ARTICLE |
Correspondence to: Hiroshi Kimura, Dept. of Forensic Medicine, Kurume U. School of Medicine, Kurume, Fukuoka 830, Japan.
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Summary |
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We have examined the immunohistochemical distribution of H Type 1 and of H Type 2 substances of the ABO blood group system in human submandibular gland using either of the two anti-H monoclonal antibodies MAb 1E3 and MAb 3A5. MAb 3A5 was specific for H Type 2, and MAb 1E3 reacted with each of H Type 1-H Type 4 artificial antigens. We have developed a competitive inhibition method against H Type 2 and have obtained MAb 1E3, which is fairly specific for H Type 1 under certain conditions. Mucous cells from secretors were strongly stained by 1E3 and weakly by 3A5, whereas those from nonsecretors showed no reaction with 1E3 and 3A5. Serous cells from both secretors and nonsecretors were stained neither by 1E3 nor by 3A5. Striated and interlobular duct cells were strongly stained by 1E3 and by 3A5, regardless of the secretor status. These results indicated that the expressions of the H Type 1 and H Type 2 in different cell types of the submandibular gland were controlled by different genes. In addition, we have determined the acceptor specificity of two (1,2)fucosyltransferases (H and Se enzymes) after transient expressions of the FUT1 and FUT2 in COS7 cells, and found that the H enzyme activity was similar for both Type 1 and Type 2 precursors, and that Se enzyme activity with the Type 1 precursor was higher than that with the Type 2 precursor. Expression of the H Type 1 antigen in mucous cells was found to be dependent on the Se gene, whereas expressions of the H Type 1 and H Type 2 antigens in striated and interlobular duct cells were dependent on the H gene. (J Histochem Cytochem 46:69-76, 1998)
Key Words:
ABO, (1,2)fucosyltransferase, anti-H antibody, FUT1, FUT2, H antigens, immunohistochemistry, submandibular gland, UEA-I
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Introduction |
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The histo-blood group ABO-related antigens are carried by at least four different core structures (Type 1 chain, Galß1-3GlcNAcß1-; Type 2 chain, Galß1-4GlcNAcß1-; Type 3 chain, Galß1-3GalNAc1-; Type 4 chain, Galß1-3GalNAcß1-) (
(1,2)fucosyltransferases, which form the H antigen, are present in human tissues and regulate the expression of ABO antigens (
In the present study, we compared the stainings of the H antigens in cell types of human submandibular gland by two anti-H MAbs (MAb 3A5 and MAb 1E3, which are specific for H Type 2 and for H Types 1-4, respectively) ((1,2)fucosyltransferases, H enzyme and Se enzyme, for interpreting our immunohistochemical findings.
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Materials and Methods |
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Blood Group Phenotypes and FUT2 Genotypes
The ABO and Lewis blood groups of tissue donors were determined on red blood cells by conventional hemagglutination techniques using mouse MAbs against histo-blood group antigens, A, B (Kokusai; Kobe, Japan), Lea, and Leb (Ortho Diagnostic; Tokyo, Japan), respectively. Because the secretor status in saliva of ABO substances is closely related to the Lewis phenotypes on red blood cells, an individual with Le(a-b+) was recognized to be a secretor, with Le(a+b-) to be a nonsecretor, or with Le(a+b+) to be a weak secretor (
Antibodies and Lectin
Two anti-H MAbs, 3A5 and 1E3, were used in the present study. The specificity of MAb 3A5 has been demonstrated to be specific for the H Type 2 structure using a series of synthetic oligosaccharides (1-2Gal disaccharide and H Type 1-H Type 4 oligosaccharides (
Absorption of MAb 1E3 with Sonicated Red Blood Cell Ghosts
Saliva and sonicated erythrocyte membranes (ghosts) from O blood group individuals were prepared as described previously (
Immunohistochemistry and Lectin Histochemistry
Submandibular glands from individuals with blood group O were obtained from autopsy cases (five secretors and four nonsecretors). The glands were fixed in 10% formalin at room temperature (RT), embedded in paraffin, and sectioned serially at 3-4 µm. Paraffin sections were mounted on gelatin-dichromate-coated glass slides. Sections were deparaffinized, rehydrated, and pretreated with 3% hydrogen peroxide for 30 min at RT to quench endogenous peroxidase activity. Then the pretreated sections were used for immunohistochemistry or lectin histochemistry.
Sections were incubated with 10% normal rabbit serum for immunohistochemistry or with 1% BSA for lectin histochemistry for 30 min at RT to block nonspecific sites, then incubated with the primary MAb (anti-H 3A5, anti-H 1E3, or anti-Leb) or HRP-conjugated UEA-I at 4C overnight in a moist chamber. After washing three times in PBST, the sections for immunohistochemistry were incubated with biotiny-lated rabbit anti-mouse immunoglobulins (IgG+IgM+IgA) (Nichirei; Tokyo, Japan) for 30 min at RT, followed by rinsing in PBST, and were then incubated with peroxidase-conjugated streptavidin (Nichirei). Positive sites were developed with 200 µg/ml of 3,3'-diaminobenzidine containing 0.04% hydrogen peroxide in PBS, and finally the sections were counterstained with hematoxylin. Control sections, incubated as above but without the primary antibody or UEA-I, showed no staining.
Transient Expression of FUT1 (H gene) and FUT2 (Se gene) in COS7 Cells and Assay of (1,2)fucosyltransferase Activity
The wild-type allele of the FUT1 or the FUT2 was subcloned into a mammalian expression vector pRc/CMV (Invitrogen; San Diego, CA) (pRc/CMV-H and pRc/CMV-Se, respectively), as described previously ((1,2)fucosyltransferase activity in cell extracts was also determined using lacto-N-biose I as a Type 1 acceptor or N-acetyl-lactosamine as a Type 2 acceptor (
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Results |
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The FUT2 genotypes of all four individuals with Le(a+b+) were identified to be homozygous for se2 (A385T), which is one of the Se enzyme-deficient alleles (
Although MAb 1E3 has been shown to be reactive with the H Type 1-H Type 4 synthetic oligosaccharides (
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The majority of mucous cells of acini from secretors were stained intensely by MAb 1E3 (Figure 4A) and weakly by MAb 3A5 (Figure 4B). Most mucous cells from nonsecretors were negative by 1E3 and 3A5 (Figure 4D and Figure 4E), but some mucous cells from nonsecretors still displayed a weak positive reaction with MAb 1E3, probably due to the weak secretor phenotype. However, mucous acini from nonsecretors were stained by MAb anti-Leb (not shown) despite the lack of staining of H Type 1 on these cells. These results suggested that the major H substance in the mucous cells was H Type 1 rather than H Type 2 and that its expression was closely related to the secretor status. Serous acini from both secretor and nonsecretor individuals were not labeled by MAbs anti-H, 1E3, and 3A5. The staining of some intercalated cells by anti-H was the same as that of mucous cells, dependent on the secretor status. Interestingly, striated and interlobular duct cells were stained by both 3A5 and 1E3, which was independent of the secretor status of the donors, indicating the presence of both H Type 1 and H Type 2 antigens.
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As shown in Figure 4C and Figure 4F, almost all cells, including mucous cells, serous cells, and duct cells in tissue sections, except for the serous cells from nonsecretors, were uniformly stained by UEA-I lectin. The staining of the serous cells by UEA-I was dependent on the secretor status of donors, whereas that of the mucous and duct cells was independent of the secretor status. All results are summarized in Table 1.
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The H enzyme activity in pRc/CMV-H-transfected cell extracts was similar with lacto-N-biose I (Type 1 chain) and with N-acetyl-lactosamine (Type 2 chain) as acceptors, whereas the Se enzyme activity in pRc/CMV-Se-transfected cell extracts showed about six-fold higher activity with lacto-N-biose I than with N-acetyl-lactosamine (Table 2). In addition, COS7 cells transfected by the Se gene (FUT2) displayed strong fluorescence with 1E3 but very weak fluorescence with 3A5, whereas cells transfected by the H gene (FUT1) showed strong fluorescence with both monoclonal anti-H antibodies (not shown), suggesting similar reactivities of the H enzyme with both Type 1 and Type 2 acceptors and higher reactivity of the Se enzyme with the Type 1 acceptor than with the Type 2 acceptor in cells.
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Discussion |
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Many immunohistochemical studies of the ABO substances in the salivary gland have been reported (
In the light of the present study, we concluded that the expression of H Type 1 in the mucous cells of submandibular glands was a consequence of the activity of the Se enzyme encoded by the Se gene (FUT2), whereas the expressions of the H Type 1 and the H Type 2 in striated and interlobular duct cells were a consequence of the H enzyme encoded by the H gene (FUT1). However, the possibility of Se enzyme expression in duct cells has not been completely eliminated, in that the stainings of duct cells from nonsecretors by 1E3 absorbed with ghosts were weaker than those from secretors. Previously, the Se enzyme was believed to use both the Type 1 and the Type 2 structures as acceptors but preferred the Type 1, and the H enzyme used only the Type 2 precursor as an acceptor ((1,2)fucosyltransferase, and obtained similar activities against the Type 1 and Type 2 precursors. Our results were similar to that of
UEA-I has been known to be and is still used as a lectin specific for H Type 2 (
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Acknowledgments |
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Supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
We are grateful to Drs K. Furukawa and S. Yazawa, Department of Legal Medicine, Gunma University School of Medicine, for the kind gift of the MAb anti-H 1E3, and to Mr Shigeo Kamimura for making serial thin sections.
Received for publication April 8, 1997; accepted July 10, 1997.
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