Vol. 60, No. 10

INFECrION AND IMMUNITY, OCt. 1992, p. 4015-4023

0019-9567/92/104015-09$02.00/0 Copyright © 1992, American Society for Microbiology

Immunoelectron Microscopic Studies Reveal Differences in Distribution of Sialo-Oligosaccharide Receptors for Mycoplasma pneumoniae on the Epithelium of Human and Hamster Bronchi R. W. LOVELESS,1 S. GRIFFITHS,2 P. R. FRYER,2 C. BLAUTH,3t AND T. FEIZI1* Glycoconjugates Section' and Electron Microscopy Group, 2 MRC Clinical Research Centre, Watford Road, Harrow, Middlesex, HAl1 3UJ, and Harefield Hospital, Harefield, Middlesex, UB9 6JH, 3 United Kingdom Received 12 March 1992/Accepted 9 July 1992

Long-chain sialo-oligosaccharides with poly-N-acetyllactosamine backbones (Ii antigen type) are maijor host cell receptors for the human pathogen Mycoplasma pneumoniae. Previous immunofluorescence studies of the human bronchial epithelium, using sequence-specific monoclonal antibodies to the branched I-type and linear i-type backbones, have indicated that sialylated and nonsialylated long-chain sequences of both types are richly expressed on the ciliated cells, where they are polarized at the apical aspects. These sequences are lacking in the goblet cells. In the present study, the display of these oligosaccharides has been investigated by electron microscopy (immunogold labelling) in the human bronchial epithelium and in that of the hamster, an animal model commonly used for M. pneumoniae infection. In the human bronchial epithelium, the long-chain branched sequences have been detected along the entire length of the cilia and on microvilli, whereas the linear sequences are confined to the microvilli and the basal aspects of the cilia. On the ciliated epithelial cells of the hamster, by contrast, the branched and linear sequences (sialo- and asialo-) have been detected exclusively on microvilli. A further striking difference is that in the hamster these structures are expressed in abundance on the goblet cells and in the intracellular globules. We suggest that the latter finding may partly explain the relatively large doses of M. pneumoniae required to establish experimental infection in the hamster, as the receptor-bearing secreted mucus may have a protective role in binding to the microorganisms, leading to their clearance by bronchociliary action.

Mycoplasma pneumoniae is a pathogen of the human respiratory tract, and infection with this microorganism is often associated with the transient production of cold agglutinins, autoantibodies directed against the carbohydrate antigen designated I which is present on erythrocytes and other cell types (6). M. pneumoniae binds in vitro to a number of cell types, including erythrocytes (17, 22) and ciliated epithelial cells (22). The major receptors involved in M. pneumoniae binding appear to contain sialic acid, as treatment of the different cell types with sialidase markedly decreases binding (17, 22). It is not yet known whether the residual binding to sialidase-treated cells is due to incomplete removal of sialic acid (13, 19) or to other attachment sites such as sulfated glycoconjugates to which M. pneumoniae binds in vitro (12, 20). Previous studies from this laboratory (13, 14) have shown that the receptors for M. pneumoniae on human erythrocytes are a family of sialylated oligosaccharides with long-chain backbones of the poly-N-acetyllactosamine type, among which those with branched backbones (sialylated I antigen) are more strongly bound than those with linear backbones (sialylated i antigen). These carbohydrate structures occur on glycoproteins such as Band 3 (13) and on a series of glycolipids (14). These findings have raised the possibility that anti-I production is triggered by an autoimmunogenic complex formed between the host oligosaccharide receptors and the lipid-rich mycoplasma. *

Immunohistochemical light microscopic studies have shown that the I and i antigens, as well as their sialylated forms, are present in abundance at the primary site of infection, namely, the human bronchial epithelium (15), in which they occur exclusively on ciliated cells and are highly polarized at the apical surface. In the present study, we have examined more precisely by immunoelectron microscopy the display of these long-chain sialo-oligosaccharides in the human bronchial epithelium and have extended the study to include their distribution in the bronchial epithelium of the Syrian hamster, an animal model commonly used for M. pneumoniae infection.

MATERIALS AND METHODS Bronchial tissue. Samples of human bronchus were taken from three patients undergoing surgery for bronchial carcinomas. Freshly resected tissues taken from unaffected areas were cut into pieces of approximately 1 mm3 and fixed for 16 h at 4°C in 2% paraformaldehyde in PB (0.1 M phosphate buffer, pH 7.4). Tracheas from three Syrian hamsters sacrificed by asphyxiation were treated in a similar fashion. Processing of tissue. Fixed tissues were washed in PB and quenched for 30 min at 4°C with 0.5 M ammonium chloride in PB. Samples were dehydrated in graded methanol, 1 h each in 30 and 50% (by volume) methanol at 4°C and in 80 and 90% methanol at -20°C, and infiltrated with a graded series of Lowicryl K4M resin diluted in 90% methanol at -20°C (1). After infiltration for a minimum of 36 h, the resin was polymerized under UV lamp for 36 h at -30°C. Polymerized blocks were stored in a desiccator until sectioned

Corresponding author.

t Present address: Guys Hospital, London, SE1, United King-

dom.

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LOVELESS ET AL.

INF-Ecr. IMMUN. TABLE 1. Oligosaccharide sequences investigateda

Antigen(s)

I(Ma)

Oligosaccharide sequence

Means of detection

GalP1-4GlcNAcI13

Anti-I(Ma)

Gal/GalNAc Sialosyl-I(Ma)

GalI1-4GlcNAcI1

GaVGalNAc

SA

I(Step)

Enhanced anti-I(Ma) reactivity following

treatment \ al/GaINAc6sialidase 6

... GlcNAcpl

Anti-I(Step) 6

GalP1-4GlcNAcP1/ Sialosyl-I(Step)

...

GlcNAcpl\

3 Gal,P1-4GlcNAcP1-

6Gal31-4GlcNAcI1-

Enhanced anti-I(Step) reactivity following sialidase treatment

GalP1-4GlcNAcI1 SA

i(Den)

GalIl-4GlcNAcI31-3GalP1-4GlcNAcP1-3GalP1-4GlcNAcP1-

Anti-i(Den)

Sialosyl-i(Den)

GalPl-4GlcNAcI1-3GalPl-4GlcNAcP1-3Gal41-4GlcNAcI31-

Enhanced anti-i(Den) reactivity following sialidase treatment

SA VIM-2

GalI1-4GlcNAcP1-3GalP1-4GlcNAcI31-3GalI31-4GlcNAcP12-3 SAa

FC10.2 and Woo Sialosyl-FC10.2 and

1-3 Fuca

Galp1-3GlcNAcp1-3Galf1-4Glc/GlcNAc

Gal41-3GlcNAcI31-3Gal$1-4Glc/GlcNAc

Sialosyl-Woo

VIM-2 antibody; reactivity abolished by sialidase

FC10.2 antibody and IgMW°° macroglobulin Enhanced FC10.2 and IgMw°° reactivity following sialidase treatment

SA a Abbreviations: Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Glc, glucose; GlcNAc, N-acetylglucosamine; SA, sialic acid. For the sialosyl-I and sialosyl-i sequences, the form and linkage of sialic acid are not specified; for the oligosaccharides reactive with VIM-2 antibody, the presence of a2-3-linked N-acetylneuraminic acid is inferred.

with a Reichert Ultracut E ultramicrotome and a Diatome diamond knife. Sections were placed on Formvar-carboncoated 200-mesh copper grids and stored at 4°C in the dark until labelled. Carbohydrate sequence-specific antibodies. The oligosaccharide sequences investigated are shown in Table 1. The anti-I and anti-i antibodies are human monoclonal immunoglobulin M (IgM) autoantibodies (6). Anti-I(Ma) recognizes the terminal trisaccharide of the 31-6 branch which occurs both on short oligosaccharides of the N-acetyllactosamine (GalI1-4GlcNAc) type and on long-chain oligosaccharides of the poly-N-acetyllactosamine type. Anti-I(Step) recognizes the more extended structure of the branched I antigen whereas anti-i(Den) recognizes the corresponding linear i antigen containing at least three N-acetyllactosamine units (7). Since substitutions at the nonreducing terminal galactose with sialic acid or other monosaccharides mask reactivities with these antibodies (6), an increase in intensity of staining or the appearance of new immunoreactivities following sialidase treatment of tissues is taken as an indication of the presence of sialyl-I and sialyl-i antigens. The mouse hybridoma IgM antibody VIM-2 (gift of W. Knapp, Institute for Immunology, University of Vienna) binds specifically to the linear poly-N-acetyllactosamine backbone when substituted both with N-acetylneuraminic acid a2-3 linked to the nonreducing terminal galactose and with fucose al-3 linked at an internal N-acetylglucosamine residue (16). A Waldenstrom macroglobulin, IgMw°, and the mouse hybridoma IgM antibody FC10.2 (gift of R. A. J. McIlhinney, MRC Anatomical Neuropharmacology Unit, Oxford, United Kingdom), both of which recognize the isomeric tetrasaccharide se-

quence Gal1-3GlcNAcpl-3Gal$1-4Glc/GlcNAc (8, 11), were used as negative controls for the human and mouse monoclonal antibodies, respectively. Normal human blood group AB serum supplemented with an unrelated Waldenstrom IgM protein (4 mg/ml) was used as an additional control for the samples of human bronchus. Immunolabelling. Ultrathin sections on copper grids were incubated before or after treatment for 1 h at 37°C with sialidase (EC 3.2.1.18) (200 mU/ml) from Vibrio cholerae (Behringwerke, Marburg, Germany), diluted in 0.15 M sodium acetate buffer (pH 5.5) containing 1% bovine serum albumin (BSA). Control sections were incubated with buffer alone. Nonspecific binding was blocked by incubating sections for 1 h with 3% BSA in 10 mM phosphate buffer in 0.85% NaCl, pH 7.3 (phosphate-buffered saline [PBS]). Grids were immunolabelled for 1 h each with primary antibody, rabbit anti-human IgM, or rabbit anti-mouse IgM

(Dako Immunoglobulins, Weybridge, Surrey, United Kingdom) as appropriate and then with protein A (Pharmacia Ltd., Milton Keynes, Buckinghamshire, United Kingdom) labelled with 20-nm-diameter gold by the method of Roth (21). Sections were washed before each step with PBS containing 1% BSA (PBS-BSA; three washes of 5 min each), and all labelling was carried out at 4°C. Unless stated otherwise, antibody dilutions, carried out with PBS-BSA, were 1/250 for anti-I(Ma) serum, VIM-2, and FC10.2 ascites and 1/700 for anti-I(Step) serum and anti-i(Den) serum. In order to demonstrate an increase in staining intensity and to visualize new immunoreactivities after treatment of sections with sialidase, it was in most cases necessary to use high dilutions of anti-I(Ma) (1/500 to 1/1,000) and of anti-I(Step)

TOPOGRAPHY OF CARBOHYDRATE RECEPTORS FOR M. PNEUMONME

VOL. 60, 1992

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TOPOGRAPHY OF CARBOHYDRATE RECEPTORS FOR M. PNEUMONIAE

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