ARTICLE |
Correspondence to: Naotaka Hamasaki, Dept. of Clinical Chemistry and Laboratory Medicine, Faculty of Medicine, Kyushu Univ., 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
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Summary |
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Hakata antigen was first reported as a serum protein that reacted with an autoantibody from patients with systemic lupus erythematosus. Recently, it has been found that Hakata antigen is a new member of the ficolin/opsonin p35 family, which is a distinct lectin family, on the basis of homology of structures and the common characteristic of possessing lectin activity. In this study we analyzed the tissue distribution of Hakata antigen. Hakata antigen mRNA and protein were generated in the lung and liver. In the lung, Hakata antigen was produced by both ciliated bronchial epithelial cells and Type II alveolar epithelial cells and was secreted into the bronchus and alveolus. In the liver, Hakata antigen was produced by bile duct epithelial cells and hepatocytes and was also secreted into the bile duct. These results demonstrate that Hakata antigen is a unique lectin protein that exists not only in serum but also in bronchus/alveolus and bile, and indicate that Hakata antigen plays a role in bronchus/alveolus and bile under physiological conditions. (J Histochem Cytochem 47:777785, 1999)
Key Words: Hakata antigen, lectin, ficolin, opsonin p35, bronchus, alveolus, bile
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Introduction |
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Hakata antigen was first reported by
We have recently identified the cDNA encoding Hakata antigen, revealing that Hakata antigen consists of a collagen-like domain in the middle section and a fibrinogen-like domain in the C terminus, both of which are homologous to two lectin activity-possessing proteins, human ficolin-1 and opsonin p35 (
Our present analysis of the tissue distribution of Hakata antigen reveals that it is produced by ciliated bronchial epithelial cells and Type II alveolar epithelial cells in the lung and by bile duct epithelial cells and hepatocytes in the liver, which are secreted into bronchus/alveolus and bile duct, respectively.
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Materials and Methods |
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Northern Blotting Analysis
A membrane filter blotted with 2 µg of poly(A)+ mRNA from human tissues (Clontech Labs; Palo Alto, CA) was hybridized with 32P-labeled Hakata antigen cDNA fragment (nucleotides 448864) overnight at 42C in a solution containing 50% formamide, 5 x Denhardt's solution, 5 x SSPE, 0.5% sodium dodecyl sulfate (SDS), and 200 µg/ml salmon sperm DNA. After two extensive washings at room temperature (RT) for 10 min in a 0.1 x standard saline citrate (SSC)/0.1% SDS solution, the filter was exposed to an autoradiographic imaging system and the image was then read using a bioimaging analyzing system (BAS 1000; Fuji Photo Film, Minamiashikaga, Japan).
Preparation of Polyclonal and Monoclonal Antibodies Against Hakata Antigen
A polyclonal antibody against Hakata antigen was produced as described previously (
Western Blotting Analysis
Fresh human lung and liver tissues were taken after informed consent from donors undergoing surgery, and the tissues were immediately frozen. Each frozen tissue was homogenized with 50 mM Tris-HCl (pH 8.0) solution containing 8 M urea, 4% SDS, and 5 mM EDTA, and the supernatant was collected after centrifugation. Hakata antigen in serum was purified according to the method used by
Total homogenates of the human lung and liver tissues, purified Hakata antigen, BAL fluids, and bile were applied to SDS-PAGE and transferred electrophoretically to PVDF membranes (Millipore; Bedford, MA). The membranes were probed with either a polyclonal or a monoclonal antibody against Hakata antigen and were visualized by enhanced chemiluminescence (Tropix; Bedford, MA).
Immunohistochemical Analysis
Autopsy specimens of normal human lung and liver were fixed with 10% formalin solution for about 2 weeks and then embedded in paraffin. The sections were pretreated with 0.1% trypsin for 30 min at 37C and then probed with Pr19 by the biotinstreptavidin method as described (
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Results |
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Expression of Hakata Antigen mRNA
To determine which tissues express Hakata antigen, we first performed Northern blotting using a complementary oligonucleotide to Hakata antigen (nucleic acids 448864 from the translation starting site) as a probe. This oligonucleotide hybridized mRNAs derived from the lung and liver (Figure 1). No band was detected in mRNAs of spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes, heart, brain, placenta, skeletal muscle, or kidney (Figure 1; data not shown). The sizes of the bands were about 1.3 KB. BANDS CORRESPONDING TO GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE WERE DETECTED IN ALL TISSUES (NOT SHOWN). THIS RESULT INDICATES THAT THE LUNG AND LIVER ARE THE PRINCIPAL ORGANS THAT PRODUCE HAKATA ANTIGEN. WE THEREFORE FOCUSED ON THE LUNG AND LIVER TO ANALYZE THE EXPRESSION OF HAKATA ANTIGEN PROTEIN.
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Western Blotting of Hakata Antigen in Lung and Liver
To confirm that the lung and liver express Hakata antigen protein as well as mRNA, we next carried out Western blotting of Hakata antigen using total homogenates of lung and liver. Using the polyclonal antibody against Hakata antigen, the homogenates of both lung and liver gave rise to sharp bands whose sizes were identical to that of Hakata antigen purified from serum, although some bands of higher molecular weight were also detected in the case of the liver (Figure 2). It is unlikely that these bands are polymeric forms because Hakata antigen exists as a monomer under reducing conditions. These results suggest that the lung and liver are rich in Hakata antigen protein, whose molecular size is almost the same as that in serum.
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Generation of a Monoclonal Antibody Against Hakata Antigen (Pr19)
We have previously shown that Hakata antigen exists as a polymer consisting of disulfide bonds in serum and that polyclonal rabbit antiserum against Hakata antigen recognizes both monomeric and polymeric Hakata antigens (
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Expression of Hakata Antigen in Lung
To localize Hakata antigen protein expressed in human lung, we carried out immunohistochemical analyses using Pr19. In the alveolar region, Type II alveolar epithelial cells, characterized by their round shape and plump cytoplasm, showed strong cytoplasmic staining with Pr19 (Figure 4A and Figure 4B), whereas Type I alveolar epithelial cells, alveolar macrophages, and endothelial cells were not stained. To confirm that Type II alveolar epithelial cells were stained by Pr19, we stained two sequential sections with either Pr19 (Figure 4B) or anti-SP-A antibody (Figure 4C) because SP-A is known to be secreted from Type II alveolar epithelial cells (
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To clarify whether Hakata antigen is secreted into bronchus/alveolus, we subjected BAL fluids to Western blotting of Hakata antigen. In nonreducing conditions, a polymeric Hakata antigen with the same mobility as that in serum was detected in all BAL fluids from eight donors (Figure 5A), whereas in reducing conditions these polymeric Hakata antigens in BAL fluid dissociated to monomeric ones (35 kD; Figure 5B). These results demonstrated that the Hakata antigen generated in bronchial epithelial cells and Type II alveolar epithelial cells in human lung is secreted as a polymer into bronchus/alveolus.
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Expression of Hakata Antigen in Liver
In liver, hepatocytes showed diffuse and weak staining of the cytoplasm by Pr19 (Figure 6A and Figure 6B), and bile duct epithelial cells were strongly stained (Figure 6A and Figure 6C). Kupffer cells, sinusoidal capillaries around hepatocytes, and Glisson's sheath around bile ducts were not stained. These results demonstrated that monomeric Hakata antigen protein is produced in both hepatocytes and bile duct epithelial cells, but to a much greater extent in the latter. The staining pattern was the same in liver tissue derived from seven different individuals (not shown).
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To clarify whether Hakata antigen is secreted into bile ducts, we subjected bile samples to Western blotting. In nonreducing conditions, polymeric Hakata antigen protein, which has the same mobility as that in serum, was detected in all samples, whereas in reducing conditions only monomeric Hakata antigen was detected in bile as in BAL fluid (Figure 7). Although the intensity of the bands was very weak in Lanes 3 and 7, longer exposure of the film gave rise to definite bands. Therefore, Hakata antigen, which is formed in bile duct epithelial cells, is secreted as a polymer into bile ducts.
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Discussion |
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It has recently been shown that Hakata antigen is a new member of the ficolin/opsonin p35 family by cloning of its cDNA and by functional analyses (
We have demonstrated in this study that Hakata antigen as well as SPs was secreted into bronchus and alveolus. SP-A and SP-D are known to be water-soluble and to sustain lectin activity (
In previous studies, it has been verified that Hakata antigen is a novel serum protein with a range of 723 µg/ml (
Bile duct epithelial cells are known to modify hepatic bile by both secretory and absorptive processes, producing as much as 40% of the daily output of bile (2-macroglobulin, hemopexin, immunoglobulin heavy chains, and the ß-chain of haptoglobulin (
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Acknowledgments |
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Supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
Dr Yae passed away in August of 1996. This work is dedicated to Dr Yae's collaborative efforts in scientific research in a career spanning nearly a quarter of a century.
We thank Ms K. Hatanaka for skillful help and Dr S. Hashimoto for providing antibodies against SP-A and CC10. We also thank Dr Dovie R. Wylie for critical review of this manuscript.
Received for publication August 26, 1998; accepted January 12, 1999.
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