to detect the "traditional" avian influenza receptor, MAA1 is more specific towards SAα2,3Galβ1,4GlcNAc [13]. When an analysis of sequential sections from the upper and lower respiratory tract was performed a number of consistent findings were observed. Firstly, SNA binding was more widespread in the upper than the lower respiratory tract and this was more pronounced in the adult tissues (Fig 2A,D,G,J,M). It was also present in the mucus secreting cells as well as the ciliated cells with a strong intensity (++). In the adult lung the pneumocytes showed only weak binding (+) of SNA (Fig 2G) but there appeared to be stronger binding (++) to the epithelium of the lungs of children (Fig 2M). Secondly, MAA1 was widely bound throughout the all the respiratory tract of children and adults and did not vary with age after delivery (Fig 2B,E,H,K,N). It bound to ciliated cells as well as the mucus secreting cells. In the paediatric bronchus it also bound to the basal cells (Fig 2K). Thirdly, MAA 2 binding appeared to be limited only to pneumocytes (+ – ++) and did not bind to cells in the nasopharynx or the bronchial epithelium (Fig 2F,O).

Bottom Line: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract.In addition we found that there were differences in tissue distribution of the alpha2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used.This finding is important if conclusions about the potential binding sites of SAalpha2,3 binding viruses, such as influenza or human parainfluenza are to be made.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pathology Department, The University of Hong Kong, Pok Fu Lam, Hong Kong, Hong Kong SAR. nicholls@pathology.hku.hk

ABSTRACT

Background: Influenza virus binds to cell receptors via sialic acid (SA) linked glycoproteins. They recognize SA on host cells through their haemagglutinins (H). The distribution of SA on cell surfaces is one determinant of host tropism and understanding its expression on human cells and tissues is important for understanding influenza pathogenesis. The objective of this study therefore was to optimize the detection of alpha2,3-linked and alpha2,6-linked SA by lectin histochemistry by investigating the binding of Sambucus nigra agglutinin (SNA) for SAalpha2,6Gal and Maackia amurensis agglutinin (MAA) for SAalpha2,3Gal in the respiratory tract of normal adults and children.

Methods: We used fluorescent and biotinylated SNA and MAA from different suppliers on archived and prospectively collected biopsy and autopsy specimens from the nasopharynx, trachea, bronchus and lungs of fetuses, infants and adults. We compared different methods of unmasking for tissue sections to determine if these would affect lectin binding. Using serial sections we then compared the lectin binding of MAA from different suppliers.

Results: We found that unmasking using microwave treatment in citrate buffer produced increased lectin binding to the ciliated and glandular epithelium of the respiratory tract. In addition we found that there were differences in tissue distribution of the alpha2,3 linked SA when 2 different isoforms of MAA (MAA1 and MAA2) lectin were used. MAA1 had widespread binding throughout the upper and lower respiratory tract and showed more binding to the respiratory epithelium of children than in adults. By comparison, MAA2 binding was mainly restricted to the alveolar epithelial cells of the lung with weak binding to goblet cells. SNA binding was detected in bronchial and alveolar epithelial cells and binding of this lectin was stronger to the paediatric epithelium compared to adult epithelium. Furthermore, the MAA lectins from 2 suppliers (Roche and EY Labs) tended to only bind in a pattern similar to MAA1 (Vector Labs) and produced a different binding pattern to MAA2 from Vector Labs.

Conclusion: The lectin binding pattern of MAA may vary depending on the supplier and the different isoforms of MAA show a different tissue distribution in the respiratory tract. This finding is important if conclusions about the potential binding sites of SAalpha2,3 binding viruses, such as influenza or human parainfluenza are to be made.

Show MeSH
Related in: MedlinePlus