ARTICLE |
Correspondence to: Hiroshi Kimura, Dept. of Forensic Medicine, Kurume U. School of Medicine, Kurume 830-0011, Japan. E-mail: hkimura@med.kurume-u.ac.jp
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Summary |
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We used three anti-H monoclonal antibodies (MAbs) specific for H Type 1, H Type 2, and H Type 3/4 antigens to investigate the distribution of H Type 1H Type 4 chains of the ABO(H) histo-blood group in the human respiratory system. Strong staining of H Type 1 chain and weak staining of H Type 2 chain were observed in mucous cells of submucosal glands of bronchial epithelium, which were dependent on the secretor status. No H Type 3/4 chains were detected in mucous cells. Serous cells of submucosal glands of respiratory system showed no staining by three anti-H antibodies. H Type 1 and H Type 3/4 antigens were detected heterogeneously in apical surfaces of bronchial epithelium from secretors but not from nonsecretors. In contrast, basal cells of bronchial epithelium expressed H Type 2 irrespective of the secretor status, probably regulated by the H gene. Some alveolar Type II cells contained only H Types 3/4, which were dependent on the secretor status, whereas alveolar Type I cells had no H antigens. Our results indicated that different cell types in respiratory epithelium expressed different types of carbohydrate chains of histo-blood group antigens under the control of the H or the Se gene. (J Histochem Cytochem 48:16491655, 2000)
Key Words: ABO, H antigen, respiratory epithelium, fucosyltransferase, secretor
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Introduction |
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Although the ABO blood group was initially found in red blood cells, it is well-known that tissues expressing abundant ABO antigens are the digestive and respiratory organs (ABO histo-blood group) (
The epitope of the H antigen, the precursor of A, B, LeY, and Leb antigens of ABO and Lewis histo-blood groups, is carried by at least four different types of internal carbohydrate backbones (Type 1, Galß1-3GlcNAcß1-R; Type 2, Galß1-4GlcNAcß1-R; Type 3, Galß1-3GalNAc1-R; and Type 4, Galß1-3GalNAc- ß1-R) (
The aim of this study was to investigate the cell type distribution of H Type 1, H Type 2, and H Type 3/4 substances in the human respiratory system by an immunohistochemical method using three anti-H MAbs specific for H Type 1 chain (1E3), H Type 2 (3A5), and H Type 3/4 chains (MBr1). These data are useful for a better understanding of the biology of secretory cell types and of several pathological conditions that are characterized by altered secretion.
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Materials and Methods |
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Twenty-two human lung tissue specimens were obtained from autopsies in our department. The ABO and Lewis histo-blood groups of tissue donors were assessed by routine blood agglutination methods. The secretor status was determined according to the Lewis phenotypes. Individuals with Le(a+b-) or Le(a+b+) phenotype were classified as non-secretors, while those with Le(a-b+) phenotype were classified as secretors. This study included six samples with blood group A, six with blood group B, and 10 with blood group O; 12 of these individuals were secretors and 10 were nonsecretors.
Deparaffinized serial sections (3 µm) of 10% formalin-fixed, paraffin-embedded human lungs were pretreated with 3% hydrogen peroxide for 30 min to quench endogeneous peroxidase activity. Then the pretreated sections were immunostained using a streptavidinbiotin complex immunoperoxidase method as described previously (
Commercial anti-A or anti-B can not discriminate Type 1Type 4 carbohydrate chains. Therefore, a panel of specific anti-H MAbs was used in this study. MAb 1E3, which was raised after immunization of mixed saliva of blood group O individuals, was a kind gift from Drs. Ken Furukawa and Shin Yazawa (Department of Legal Medicine, Gunma University School of Medicine). Although anti-H 1E3 has been demonstrated to be reactive with H Type 1H Type 4 synthetic trisaccharides (1-2Galß1-3GlcNAcß1-) after absorption with red cell ghosts (
1-2Galß1-4GlcNAcß1-) (
1-2Galß1-3GalNAc
-) from H Type 4 (Fuc
1-2Galß1-3GalNAcß-). Ulex europaeus agglutinin I (UEA-I) conjugated with horseradish peroxidase (EY Laboratories; San Mateo, CA) was used to compare with staining by anti-H antibodies. Anti-factor VIII (Dako; Carpinteria, CA) and anti-SP-A (Dako) were also used to identify alveolar Type I and Type II cells, respectively.
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Results |
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In this study, we used specimens mainly from blood group O individuals to avoid an effect of metabolic conversion of H carbohydrate chains to A and B chains.
Fig 1 shows staining of submucosal glands of bronchial epithelium from O secretors by anti-H MAbs 1E3, 3A5, and MBr1. MAb 3A5, specific for H Type 2 antigen, strongly stained capillary endothelial cells and erythrocytes (Fig 1B), whereas no staining was observed with erythrocyte-absorbed anti-H 1E3 (Fig 1A), indicating that MAb 1E3 was specific for H Type 1 chain under certain conditions. In secretors, the cytoplasm of mucous cells of submucosal glands of bronchial epithelium was strongly stained with 1E3 (Fig 1A) and UEA-I (Fig 1D), or by anti-A and anti-B (not shown), and was weakly stained with 3A5 (Fig 1B), but not with MBr1 (Fig 1C). In nonsecretors, no staining of mucous cells with any of the anti-H MAbs and with UEA-I was observed (not shown). Serous cells did not react with any of the anti-H MAbs, in contrast to serous cells of secretory acini of the submandibular gland, which were strongly stained by MAb MBr1 (
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The bronchial epithelial cells showed heterogeneous staining with the anti-H MAbs according to cell type. Luminal surfaces of bronchial epithelium were labeled heterogeneously by anti-H 1E3 and MBr1, depending on the secretor status (Fig 2A and Fig 2C). However, the cytoplasm of columnar cells and cuboidal cells of bronchial epithelium completely lacked positive staining by 1E3 and MBr1. Anti-H MAbs did not stain a majority of goblet cells, but a few goblet cells from secretors reacted with 1E3 and MBr1. The contents of bronchioles were strongly stained with 1E3 and MBr1 and weakly with 3A5 (Fig 2B and Fig 3B), suggesting that carbohydrate chains of mucins consisted of various type of H-active chains. Cytoplasm of basal cells from both secretors and nonsecretors was labeled with 3A5 (Fig 2B and Fig 2D) or with anti-A or anti-B (not shown), corresponding to the ABO blood groups. However, UEA-I did not stain basal cells (not shown), suggesting that the substances positive for anti-H 3A5 and for UEA-I were different, as reported previously (
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Type I cells (squamous alveolar cells) of alveoli from both secretors and nonsecretors did not react with any of the anti-H antibodies (not shown). However, about 15% of alveolar Type II cells (great alveolar cells) reacted with MBr1 only from secretors but not from nonsecretors (Fig 4). The MBr1-positive cells were stained with anti-SP-A (not shown). All results are summarized in Table 1.
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Discussion |
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Although many studies have investigated the distribution of ABO and Lewis antigens in normal and malignant tissues of the digestive system, studies on the distribution of these antigens in the respiratory system have been few.
Although the ABO histo-blood group was identified 100 years ago and has contributed enormously to transfusion medicine, its function is still an enigma. However, there is increasing evidence that the remarkable diversity of carbohydrate chains of respiratory mucins allows many interactions with microorganisms and may be an important factor in maintaining the sterility of the respiratory tree (1-anti-trypsin gene has been established, the associations of pulmonary diseases and lung function with other genetic risk factors, including glycosyltransferases for synthesis of histo-blood group antigens, were also suggested (
It is known that many carbohydrate structures are recognized by microorganisms (
It is plausible that carbohydrate chains of mucins play an important role in binding of pathogens in the respiratory organs. However, further studies are required to clarify the relation between carbohydrate structures and binding of each pathogen or in pulmonary diseases.
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Acknowledgments |
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Supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan.
We thank Drs Ken Furukawa and Shin Yazawa (Department of Legal Medicine, Gunma University School of Medicine) and Dr Maria Ines Colnaghi (Division of Experimental Oncology E; National Institute for Tumor, Milan) for the kind gifts of the anti-H 1E3 and the anti-H MBr1 monoclonal antibodies, respectively. We also thank Mr Shigeo Kamimura and Ms Yasuko Noguchi for technical assistance.
Received for publication March 28, 2000; accepted June 22, 2000.
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