Oct 31, 1988 - The occurrence and distribution of the sialo-oligosaccharide receptors (sialosyl-I and sialosyl-i) for. Mycoplasma pneumoniae as well as other ...
Vol. 57, No. 4
INFECTION AND IMMUNITY, Apr. 1989, p. 1285-1289 0019-9567/89/041285-05$02.00/0 Copyright C) 1989, American Society for Microbiology
Sialo-Oligosaccharide Receptors for Mycoplasma pneumoniae and Related Oligosaccharides of Poly-N-Acetyllactosamine Series Are Polarized at the Cilia and Apical-Microvillar Domains of the Ciliated Cells in Human Bronchial Epithelium R. W. LOVELESS AND T. FEIZI*
Section of Glycoconjugate Research, MRC Clinical Research Centre, Harrow, Middlesex, HAI 3UJ, United Kingdom Received 31 October 1988/Accepted 4 January 1989
The occurrence and distribution of the sialo-oligosaccharide receptors (sialosyl-I and sialosyl-i) for Mycoplasma pneumoniae as well as other related oligosaccharide structures of poly-N-acetyllactosamine type, and their short-chain analogs based on galactose linked ,11-4 or ,11-3 to N-acetylglucosamine (Galo1l-4GlcNAc or Galoll-3GlcNAc, respectively) were investigated in the human bronchial epithelium by histochemistry by using sequence-specific monoclonal antibodies and lectins. Among the mature epithelial cells, only ciliated cells were found to express the long-chain antigens, whereas mucus-secreting cells contained the short-chain antigens associated with mucus globules. The long-chain oligosaccharides were found to be highly polarized at the luminal aspects of the ciliated cells where the branched structures (I and sialosyl-I antigens) were detected both at the apical-microvillar border and on the cilia, but the linear structures (i, sialosyl-i, and VIM-2 antigens) were detected exclusively at the apical-microvillar border. These observations provide the first in situ visualization of the receptor structures for M. pneumoniae at the primary site of infection. The lack of sialo-oligosaccharide receptors in secretory cells and the mucus they produce provides a biochemical basis for evasion by this microorganism of the secreted mucus barrier.
antibodies, Ulex europaeus lectin, and concanavalin A. We report here that several sialo-oligosaccharides of the polyN-acetyllactosamine series as well as their asialo forms are abundantly expressed and highly polarized at the luminal aspects of the ciliated cells, whereas their short-chain analogs occur in the mucus-secreting cells of the human bronchial epithelium.
Mycoplasma pneumoniae causes a spectrum of acute respiratory disease in humans associated with the transient production of autoantibodies to a carbohydrate antigen, I, which occurs on erythrocytes and other tissues (4). This infection is therefore an excellent natural model for studying host-parasite interactions and the pathogenesis of autoimmunity in humans. M. pneumoniae adheres to human erythrocytes, the ciliated bronchial epithelium and a variety of other cell types (9). Investigations of the erythrocyte receptors for this agent have shown that they are long-chain sialo-oligosaccharides of I and i antigen type, i.e., sialic acid joined by cx2-3 linkage to penultimate galactose residues of oligosaccharides of poly-N-acetyllactosamine type (6, 7). These carbohydrate receptor structures occur on glycoproteins such as band 3 protein (6), and on a series of glycolipids (7). Sialo-oligosaccharide antigens of this family have also been shown to occur on human lymphocytes, monocytes, and granulocytes (1, 8, 15). These findings have led us to propose that in the inflamed areas of the respiratory tissues, receptor-mediated complexes formed between the host oligosaccharides and the lipid-rich mycoplasma (functioning as an adjuvant) may serve as a trigger for autoimmunization (7). Moreover, the occurrence of the candidate receptor structures on monocytes (accessory cells with important roles in antigen presentation) may potentiate the immunogenic stimulus and account for the frequent occurrence of the autoimmune response after infection (3). However, there is no information to date on the distribution of the long-chain sialo-oligosaccharides of I and i antigen type at the primary target of infection, namely, the human bronchial epithelium. The present investigation was undertaken in order to visualize these and related long- and short-chain oligosaccharides by histochemistry with sequence-specific monoclonal *
MATERIALS AND METHODS Bronchial tissue. Samples of human bronchi in neutral buffered Formalin solution were kindly provided by A. Pomerance, Mount Vernon Hospital, Middlesex. Histologically normal segments were selected from bronchi resected from patients receiving heart-lung transplants (three cases) or undergoing surgery for bronchial carcinoma (four cases). Examination of secretor status. Secretor status of the patients was determined by examination of the saliva for the presence of blood group A, B, or H antigens as described previously (11). Four of the patients were typed as secretors and three as nonsecretors. Carbohydrate sequence-specific reagents. The anti-I and anti-i antibodies used were human monoclonal autoantibodies (2). Anti-I(Ma) recognizes the trisaccharide branch-point sequence which occurs on both short and long oligosaccharides of poly-N-acetyllactosamine type (Table 1). AntiI(Step) recognizes the extended backbone domain (indicated in Table 1) on branched oligosaccharides of poly-N-acetyllactosamine type, whereas anti-i(Den) recognizes the corresponding linear structures consisting of at least three N-acetyllactosamine (galactose linked p1-4 to N-acetylglucosamine [Galp1-4GlcNAc]) units. Glycosylation at nonreducing terminal galactose residues to form the blood group H antigen, and certain sialic acid substitutions at these galactose residues mask reactivities of the anti-I and -i antibodies (2). Enhanced immunoreactivities atter sialidase treatment
Corresponding author. 1285
TABLE 1. Oligosaccharide sequences investigated" Designation
I(Ma)
Oligosaccharide sequence
Means of detection
Galp1-4GlcNAcpl
Anti-I(Ma)
6Gal/GalNAc
Sialosyl-I(Ma)
Galp1-4GlcNAcp1
Enhanced anti-I(Ma) reactivity following neuraminidase treatment
11
6Gal/GaINAc SA
I(Step)
...
GlcNAcpl
Anti-I(Step) 6 GalI3l-4GlcNAcp13
Gal,Bl-4GlcNAcp1 Sialosyl-I(Step)
...
/
GlcNAcpl 6 3
GalI3l-4GlcNAco1
Enhanced anti-I(Step) reactivity following neuraminidase treatment
Galp1-4GlcNAc,l-
/
SA
i
Gal,1-4GlcNAc,1-3Galp1-4GlcNAcp1-3Galp1-4GlcNAcp1-
Anti-i(Den)
Sialosyl-i
GalPl-4GlcNAcPl-3GalIl-4GlcNAcP1-3Galp1-4GIcNAcP1-
Enhanced anti-i(Den) reactivity following neuraminidase treatment
SA
VIM-2
Gal,1-4GlcNAc,1-3Gal,1-4GlcNAcp1-3Ga431-4GlcNAcp12-3 SAa
Gd
VIM-2 antibody. Reactivity abolished by neuraminidase
1-3 Fuca
Galp1-4GlcNAc,B1-
Anti-Gd. Reactivity abolished by neuraminidase
2-3
SAa L Galp1-4GlcNAcI11-2 Fuca
Ulex europaeus agglutinin 1. Reactivity inhibited in the presence of fucose
H
Galpl-4GlcNAcp1-3Gal41-4Glc/GlcNAc
HF39 antibody
1,2 Fuca
Lea-related
Galpl-3GlcNAcp1-3Galpl-4Glc/GlcNAc
115E6 antibody
1-4
Fuca Leb-related
Galpl-3GlcNAcpl-3Galpl1-2
Fucoa Core regions of certain N-linked glycoprotein oligosaccharides
115D11 antibody
1-4
Fuca Manal
(Mana1-2)0_4 6
/3
Manotl
Manal
\63 Manpl-4GlcNAcpl-4GlcNAc
Concanavalin A. Reactivity inhibited in the presence of amethyl mannoside
ManoLu or
±(Rl) Manal\ 3
-L
V.-
ru1a
Manp1-4GlcNAcp1-4GlcNAc
±(R2) Manal / a Abbreviations: Fuc, fucose; Gal, galactose; GaINAc, N-acetylgalactosamine; GIcNAc, N-acetylglucosamine; Man, mannose; RI, Manotl-3 or Mancsl-3 and Manal-6 as in hybrid type oligosaccharides or . . . GlcNAc13l-2 as in biantennary oligosaccharides; R2, . . . GlcNAc,1-2 as in biantennary and hybrid type oligosaccharides; SA, sialic acid. In the case of sialosyl-I and sialosyl-i structures investigated, the form and linkage of sialic acid are not specified; in the case of oligosaccharides reactive with antibodies VIM-2 and anti-Gd, the presence of (x2-3-linked N-acetylneuraminic acid is inferred.
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OLIGOSACCHARIDE POLARIZATION IN EPITHELIAL CELLS
of tissues is therefore taken as evidence for the presence of the corresponding sialosyl-I and sialosyl-i structures. The epitopes recognized by the mouse hybridoma antibody VIM2 (gift of W. Knapp, Institute for Immunology, University of Vienna) and the human monoclonal antibody anti-Gd (gift of D. Roelcke, Institute for Immunology, University of Heidelberg) involve N-acetylneuraminic acid joined by a2-3 linkage to N-acetyllactosamine. Antibody Gd does not have an absolute requirement for the repeating N-acetyllactosamine sequence (17) but can react with shorter backbones as indicated in Table 1. Antibody VIM-2, however, has an absolute requirement for the repeating N-acetyllactosamine sequence and, in addition, it requires al-3 fucosylation at an internal N-acetylglucosamine (8). The mouse hybridoma antibodies 115E6 and 115D11 (5) recognize the fuco-oligosaccharide sequences shown in Table 1. These structures, which terminate in galactose linked p1-3 to N-acetylglucosamine (Galpl-3GlcNAc), are related to the blood group Lea and Leb antigens, respectively. The U. europaeus agglutinin 1 recognizes the short-chain blood group H structure carried on Galpl-4GlcNAc but not on the Galpl-3GlcNAc sequence (13), whereas antibody HF39 (gift of D. Dobbie, Blood Group Reference Laboratory, Oxford) recognizes the longchain blood group H structure as shown in Table 1 (J. Picard and T. Feizi, unpublished observations). Concanavalin A binds to oligosaccharides which have at least two a-mannosyl residues not substituted at the C-3, C-4, and C-6 positions (10). Examples are given in Table 1. Immunofluorescence. Paraffin-embedded sections (4 ,um) were stained by indirect immunofluorescence before or after sialidase treatment as described previously (16), with minor modifications. For desialylation, sections were treated for 16 h at 37°C with Vibrio cholerae sialidase diluted in 0.15 M sodium acetate buffer, pH 5.5, containing 1 mM calcium chloride. Human monoclonal antibodies were used as plasma dilutions of 1/10 (anti-Gd), 1/100 (anti-I[Ma]) or 1/700 (anti-I[Step] and anti-i[Den]). Appropriate dilutions of normal human serum supplemented with a Waldenstrom macroglobulin were used as negative controls. Mouse hybridoma antibodies (VIM-2, 115E6, and 115D11) were used as ascites at a 1/100 dilution; an irrelevant ascites (anti-dust mite) was used as a negative control. Antibody HF 39 was used undiluted as a hybridoma culture supernatant; Eagle minimum essential medium (Flow Laboratories Ltd., Ayrshire, Scotland) supplemented with 10% fetal calf serum (Biological Industries, Glasgow, Scotland) was used as a negative control. Fluorescein-labeled U. europaeus agglutinin 1 and concanavalin A (Vector Laboratories Inc., Burlingame, Calif.) were used for direct immunofluorescence at a 1/80 dilution. As negative controls, the U. europaeus lectin and concanavalin A were used in the presence of 20 mM fucose or 100 mM a-methyl mannoside, respectively. All staining was carried out at 4°C. RESULTS In all cases examined there was a striking difference in the distribution of the long- and the short-chain oligosaccharide antigens in the ciliated and the mucus-secreting cells lining the bronchial epithelium as detected by reaction with antiI(Step), anti-i(Den), and VIM-2 antibody for the long-chain sequences, and anti-I(Ma), anti-Gd, antibodies 115E6 and 115D11, and U. europaeus lectin for the short-chain sequences (see Table 1 for structures). The long-chain oligosaccharides of poly-N-acetyllactosamine type are detected on the ciliated cells. The long-chain anti-
laI.N,i -
.
'.
k
I..
1W
or,.r
9
dbW
vuov. '*k it.
1287
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OW
at"KA
A Nit.4I
FIG. 1. Immunofluorescence of sections of the bronchial epithelium showing polarized distribution of antigens of poly-N-acetyllactosamine type at the apical aspects of the ciliated cells, contrasting with the pericellular and intracellular distribution of oligosaccharide structures reactive with concanavalin A. These sections are from a nonsecretor. Similar results were obtained with epithelia from other subjects irrespective of secretor status. Panels: A, C, and E, immunofluorescence (A', C' and E', corresponding phase-contrast micrographs) with anti-I(Step), anti-i(Den), and VIM-2 antibody, respectively; B and D, enhancement of immunofluorescence; F, abolition of immunofluorescence with these three antibodies, respectively, after treatment of the sections with neuraminidase; G, immunofluorescence (G', corresponding phase-contrast micrograph) with concanavalin A; H, lack of immunofluorescence with concanavalin A in the presence of 100 mM a-methyl mannoside. Scale bar, 10 ,um.
gens, I(Step), i(Den), and VIM-2, were detected on the ciliated cells but were lacking in the mucus-secreting cells. The branched antigens I(Step) and sialosyl-I(Step) were the most abundantly expressed; they were highly concentrated
1288
LOVELESS AND FEIZI
INFECT. IMMUN.
~
~Aj
A
4# Immunofluorescence of sections of the bronchial epitheshowing presence of Le'-related antigen (reactive with antibody 115D11) and Gd antigen in the intracellular mucus of secretory FIG. 2.
lium
cells but
no
fluorescence of ciliated cells.
europaeus lectin
Fluorescence with
parallelled that of antibody
shown). The sections
are
from
a
U.
(results
not
secretor; in nonsecretors there
was
115D11
antibody 115D11 and U. europaeus lectin, but instead a strong immunofluorescence was obtained with antibody 115E6 which recognizes the antigen. Panels: A and B, immunofluorescence with the antibody and anti-Gd A' and B', corresponding phase-contrast micrographs. Scale bar, 10 p.m. a
lack of immunofluorescence with
_
2r
*,: S ,
_W
Lea-related
115Ds11
respectively;
polarized on domains of these and
cilia
cells
(Fig. 1, A and B). There of the trisaccharide branch
coincident expression
epitopes shown),
chains
apical-microvillar
the
and
at
I(Ma) and sialosyl-I(Ma) in
indicating that there
was
an
the
these
was
a
point
areas (results not
abundance of
short
side
oligosaccharides. In contrast to the branched antigens, the long linear antigens i(Den), sialosyland VIM-2 were restricted to the apical-microvillar the branched
on
i(Den), margins of the ciliated cells (Fig. 1, C through E). The pronounced polarization of the poly-N-acetyllactosamine
chains basal
was
cells
extent
clearly
differentiation-related event since in the
a
sialosyl-i(Den), and to a lesser located pericellularly. oligosaccharides of N-acetyllactosamine
sialosyl-I(Step),
their asialo forms were
The short-chain
mucus-secreting cells. The short-chain (reactive with U. europaeus lectin and but not with antibody HF39) and Gd, as well as the but antigens, were lacking in the ciliated were associated with mucus in the secretory cells of the
type
detected in
are
structures, blood
group H
Leap cells,
Lembrelated
bronchial epithelium (Fig. the lamina propria (Fig. 3).
2) and in the bronchial glands of The
sialo-oligosaccharide antigen
subpopulation of the mucus cells in all individuals examined but the expression of the blood group H and Le a-and antigens was in accordance with
Gd
was
found in
a
Le'related expected (14). Thus, in and Lewbantigens detected with little secretor
status,
as
secretors the
or no
were
whereas in nonsecretors the Lea antigen expressed, and there was a lack of H and Leb
ity,
bronchial
antigens
glands of the I(Step) and
short-chain
FIG. 3. Immunofluorescence of sections of bronchial glands in the lamina propria showing the presence of the Le b-related antigen (reactive with antibody 115D11), Gd antigen, and sialosyl-I(Ma). The sections were from the same secretor subject as in Fig. 2. In nonsecretors, there was a lack of immunofluorescence with antibody 115D11 but a strong reactivity with antibody 115E6 which recognizes the Lea-related antigen. Immunofluorescence with U. europaeus lectin parallelled that with antibody 115D11 (results not shown). Panels: A and B, immunofluorescence with antibody 115D11 and anti-Gd, respectively; C, immunofluorescence of sialidase-treated section with anti-I(Ma); A', B', and C', the respective phase-contrast micrographs. Scale bar, 10 p.m.
lamina propria
lacked
sialosyl-I(Step),
antigens sialosyl-I(Ma)
but
Lea
was
H
reactivstrongly
antigens. The the long-chain contained the
(Fig. 3C) and
to
extent I(Ma) (data not shown) predominantly in intracellular juxtanuclear granules. Mannose-containing glycoprotein oligosaccharides reactive with concanavalin A detected peri- and intracellularly in all epithelial cell types. There was evidence that not all glycans are polarized in the apical aspects of the ciliated cells or concentrated intracellularly in the secretory cells. Mannosecontaining oligosaccharides reactive with concanavalin A were widely distributed both pericellularly and intracellularly in the majority of cells of the bronchial epithelium, i.e., among the mature epithelial cells as well as the intermediate and the basal cells (Fig. 1G).
a lesser
DISCUSSION The salient conclusions that can be drawn from these results are (i) that long-chain sialo-oligosaccharides of polyN-acetyllactosamine type which are attachment factors for M. pneumoniae, as well as their asialo analogs, are highly
VOL. 57, 1989
OLIGOSACCHARIDE POLARIZATION IN EPITHELIAL CELLS
concentrated at the luminal aspects of the ciliated cells of the bronchial epithelium, but are lacking in the secretory cells and the mucus they produce, and (ii) that the branched and linear oligosaccharides of this series are differentially distributed; the latter are confined to the apical-microvillar domain while the former occur in both this region and on the cilia. Previous investigations have suggested that during infection, M. pneumoniae organisms evade the secreted mucus and adhere to the ciliated epithelia (12). Our results provide for the first time a biochemical basis for this selective attachment. The lack of the candidate long-chain receptor structures in the secreted mucus would be an important permissive factor favoring adhesion to the ciliated cells rather than clearance by ciliary action on the secreted mucus.
The autoantibodies in patients with M. pneumoniae infection are directed at the I antigen (2). The codistribution of this antigen with the sialylated analog on the ciliated cells raises the possibility that these structures are carried on the same carbohydrate chains, or on closely spaced chains on neighboring glycoproteins and glycolipids. Further biochemical investigations are required to evaluate these possibilities. However, our findings now suggest that the potentially immunogenic complexes of mycoplasma with sialo-oligosaccharide receptors on the host-cell membrane may also include oligosaccharides with the exposed I-active backbone structures. Therefore, desialylation of the receptors need not be invoked as a mechanism for increased immunogenicity of the I-active host oligosaccharides after interaction with M. pneumoniae. The reason for the occurrence of anti-I rather than anti-i autoantibodies after infection requires investigation, but we suggest that it may be a reflection of the relative abundance and greater accessibility of the I-related carbohydrate structures on the bronchial epithelium. ACKNOWLEDGMENTS R.W.L. was supported by the Arthritis and Rheumatism Council. The authors are grateful to A. Pomerance for providing bronchial material, the histology department of the Northwick Park Hospital for providing the tissue sections, and Maureen Moriarty for typing the manuscript. LITERATURE CITED 1. Childs, R. A., and T. Feizi. 1981. Differences in carbohydrate moieties of high molecular weight glycoproteins of human lymphocytes of T and B origins revealed by monoclonal autoantibodies with anti-I and anti-i specificities. Biochem. Biophys. Res. Commun. 102:1158-1164. 2. Feizi, T. 1981. The blood group Ii system: a carbohydrate antigen system defined by naturally monoclonal or oligoclonal autoantibodies of man. Immunol. Commun. 10:127-156.
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3. Feizi, T. 1987. Significance of carbohydrate components of cell surfaces, p. 43-58. In D. Evered (ed.), Autoimmunity and autoimmune disease, Ciba Symposium no. 129. John Wiley & Sons, Ltd., Chichester, United Kingdom. 4. Feizi, T., and N. Hadler. 1983. Autoantibodies and disease, p. 656-692. In R. S. Elkeles and A. S. Tavill (ed.), Biochemical aspects of human disease. Blackwell Scientific Publications, Ltd., Oxford. 5. Gooi, H. C., N. J. Jones, J. Hilkens, J. Hilgers, and T. Feizi. 1985. Lewis blood group-related specificities of monoclonal antibodies designated MAM-3a, b and c against human milk-fat globule membranes. Glycoconjugate J. 2:409-420. 6. Loomes, L. M., K.-I. Uemura, R. A. Childs, J. C. Paulson, G. N. Rogers, P. R. Scudder, J.-C. Michalski, E. F. Hounsell, D. Taylor-Robinson, and T. Feizi. 1984. Erythrocyte receptors for Mycoplasma pneumoniae are sialylated oligosaccharides of Ii antigen type. Nature (London) 307:560-563. 7. Loomes, L. M., K.-I. Uemura, and T. Feizi. 1985. Interaction of Mycoplasma pneumoniae with erythrocyte glycolipids of I and i antigen types. Infect. Immun. 47:15-20. 8. Macher, B. A., J. Buehler, P. Scudder, W. Knapp, and T. Feizi. 1988. A novel carbohydrate, differentiation antigen on fucogangliosides of human myeloid cells recognised by monoclonal antibody VIM-2. J. Biol. Chem. 263:10186-10191. 9. Manchee, R. J., and D. Taylor-Robinson. 1968. Haemadsorption and haemagglutination by mycoplasmas. J. Gen. Microbiol. 50:465-468. 10. Ogata, S., T. Muramatsu, and A. Kobata. 1975. Fractionation of glycopeptides by affinity column chromatography on concanavalin A-Sepharose. J. Biochem. 78:687-696. 11. Picard, J., E. D. Waldron, and T. Feizi. 1978. Changes in the expression of the blood group A, B, H, Lea and Leb antigens and the blood group precursor associated I(Ma) antigen in glycoprotein-rich extracts of gastric carcinomas. J. Clin. Lab. Immunol. 1:119-128. 12. Razin, S. 1986. Mycoplasmal adhesins and lectins, p. 217-235. In D. Mirelman (ed.), Microbial lectins and agglutinins: properties and biological activity. John Wiley & Sons, Inc., New York. 13. Sugii, S., E. A. Kabat, and H. H. Baer. 1982. Further immunochemical studies on the combining sites of Lotus tetragonolobus and Ulex europaeus I and II lectins. Carbohydr. Res. 99:99-101. 14. Szulman, A. E. 1966. Chemistry, distribution, and function of blood group substances. Annu. Rev. Med. 17:307-322. 15. Thorpe, S. J., and T. Feizi. 1984. Species differences in the expression of carbohydrate differentiation antigens on mammalian blood cells revealed by immunofluorescence with monoclonal antibodies. Biosci. Rep. 4:673-685. 16. Thorpe, S. J., R. Bellairs, and T. Feizi. 1988. Developmental patterning of carbohydrate antigens during early embryogenesis of the chick: expression of antigens of the poly-N-acetyllactosamine series. Development 102:193-210. 17. Uemura, K., D. Roelcke, Y. Nagai, and T. Feizi. 1984. The reactivities of human erythrocyte autoantibodies anti-Pr2, antiGd, Fl and Sa with gangliosides in a chromatogram binding assay. Biochem. J. 219:865-874.
