ARTICLE |
Correspondence to: Eleftherios P. Diamandis, Dept. of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. E-mail: ediamandis@mtsinai.on.ca
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Summary |
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The human tissue kallikrein 13 gene (KLK13), encoding for hK13 protein, was recently cloned and characterized. Here we describe the immunohistochemical (IHC) localization of hK13 in normal human tissues and compare it with the expression of two other kallikreins, hK6 and hK10. We performed the streptavidinbiotin IHC method on 204 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue, using a polyclonal and a monoclonal hK13 antibody. The staining was cytoplasmic and both antibodies yielded similar results. The hK13 protein was revealed in a variety of tissues, mainly in glandular epithelia. Other epithelia that expressed hK13 included the urothelium, the spermatic epithelium, and the epithelium of the choroid plexus. hK13 was intensely immunoexpressed by some endocrine organs, such as the adenohypophysis, the thyroid gland, the parathyroid glands, the adrenal medulla, the Leydig cells of the testis, and the cells of the endocrine pancreas. Immunoreactivity was also observed in the primordial follicles, the corpus luteum, and sparse luteinized cells in the stroma of the ovary, the trophoblastic cells of the placenta, the Hassall's corpuscles of the thymus, and chondrocytes. Nerves and ganglia of the peripheral nervous system, and both neurons and glial cells in the central nervous system, were positive. In short, hK13 was expressed by many glandular epithelia, some endocrine organs, and some specialized epithelia and cells. Comparison of these data with hK6 and hK10 expression suggests that the three kallikreins have a similar IHC pattern in normal human tissues. (J Histochem Cytochem 51:493501, 2003)
Key Words: human kallikreins, human kallikrein 13, immunohistochemical, expression, serine proteases, cancer biomarkers, epithelial cell markers
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Introduction |
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HUMAN TISSUE KALLIKREINS are members of a large multigene family of 15 serine proteases (
Very few studies have examined human kallikrein expression by IHC in normal or diseased tissues. In our previous reports, we described hK6 and hK10 expression in a variety of normal tissues by IHC (
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Materials and Methods |
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Representative samples from almost all normal human tissues were studied by IHC. Parts of an organ with different histology (e.g., stomach fundus, body, antrum) were examined separately. Tissues that exist in several organs (e.g., fat, muscle, vessels, peripheral nerves, ganglia) were not studied separately. A total of 204 paraffin blocks were examined. From these, 170 represented archival or current material from 137 cases and the other 34 samples were autopsy material from two cases. The archival and the current material were biopsy or surgical specimens (Table 1). Most but not all of these samples were examined previously for hK6 and hK10 immunoexpression (
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The IHC staining was performed on 4-µm-thick paraffin sections of tissues fixed in buffered formalin, according to a streptavidinbiotinperoxidase protocol using the DAKO LSAB+Kit Peroxidase (DAKO; Glostrup, Denmark). An hK13-specific rabbit polyclonal antibody (1:500) and two hK13 (2-17 and 11C1) mouse monoclonal antibodies (MAbs: 1:150 and 1:50, respectively) raised against full-length recombinant hK13 protein produced in yeast were used. The recombinant hK13 protein was produced and purified by HPLC as previously described for hK10 protein (
In selected tissues the primary polyclonal antibody was replaced by a nonimmune rabbit serum (1:500) in 3% BSA to evaluate nonspecific binding. For the same reason, an immunoabsorption test was also performed in which the primary hK13 antibody was incubated for 1 hr at RT with an excess of recombinant hK13 antigen.
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Results |
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The hK13 immunoreactivity, using the polyclonal antibody and the two MAbs, was generally localized in the cytoplasm. All antibodies revealed the same distribution of the antigen in all tissues. Replacement of the primary polyclonal antibody with nonimmune rabbit serum and the immunoabsorption test resulted in disappearance of the immunostaining in all tissues examined.
In our previous studies we did not find any major differences between the immunolocalization of hK6 and hK10 in normal human tissues. It is worth mentioning that, in the present study, hK13 had, in general, a similar IHC distribution as the other two kallikreins. The protein was revealed in a variety of tissues, indicating that it is not tissue-specific.
The hK13 protein was immunoexpressed mainly by glandular epithelia. The gastric mucosa expressed the kallikrein focally in all cell types (Fig 1A). A characteristic supranuclear, droplet-like, and brush border localization was observed in the duodenum (Fig 1B) and the other parts of the small intestine (Fig 1C). All parts of the large intestine (Fig 1D) and the appendix (Fig 1E) showed strong and diffuse immunoexpression in both the enterocytes and the goblet cells. The major and minor salivary glands (Fig 1F), as well as the submucosal glands of the pharynx and the esophagus, expressed the antigen. The epithelium of the pancreatic (Fig 1G) and bile duct system (Fig 1H), and also of the gallbladder (Fig 1I), was positive. Strong cytoplasmic staining was observed in the epithelium of the bronchial tree (Fig 1J). All glands of the upper and lower respiratory system (nasal, sinusoidal, laryngeal, tracheal, and bronchial) were strongly positive (Fig 1K). The epithelium of the eccrine and apocrine glands of the skin was also strongly positive (Fig 1L). The glandular epithelia in the genitourinary tract and in the male and female reproductive systems expressed hK13. The urinary tubuli of the kidney expressed the protein diffusely (Fig 2A). Immunoexpression in the epithelium of the prostate (Fig 2B), the ejaculatory ducts, the seminal vesicles, the rete testis, the epididymis (Fig 2C), and the spermatic duct (Fig 2D) was diffuse and strong. The staining in the surface epithelium of the ovary, epithelium of the fallopian tubes, breast (Fig 2E and Fig 2F), and endometrium (Fig 2G) was strong, but in some cases focal. No differences were observed during the course of the menstrual cycle. The columnar epithelium of the endocervix showed weaker and more focal distribution, and foci of immature squamous metaplasia also expressed the antigen (Fig 2H).
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Weak and diffuse immunoexpression of hK13 by the squamous epithelia in the different organs of the human body was not considered as positive. Other than glandular epithelia, epithelia that expressed hK13 were the urothelium, with immunolocalization mainly in the superficial cells (Fig 2I), the spermatic epithelium (Fig 2J), and the epithelium of the choroid plexus (Fig 2K).
The hK13 protein was intensely immunoexpressed by some endocrine organs and showed a similar distribution to that of the other two kallikreins. The lactotrophs and the corticotrophs of the anterior lobe of the pituitary (Fig 2L), the epithelium of the thyroid follicles, mainly in foci of hyperplasia oxyphilic metaplasia (Fig 3A), the cells, mainly oxyphilic, in the parathyroid glands (Fig 3B), the adrenal medulla, the Leydig cells of the testis (Fig 3C), and the cells of the endocrine pancreas islets of Langerhans and scattered endocrine cells in the exocrine pancreas (Fig 3D) also expressed hK13.
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A clear immunoreaction was also observed in the primordial follicles, the corpus luteum, and in sparse luteinized cells in the stroma of the ovary (Fig 3E). The trophoblastic cells of the placenta expressed this kallikrein in all phases of their development (Fig 3F). The decidual cells in the endometrium during pregnancy and in certain endocrine disorders were also positive (Fig 3G).
The Hassall's corpuscles of the thymus (Fig 3H) and the chondrocytes (Fig 3I) also expressed the kallikrein. Nerves and ganglia of the peripheral nervous system (Fig 3J) were strongly positive. In the central nervous system, both neurons and glial cells were positive (Fig 3K). Among the inflammatory cells, neutrophils expressed hK13 strongly (Fig 3L).
In short, hK13 was expressed by many glandular epithelia, by some endocrine organs, and by some specialized epithelia and cells.
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Discussion |
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The human kallikrein 13 gene (KLK13) was cloned by using the positional candidate approach (
We report here that hK13 is expressed mainly by glandular epithelia, further suggesting that this protein is secreted. The finding of hK13 protein in seminal plasma, amniotic fluid, milk of lactating women, follicular fluid, and cerebrospinal fluid further supports this hypothesis. Among other kallikreins that have been identified, all are found in biological fluids, an indication that all members of this family are secreted serine proteases.
In the testis, we have demonstrated that Leydig cells and the spermatic epithelium express hK13 protein. In our previous work we described the identification of five new mRNA KLK13 splice variants, which are expressed only in human testis (
The hK13 protein is expressed at high levels in the pituitary, especially in lactotrophs and corticotrophs, and in epithelium-lined spaces of the intermediate lobe remnant, which are prolactin-immunoreactive. The same distribution was observed with hK6 and hK10 expression in our previous studies (
The expression of a few kallikreins in the CNS has been previously reported (
We have previously reported expression of hK6 and hK10 in oxyphylic cells in the thyroid and parathyroid glands. The expression of hK13 in these tissues is similar. Previously, the presence of hK2 and hK3 in the thyroid has been reported (
Many kallikreins have found important applications as cancer biomarkers, including hK2 and hK3 for prostate cancer (
In summary, we describe for the first time the IHC localization of hK13 in various human tissues. Further studies will be necessary to examine the physiological and pathobiological role of this protein in these tissues.
Received for publication July 26, 2002; accepted November 6, 2002.
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