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

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.

Figure 1

Effect of different retrieval techniques on lectin binding. Single labelling of paediatric respiratory mucosa by FITC- conjugated Sambucus nigra agglutinin (SNA) (A-F) and Maackia amurensis agglutinin (MAA) (G-J) using different methods of retrieval. No antigen retrieval, (A) and (F), Citrate buffer (B) and (G), EDTA (C) and (H), Pronase (D) and (I), and Trypsin (E) and (J). Stain intensity was graded as strong (++) and a weaker pattern as +. In the absence of unmasking techniques there was minimal to weak (-/+) SNA binding and weak (+) MAA binding in the basal epithelium and epithelial cells of the bronchial mucosa of paediatric tissues. All forms of retrieval enhanced the lectin staining of the surface epithelial cells and mucus containing cells for both SNA and MAA. Examination with dual FITC/Rhodamine filter. Magnification × 200.

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.

Figure 3

Lectin binding to bronchial epithelium using different detection methods. Serial sections for comparison of detection methods for lectin binding of Sambucus nigra agglutinin (SNA) to SAα2,6 and Maackia amurensis (MAA) agglutinin for binding to SAα2,3 in adult bronchial epithelium. Double fluoresecence for FITC labelled SNA and TRITC labelled MAA shows a heterogeneous pattern (A), with more binding to the basal epithelium with MAA (B) than SNA (C). HRP labelling of MAA (D) and SNA (E) shows a similar pattern of binding to the fluorescent labelled lectin. 200 × magnification.

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.