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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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Comparison of MAA binding using MAA from different suppliers. Serial sections of adult lung tissue for comparison of lectin binding of Maackia amurensis (MAA) for SAα2,3Gal. Biotin conjugated MAA1(also known as MAL) from Vector Laboratories (A) and (E), Biotin conjugated MAA2 (also known as MAH) from Vector Laboratories (B) and (F), Digoxigenin conjugated MAA from Roche (C) and (G) and HRP conjugated MAA from EY Laboratories (D) and (H). Orange arrows indicate alveolar macrophages, Green arrows indicate alveolar pneumocytes and blue arrows indicate bronchiolar epithelium. Haematoxylin counterstain 200 × magnification.
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Figure 4: Comparison of MAA binding using MAA from different suppliers. Serial sections of adult lung tissue for comparison of lectin binding of Maackia amurensis (MAA) for SAα2,3Gal. Biotin conjugated MAA1(also known as MAL) from Vector Laboratories (A) and (E), Biotin conjugated MAA2 (also known as MAH) from Vector Laboratories (B) and (F), Digoxigenin conjugated MAA from Roche (C) and (G) and HRP conjugated MAA from EY Laboratories (D) and (H). Orange arrows indicate alveolar macrophages, Green arrows indicate alveolar pneumocytes and blue arrows indicate bronchiolar epithelium. Haematoxylin counterstain 200 × magnification.

Mentions: Because we found that there was a different binding pattern in the upper and lower respiratory tract for MAA1 and MAA2 using these lectins from Vector labs we then investigated whether the MAA from 2 other suppliers – EY Laboratories and Roche would identify one or both of the MAA isoforms. We therefore used serial sections from adult lung tissue and stained them using the Vector MAA1 and MAA2, digoxigenin labelled MAA (Roche) and biotinylated MAA from EY Laboratories. While EY Laboratories state that their MAA is a combination of 2 isoforms, this information is not evident from Roche. As expected using MAA1 (Fig 4A,E) the alveolar macrophages (orange arrows) and bronchial cells (blue arrow) bound MAA but pneumocytes (green arrow) showed no binding. Conversely MAA2 (Fig 4B,F) bound to pneumocytes (green arrows). The digoxigenin labelled MAA from Roche (Fig 4C,G) mainly bound to macrophages (orange arrows) and bronchial cells (blue arrow) but there was minimal binding to pneumocytes (green arrows) indicating that this MAA is mainly identifying MAA1 rather than MAA2. The MAA from EY Laboratories (Fig 4D,H) showed a weak (+) binding to the pneumocytes (green arrows) as well as strong binding (++) to bronchial epithelial cells and macrophages but it should be noted that this binding to pneumocytes appeared weaker than MAA2.


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)

Comparison of MAA binding using MAA from different suppliers. Serial sections of adult lung tissue for comparison of lectin binding of Maackia amurensis (MAA) for SAα2,3Gal. Biotin conjugated MAA1(also known as MAL) from Vector Laboratories (A) and (E), Biotin conjugated MAA2 (also known as MAH) from Vector Laboratories (B) and (F), Digoxigenin conjugated MAA from Roche (C) and (G) and HRP conjugated MAA from EY Laboratories (D) and (H). Orange arrows indicate alveolar macrophages, Green arrows indicate alveolar pneumocytes and blue arrows indicate bronchiolar epithelium. Haematoxylin counterstain 200 × magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2169242&req=5

Figure 4: Comparison of MAA binding using MAA from different suppliers. Serial sections of adult lung tissue for comparison of lectin binding of Maackia amurensis (MAA) for SAα2,3Gal. Biotin conjugated MAA1(also known as MAL) from Vector Laboratories (A) and (E), Biotin conjugated MAA2 (also known as MAH) from Vector Laboratories (B) and (F), Digoxigenin conjugated MAA from Roche (C) and (G) and HRP conjugated MAA from EY Laboratories (D) and (H). Orange arrows indicate alveolar macrophages, Green arrows indicate alveolar pneumocytes and blue arrows indicate bronchiolar epithelium. Haematoxylin counterstain 200 × magnification.
Mentions: Because we found that there was a different binding pattern in the upper and lower respiratory tract for MAA1 and MAA2 using these lectins from Vector labs we then investigated whether the MAA from 2 other suppliers – EY Laboratories and Roche would identify one or both of the MAA isoforms. We therefore used serial sections from adult lung tissue and stained them using the Vector MAA1 and MAA2, digoxigenin labelled MAA (Roche) and biotinylated MAA from EY Laboratories. While EY Laboratories state that their MAA is a combination of 2 isoforms, this information is not evident from Roche. As expected using MAA1 (Fig 4A,E) the alveolar macrophages (orange arrows) and bronchial cells (blue arrow) bound MAA but pneumocytes (green arrow) showed no binding. Conversely MAA2 (Fig 4B,F) bound to pneumocytes (green arrows). The digoxigenin labelled MAA from Roche (Fig 4C,G) mainly bound to macrophages (orange arrows) and bronchial cells (blue arrow) but there was minimal binding to pneumocytes (green arrows) indicating that this MAA is mainly identifying MAA1 rather than MAA2. The MAA from EY Laboratories (Fig 4D,H) showed a weak (+) binding to the pneumocytes (green arrows) as well as strong binding (++) to bronchial epithelial cells and macrophages but it should be noted that this binding to pneumocytes appeared weaker than MAA2.

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