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 KLK6 gene is a new member of the human kallikrein gene family and encodes for a secreted protease, human kallikrein 6 (hK6; also known as zyme/protease M/neurosin). No study has as yet reported detailed immunohistochemical localization of hK6 in human tissues. Our purpose was to examine the expression of hK6 in human tissues by immunohistochemistry. We have analyzed 199 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue. We employed an hK6-specific polyclonal rabbit antibody and avidinbiotin to localize hK6 by IHC. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. The IHC expression of zyme was generally cytoplasmic. Various normal human tissues expressed the protein abundantly. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, endometrium, colon, appendix, salivary glands, bile ducts, and gallbladder. The small intestine, stomach, endocervix, Fallopian tube, epididymis, bronchus, and upper respiratory tract showed a focal expression as well. Choroid plexus epithelium, peripheral nerves, and some neuroendocrine cells (including the islets of Langerhans, cells in the anterior pituitary gland, and adrenal medulla) expressed the protein strongly and diffusely. A characteristic immunostaining was observed in the Hassall's corpuscles of the thymus, the oxyphilic cells of the thyroid and parathyroid glands, the primordial follicles of the ovary, dendritic cells mainly in the spleen, and in various cells of the placenta. (J Histochem Cytochem 49:14311441, 2001)
Key Words: human kallikrein 6, immunohistochemical, expression, normal human tissues, secreted proteins, serine proteases, cancer biomarkers
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Introduction |
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UNTIL RECENTLY, the human kallikrein gene family was believed to consist of only three genes: pancreatic/renal kallikrein (KLK1, encoding for hK1 protein), human glandular kallikrein 2 (KLK2, encoding for hK2 protein), and human kallikrein 3 (KLK3, encoding for hK3 protein or prostate-specific antigen, PSA). The latter two kallikreins, PSA and hK2, are relatively prostate-specific and have already found important applications as biomarkers for the diagnosis and monitoring of prostate cancer (
New members of the human kallikrein gene family have recently been discovered (
The KLK6 gene (encoding for human kallikrein 6, hK6) has been cloned independently by three groups of investigators and was previously given the names zyme (
Recently, we have established the genomic organization and the hormonal regulation of the KLK6 gene and studied its tissue expression by rRT-PCR (
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Materials and Methods |
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This was an IHC study on almost all normal human tissues to establish the expression of hK6. Parts of an organ with different histology (e.g., stomach, fundus, body, antrum) were examined separately. A paraffin block of three different cases for every tissue (organ, all parts with different histology) was selected. Cases with malignancy in adjacent sites of the organ were excluded to avoid phenotypic changes that may be associated with cancer. Tissues that exist in several organs (e.g., fat, muscle, vessels, peripheral nerves, ganglia, and neuroendocrine cells) were not studied separately. A total of 199 paraffin blocks were examined. A total of 165 blocks were from archival or current material from 132 cases and the rest were autopsy material from two cases (Table 1).
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The avidinbiotin complex IHC method was performed on 4-µm-thick sections using two hK6-specific antibodies, one rabbit polyclonal and one mouse monoclonal. Both antibodies were raised in-house against full-length hK6 produced recombinantly in a mammalian stable cell line system (
The staining pattern, the distribution of the immunostaining in each tissue, and the intensity of the staining were studied in detail.
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Results |
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The hK6 immunoreactivity using the polyclonal and the monoclonal antibody was generally localized in the cytoplasm. Both antibodies revealed the same distribution of the antigen in all tissues. Replacement of the primary antibody with non-immune serum or immunoabsorbtion of the primary antibody abolished the immunostaining in all tissues examined. These findings indicate that our staining procedure is specific for hK6. The protein was revealed in a variety of tissues, indicating that this is not a tissue-specific protein. hK6 is mainly expressed by glandular tissues, but our data suggested that it could also serve as a neuroendocrine marker. The distribution and the expression levels of hK6 in various tissues are described below and further summarized in Table 2.
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Central and Peripheral Nervous System
Strong and diffuse positivity in the epithelium of the choroid plexus was observed. (Fig 1A and Fig 1B ). In the cerebellum, the antigen was expressed weakly in the Purkinje cells and the stellate (basket cells), whereas the granular cells were negative. In the entire central nervous system the nerve cells showed weak immunostaining as well (Fig 1C). Glial cells showed a weaker expression focally. Staining of the peripheral nerves was intense (Fig 1D).
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Female Reproductive System
The epithelium of the breast stained positive. Cytoplasmic and brush-border distribution was observed, and luminal secretions were positive as well (Fig 2A2D). A similar immunophenotype appeared in the columnar epithelium of the endometrium and the endocervix of the uterus. A characteristic droplet-like, widely distributed expression was noted in the endometrium (Fig 3A), but staining in the endocervix was focal and paranuclear. No major differences were seen during the menstrual cycle. The myometrium was negative. Weak and focal expression by the squamous cervical epithelium could not be safely considered as positive (see Discussion). The squamous epithelium of the vagina was negative. Supranuclear cytoplasmic, brush-border, and ciliary staining was revealed in the Fallopian tubes. Characteristic was the positivity in the primordial follicles of the ovary. In the placenta the protein was localized in the endothelia, in calcifications of the villi, as well as in "X" cells and, focally, in trophoblastic cells.
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Genitourinary Tract
Strong diffuse immunostaining was observed in the prostate columnar cells. Basal cells remained unstained. Because we did not use a double-staining method, it was not possible to evaluate the positivity of the neuroendocrine cells. In general, hK6 had the same immunostaining pattern in the benign prostate gland as hK2 and hK3 (Fig 3B3D). A focal, mainly suprabasal cytoplasmic, brush-border and ciliary staining in the epithelium of the epididymis, seminal vesicles, and spermatic duct was the rule. The urinary tubuli of the kidney (Fig 4A) showed immunoexpression as well. It is doubtful if weak focal expression by the umbrella cells of the urothelium and the Leydig cells of the testis could be considered as positive. Cavernous bodies were negative.
Gastrointestinal Tract
The glandular epithelium of the large bowel and the appendix showed strong, diffuse cytoplasmic, mainly subnuclear (large intestine) and supranuclear (appendix) immunostaining in the enterocytes (Fig 4B). No clear differences were noted among the different segments of the colon. Expression in the small intestine was focal cytoplasmic, mainly subnuclear in the duodenum and mainly peri- and supranuclear in the ileum. The antrum of the stomach showed a focal cytoplasmic, mainly subnuclear and brush-border staining; the body showed a brush-border and parietal cell expression (Fig 4C). Strong positivity in foci of intestinal metaplasia in the gastric mucosa was the rule (Fig 4D). The duct epithelium of the eosophageal glands expressed the antigen as well. Expression by neuroendocrine cells throughout the gastrointestinal tract was generally obvious. The reactivity by basal cells in the epithelium of the esophagus and the anus was considered to arise from neuroendocrine cells. We found strong positivity in the cells of the islets of Langerhans in the pancreas (Fig 5A5D). The acinar cells of the exocrine pancreas were negative. Only some scattered positive cells were observed between them. The epithelium of the medium-sized pancreatic ducts showed a cytoplasmic and mainly brush-border immunostaining. Hepatocytes were negative. A cytoplasmic and brush border immunostaining was observed in the bile ducts and the gallbladder mucosa (Fig 6A).
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Respiratory Tract
A mainly brush-border staining in the respiratory epithelium of the bronchus, larynx, trachea, rhinopharynx, and paranasal sinuses was observed (Fig 6B). The staining in the ducts was cytoplasmic. The alveoli of the lung were negative.
Salivary Glands (Major and Minor) and Skin Appendages
Cytoplasmic positivity in the ductal epithelium and scattered positive cells in the alveoli were noted (Fig 6C).
Spleen, Tonsils, Lymph Nodes, and Bone Marrow
Some positive cells, possibly dendritic, in the germinal centers of the follicles, mainly in the spleen, were found. Various inflammatory cells, mainly neutrophils but also some plasma cells, also showed positivity.
Thymus
Strong positivity in the Hassall's corpuscles was characteristic (Fig 6D).
Adrenal Gland
Weak to moderate positivity was observed in the medulla.
Thyroid Gland
Focal protein immunoexpression was revealed by follicular cells, mainly in hyperplastic conditions and in oxyphilic cell metaplasia (Fig 7A).
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Parathyroids
Immunoexpression by the oxyphilic cells was noted (Fig 7B).
Pituitary Gland
In the anterior pituitary, many cells expressed the protein strongly (Fig 7C). The pituitocytes of the pars nervosa were negative.
Mesothelium (Pleura)
The positivity was variable and was observed mainly in hyperplastic conditions.
Squamous Epithelia
Squamous epithelia were generally negative. In some cases weak focal expression by keratinocytes (cervix, mouth mucosa) was observed but could not be considered safely as positivity.
Mesenchymal Tissues
In general, mesenchymal tissues, except for neural tissue, were negative. Some weak positivity was observed in some myoepithelial cells in the walls of small arteries and in chondrocytes (Fig 7D).
Diffuse Neuroendocrine System
Neuroendocrine cells in several organs expressed hK6.
In short, hK6 is expressed by many normal human tissues. Glandular epithelia constitute the main hK6 immunoexpression sites, with representative organs being breast, prostate, kidney, endometrium, colon, appendix, salivary ducts, bile ducts, and gallbladder. The small intestine, the stomach, the endocervix, the Fallopian tube, the epididymis, the bronchus, and the upper respiratory tract showed focal expression. Choroid plexus epithelium, peripheral nerves, and neuroendocrine cells (including the islets of Langerhans and adrenal medulla) expressed the protein strongly and diffusely. Characteristic immunostaining was observed in Hassall's corpuscles of the thymus, oxyphilic cells in the thyroid and parathyroid glands, in the primordial follicles of the ovary, in dendritic cells mainly in the spleen, and in various cells of the placenta.
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Discussion |
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The kallikrein 6 gene (KLK6) is one of the recently cloned members of the human tissue kallikrein gene family (1-antichymotrypsin (
In our recent work, we found the highest concentration of hK6 in milk of lactating women, followed by cerebrospinal fluid, nipple aspirate fluid, and breast cyst fluid (
This is the first report describing IHC localization of hK6 in a large variety of human tissues. Only
hK6 was localized in a large number of normal human tissues and therefore cannot be considered as a specific tissue marker, in contrast to the homologous proteins hK2 and PSA, which show prostate-restricted specificity (
Another interesting finding was the presence of hK6 in some cells of the diffuse neuroendocrine system and in nerves. The staining was relatively strong, and we believe that it could be representative of neuroendocrine differentiation. The expression of hK6 is reminiscent of CD56 (natural killer cell-associated antigen, neural cell adhesion molecule) and CD57 (Leu-7, T-cell surface marker) which are sensitive but not specific for cells and neoplasms with neuroendocrine differentiation (
The contribution of hK3 (PSA) and hK2 in the diagnosis and monitoring of prostate cancer suggests that other kallikreins may also have value as candidate biomarkers. We have already shown that serum hK6 concentration is increased in ovarian carcinoma (
KLK6 has been shown to be regulated by steroid hormones (
Received for publication January 12, 2001; accepted May 17, 2001.
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