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 normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10, KLK10) was recently cloned and encodes for a putative secreted serine protease (human kallikrein 10, hK10). Several studies have confirmed that hK10 shares many similarities with the other kallikrein members at the DNA, mRNA, and protein levels. The enzyme was found in biological fluids, tissue extracts, and serum. Here we report the first detailed immunohistochemical (IHC) localization of hK10 in normal human tissues. We used the streptavidinbiotin method with two hK10-specific antibodies, a polyclonal rabbit and a monoclonal mouse antibody, developed in house. We analyzed 184 paraffin blocks from archival, current, and autopsy material, prepared from almost every normal human tissue. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. Previously, we reported the expression of another novel human kallikrein, hK6, by using similar techniques. The IHC expression of hK10 was generally cytoplasmic and not organ-specific. A variety of normal human tissues expressed the protein. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, epididymis, endometrium, fallopian tubes, gastrointestinal tract, bronchus, salivary glands, bile ducts, and gallbladder. The choroid plexus epithelium, the peripheral nerves, and some neuroendocrine organs (including the islets of Langerhans, cells of the adenohypophysis, the adrenal medulla, and Leydig cells) expressed the protein strongly and diffusely. The spermatic epithelium of the testis expressed the protein moderately. A characteristic immunostaining was observed in Hassall's corpuscles of the thymus, oxyphilic cells of the thyroid and parathyroid glands, and chondrocytes. Comparing these results with those of hK6, we observed that both kallikreins had a similar IHC expression pattern. (J Histochem Cytochem 50:12471261, 2002)
Key Words: human kallikrein 10, immunohistochemical, expression, normal human tissues, secreted proteins, serine proteases, cancer biomarkers
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Introduction |
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The basic components of the kallikreinkinin system were discovered in the 1930s (
In the past 3 years, new members of the human kallikrein gene family have been discovered (
The normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10, KLK10) was cloned by subtractive hybridization techniques based on its downregulation in radiation-transformed breast epithelial cells (5.5 kb of genomic DNA sequence, and contains six exons (one untranslated) and five introns (
There are very few reports describing the expression of kallikreins by IHC in human normal or diseased tissues (
Here we describe the IHC localization of hK10 in diverse human tissues.
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Materials and Methods |
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This is an IHC study on almost all normal human tissues, aiming to establish the expression pattern of hK10. 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. Tissues that exist in several organs (e.g., fat, muscle, vessels, peripheral nerves, ganglia, and neuroendocrine cells) were not studied separately. A total of 184 paraffin blocks were examined. Of these, 150 concerned archival or current material from 117 cases and 34 were autopsy material from two cases. The archival and the current material were biopsy or surgical specimens. We used autopsy material to study the different parts of the brain, including the pituitary gland, because the routine material is usually from neoplastic cases. We did not study any specimens from the pineal gland and the spinal cord.
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 (Carpinteria, CA). An hK10-specific rabbit polyclonal antibody (1:400) and an hK10 mouse monoclonal antibody (Code 5D3, 1:100) were raised by immunizing with full-length recombinant hK10 produced in yeast, as described elsewhere (
The staining pattern, the distribution of the immunostaining in each tissue, and the intensity of the staining were studied in detail.
In selected tissues the primary antibody was replaced by a non-immune rabbit serum (1:500) in 3% BSA to assess nonspecific binding. For the same reason, an immunoabsorption test was also performed in which the primary polyclonal hK10 antibody was incubated for 1 hr at RT with recombinant hK10 antigen (10 µg/liter) before immunostaining.
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Results |
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The hK10 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 polyclonal antibody with non-immune rabbit serum and immunoabsorption of the antibody with hK10 antigen abolished the immunostaining in the tissues, supporting the specificity of staining. The protein was found in a variety of tissues, indicating that it is not tissue-specific. hK10 was mainly expressed by glandular tissues, but we obtained evidence that it could also serve as a neuroendocrine marker. The distribution and the quantitative expression of hK10 in various tissues are described below and are further summarized in Table 1. It is noteworthy that there were no major differences between the immunolocalizations of hK10 and hK6 in the examined tissues. Western blots confirmed the specificity of the two polyclonal antibodies for hK6 and hK10. As shown in Fig 1, no crossreactivity was detected and the two antibodies recognize only their cognate antigens.
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Central and Peripheral Nervous Systems
Our most striking finding in the CNS was the strong and diffuse positivity in the epithelium of the choroid plexus, the specialized organ that lines the ventricular system and is responsible for the production of the cerebrospinal fluid (Fig 2A). In the cerebellar cortex the antigen was expressed weakly to moderately in the Purkinje cells (neural cells), whereas the granular cells were negative (Fig 2B). In the gray matter of the cerebral cortex, neurons and glial cells were weakly to moderately immunoreactive (Fig 2C and Fig 2D). It is worth mentioning that although the majority of neurons were positive, the immunoreactivity in the glial cells was focal and concerned both protoplasmic astrocytes and oligodendrocytes. Glial cells were morphologically easily distinguishable from neurons. We verified the distinction of small neurons from protoplasmic astrocytes using synaptophysin, neurofilaments, and GFAP (glial fibrillary acidic protein) antibodies. The former cells react with synaptophysin and neurofilaments and the latter cells with GFAP. Oligodendrocytes were easily distinguished by their smaller size, their characteristic location against the cell membrane of neurons, and their CD57/synaptophysin positivity. Microglia were not easily recognized in our sections, and we could not comment about its reactivity with hK10 antibodies. In the white matter of the cerebral cortex, both fibrillary astrocytes and oligodendrocytes expressed hK10 focally. The distinction between these cell types was difficult. The nuclei of oligodendrocytes are somewhat smaller and more hyperchromatic, but usually these two cell types do not fall into two clearly defined groups. The CD57 positivity in the oligodendrocytes was helpful. No staining was identified in the meninges.
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We studied hK10 immunostaining in the peripheral nervous system, examining nerves and ganglia, which were contained in different specimens. All nerves, motor, sensory, and autonomic, showed the same strong positivity for hK10 (Fig 3A). We suggested that the immunoexpression concerned Schwann cells because the staining pattern was similar to that of S100 protein. Strong positivity for hK10 was also observed in the ganglia of the peripheral nervous system. Both the neurons and their satellite cells that have an origin similar to that of Schwann cells were immunoreactive.
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Female Reproductive System
We examined non-malignant breast specimens from women with mild fibrocystic disease. We tried to focus on almost "normal" structures. Cytoplasmic immunoexpression was identified in the columnar cells of ductal and lobuloalveolar structures. Myoepithelial cells remained unstained. Luminal secretions were also positive (Fig 3B and Fig 3C). Foci of apocrine metaplasia and apocrine cysts showed strong staining (Fig 3D). The staining in breast tissues was in most cases focal. Not every structure expressed the protein, and there were differences in the staining intensity.
We studied both layers of the endometrium, the basalis and the functionalis, during the different phases of the menstrual cycle. Strong but focal hK10 immunostaining was seen in the epithelium of the endometrium in both the proliferative and secretory phase, with cytoplasmic localization. A characteristic infranuclear droplet-like, in some areas widely distributed, expression was noted in the secretory phase (Fig 4A and Fig 4B). The staining in the functionalis layer was more diffuse and intense than in the basalis layer. Characteristic was the moderate hK10 immunoexpression in areas of decidual change of the endometrial stroma due to hormonal changes during the menstrual cycle, in cases of hormone intake for therapeutic reasons, and in pregnancy. No staining was identified in the myometrium.
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Cytoplasmic immunoexpression in the mucin-secreting epithelium of the endocervix and the tubular cervical glands was observed. A ciliar staining pattern was observed in ciliated cells. Foci of squamous metaplasia and the squamous epithelium of the exocervix were negative (data not shown), as was the squamous epithelium of the vagina.
Ovarian specimens from pre- and post-menopausal women were examined. hK10 immunostaining was observed in the surface epithelium. A clear immunoreaction was also observed in the primordial follicles, the granulosa lutein cells in the corpus luteum, and sparse luteinized cells in the stroma of the ovary (data not shown). No other cells expressed the protein. Strong, diffuse cytoplasmic and ciliar staining was revealed in the epithelium of the fallopian tubes (Fig 4C).
Genitourinary Tract
We studied specimens from the renal cortex and the renal medulla. The epithelium of all types of the urinary tubuli showed a cytoplasmic IHC expression of hK10. Stronger staining was observed in the epithelium of the proximal and distal convoluted tubuli (Fig 4D). In the proximal tubuli a brush border staining was obvious. No cell type of the renal glomeruli expressed hK10.
The urothelium of the urinary bladder, as well as the other parts of the urinary tract, showed a characteristic immunoexpression of hK10. The superficial umbrella cells and only some scattered intermediate urothelial cells expressed the protein (Fig 5A).
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Male Reproductive System
We studied hK10 immunoexpression in all zones of the prostate gland. Variable but mostly strong and diffuse hK10 immunostaining in the prostate secretory cells was observed in all parts. Although the basal cells remained unstained, hK10 was often expressed in basal cell hyperplasia. In these areas the columnar cells expressed the protein weakly or remained unstained. Another interesting finding was the most intense staining in scattered cells among the columnar epithelium. Chromogranin positivity in these cells supported their neuroendocrine nature (Fig 5B5D). The epithelium of the ejaculatory ducts revealed hK10 positivity as well.
Strong cytoplasmic, brush border, and ciliar staining was revealed in the epithelium of the efferent ductules, the epididymis, the ductus deferens, the ampulla of ductus deferens, the seminal vesicle, and the ejaculatory duct (Fig 6A).
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Strong hK10 immunostaining in the Leydig cells of the testis was observed. The spermatic epithelium showed variable expression from case to case. Weak to moderate staining throughout the epithelium was mostly observed. In cases with atrophy of the spermatic epithelium, spermatogonia and Sertoli cells expressed the protein more intensely (Fig 6B and Fig 6C). The rete testis showed cytoplasmic hK10 expression (Fig 6D).
The columnar epithelium of the penile urethra, as well as the Littre's and Cowper's glands, showed moderate hK10 expression (data not shown). The smooth muscle and the connective tissue of the corpus cavernosum and the corpus spongiosum, as well as other structures in the penis, were negative.
Gastrointestinal Tract
The non-keratinizing squamous epithelium of the esophageal mucosa was negative. The mucus-secreting esophageal glands located in the lamina propria expressed hK10, mainly in the ductal epithelium (data not shown).
Specimens from cardiac, antral, and fundic mucosa of the stomach were examined. The columnar mucus-secreting surface epithelium in all parts of the gastric mucosa showed focal hK10 cytoplasmic, mainly infranuclear staining. The cardiac and antral gastric glands, as well as the mucous neck cells in the fundus, expressed the kallikrein in the same manner. Immunoexpression in the fundic mucosa was more extensive and diffuse. Both parietal and chief cells expressed hK10, but the immunoexpression in the former cells was constant and more intense (Fig 7A). Strong positivity in foci of intestinal metaplasia in the gastric mucosa was the rule. Stronger expression of some cells in some glands could concern neuroendocrine cells, but this suggestion was not further investigated in the present study.
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We studied specimens from all parts of the small intestine. No apparent differences in hK10 expression were noted in the mucosa of the duodenum, the jejunum, and the ileum. Both the villous and the crypt epithelium expressed the kallikrein with a focal distribution. Intense cytoplasmic, mainly supranuclear, and brush border immunoexpression was observed in the absorptive cells. Goblet cells were positive to a lesser degree, expressing the protein in their cytoplasm and only rarely in the mucin droplets (Fig 7B). As in the gastric mucosa, there was a strong suggestion that endocrine cells in the crypts were positive. Staining of Paneth cells was not always obvious. Therefore, these cells require further investigation. The submucosal Brunner's glands in the bulb of the duodenum showed moderate focal immunostaining.
Specimens from all segments of the large intestine were examined. No clear differences in hK10 expression were noted in the mucosa of the different regions of the colon. Both the surface and the crypt epithelium expressed the kallikrein strongly and mostly diffusely. Cytoplasmic, mainly infranuclear immunoexpression dominated in the absorptive cells. Goblet cells also showed cytoplasmic, mainly supranuclear staining, whereas most mucin droplets remained unstained (Fig 7C).
A mainly supranuclear cytoplasmic immunostaining was expressed in the absorptive cells of the appendix (Fig 7D). A brush border pattern in the surface epithelium, covering lymphoid aggregates, concerned membranous cells. A cytoplasmic, also supranuclear, immunoexpression was revealed in goblet cells, whereas the apical mucin droplets usually remained unstained.
Pancreas
In the exocrine pancreas, cytoplasmic hK10 immunoexpression was observed in the medium and small pancreatic ducts, whereas the acinar cells were negative. We found strong positivity in the cells of the islets of Langerhans. Scattered hK10-positive cells were localized in relationship to pancreatic acinar cells (Fig 8A8D). Using a double immunostaining method, we demonstrated that all cell types in the islets revealed a co-localization of each hormone and hK10 in the same cell population. It is well known that scattered cells in the exocrine pancreas express the different hormones that are produced in the different cell types in the islets. These cells probably express hK10 as well. This finding was documented by double immunostaining techniques (our unpublished data).
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Liver, Gallbladder, and Extrahepatic Bile Ducts
Hepatocytes were negative. Cytoplasmic immunostaining was observed in the bile ducts of the portal tracts (data not shown).
Cytoplasmic immunostaining was observed in the columnar epithelium of the gallbladder, the cystic duct, and the extrahepatic bile ducts (data not shown).
Respiratory Tract
Specimens from most regions of the upper and lower respiratory system were examined. The pseudostratified ciliated columnar epithelium that covers the upper and lower respiratory tract (nose, paranasal sinuses, larynx, trachea, bronchial tree) showed cytoplasmic and ciliar hK10 staining. Intermingled goblet cells were also positive (data not shown). A characteristic immunostaining was observed in the submucosal glands in the larynx, trachea, and larger bronchi. The epithelium of the excretory ducts and the serous alveoli expressed hK10 strongly and diffusely, and mucous alveoli stained to a lesser degree (Fig 9A). The alveolar epithelium of the lung parenchyma was negative.
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Salivary Glands and Skin Appendages
We examined specimens from the major and minor salivary ducts. hK10 immunoexpression in the different cells in serous, mucous, and mixed glands was evaluated. Strong cytoplasmic positivity in the epithelium of the excretory ducts was constant in all cases (Fig 9B). Most mucous and serous alveoli were negative. In mixed glands, crescent-shaped formations of serous cells on the periphery of mucous alveoli expressed the kallikrein often.
The ductal epithelium of skin appendages expressed hK10.
Spleen, Tonsils, Lymph Nodes, and Bone Marrow
The lymphatic organs, such as the spleen and the lymph nodes, generally did not express hK10. Only characteristic positivity in the Hassall's corpuscles of the thymus was only observed (Fig 9C). In inflammatory lesions in different tissues, hK10 was expressed by leukcocytes, mainly neutrophils.
Adrenal Glands
Moderate positivity was observed in the cytoplasm of the cells in the adrenal medulla (Fig 9D).
Thyroid Gland
Focal protein immunoexpression was revealed in the follicular cells in the thyroid gland, mainly in hyperplastic conditions and in oxyphilic cell metaplasia (Fig 10A).
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Parathyroid Glands
hK10 immunoexpression by the oxyphilic cells was noted in the parathyroid glands. Chief cells remained mostly unstained (data not shown).
Pituitary Gland
In the anterior lobe of the adenohypophysis, many cells expressed the protein strongly (Fig 10B). These cells had the morphology of lactotrophs and corticotrophs. Lactotrophs usually wrap around adjacent cells and sometimes show paranuclear staining, a pattern corresponding to prolactin in the Golgi apparatus. Corticotrophs are larger, ovoid to polyhedral, have a tendency to cluster, and many have an unstained region near the nucleus. Furthermore, they reacted with prolactin and ACTH antibodies, respectively. Characteristic strong positivity was also shown in epithelium-lined follicular and ductal formations in the poorly developed (in humans) pars intermedia. (Fig 10C). It is well known that these cystic formations are lined by a single layer of cuboidal to columnar epithelium which may be non-ciliated, ciliated, mucin-producing, or granulated (endocrine). These endocrine cells show variable immunoreactivity for pituitary hormones. In our case, they were prolactin-immunoreactive. The pituicytes of the pars nervosa were negative.
Mesothelium
Specimens including pleural, pericardial, and peritoneal mesothelium were studied. The positivity for hK10 was variable, from weak to strong. In reactive processes, where the mesothelial cells undergo marked proliferative and hyperplastic changes, the staining was more prominent (data not shown).
Squamous Epithelia
Squamous epithelia (skin, uterine cervix, mouth mucosa) were generally negative. In some cases weak focal expression by keratinocytes was observed but could not be considered safely as positive.
Mesenchymal Tissues
In general, no expression was observed in mesenchymal tissues. Positivity was expressed in chondrocytes (Fig 10D) and in the wall of small vessels in some cases.
In short, hK10 is expressed by many normal human tissues. Glandular epithelia constitute the main hK10 immunoexpression sites, with representative organs being the prostate, epididymis, spermatic duct, kidney, breast, endometrium, endocervix, fallopian tube, colon, appendix, small intestine, stomach, salivary ducts, bile ducts, gallbladder, bronchus, and the upper respiratory tract. Both the Leydig cells and the spermatic epithelium in the testis also expressed hK10. Focal immunostaining in glial cells in the CNS was also observed. Diffuse and strong expression was noted in the choroid plexus epithelium, the peripheral nerves, the ganglia, cells of the anterior lobe of the pituitary, and the islets of Langerhans in the pancreas. A characteristic immunostaining was observed in the Hassall's corpuscles of the thymus, the oxyphilic cells in the thyroid and parathyroid glands, and in chondrocytes.
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Discussion |
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The KLK10 gene, initially named NES1, was cloned by subtractive hybridization from radiation-transformed breast epithelial cells (
This is the first report describing the IHC localization of hK10 in diverse normal human tissues. Similar work has been reported recently by our group for the homologous kallikrein hK6 (
The hK10 protein was localized in a large number of normal human tissues and therefore, like hK6, it cannot be considered a specific tissue marker, in contrast to the homologous proteins hK2 and PSA (hK3), which have prostate-restricted expression (
Our IHC findings correspond well with the data of hK10 quantification in tissue extracts by immunoassay (
Kallikreins are regulated by steroid hormones (
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 and hK10 concentration is increased in ovarian carcinoma (
In conclusion, we report here the expression of hK10 protein in various human tissues by immunohistochemistry. We believe that this study further illumines the role of this protein in human tissues and will help to generate hypotheses of its function and pathophysiology.
Received for publication November 16, 2001; accepted April 10, 2002.
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