Osteoclastic tartrate-resistant acid phosphatase (Acp 5): its localization to dendritic cells and diverse murine tissues.

Tartrate-resistant acid phosphatase (TRAP) is a histochemical marker of the osteoclast. It is also characteristic of monohistiocytes, particularly alveolar macrophages, and is associated with diverse pathological conditions, including hairy cell leukemia and AIDS encephalopathy. To study the biology of this enzyme, we investigated its expression and activity in mouse tissues. Confocal fluorescence studies showed that TRAP is localized to the lysosomal compartment of macrophages. In adult mice, high activities of the enzyme were demonstrated in bone, spleen, liver, thymus, and colon, with lower amounts in lung, stomach, skin, brain, and kidney. Trace amounts were detected in testis, muscle, and heart. Expression of TRAP mRNA was investigated in tissue sections by in situ hybridization and protein expression was monitored by histochemical staining or immunohistochemically. TRAP is widely expressed in many tissues, where it is associated with cells principally originating from the bone marrow, including those of osteoclast/macrophage lineage. The cellular distribution of TRAP mRNA and enzyme antigen in the tissues corresponds closely to that of cells staining with an antibody directed to the CD80 (B7) antigen. Therefore, to confirm its putative localization in dendritic cells, isolated bone marrow dendritic cells were matured in culture. These co-stained strongly for TRAP protein and the CD80 antigen. These studies demonstrate that TRAP is a lysosomal enzyme that is found in diverse murine tissues, where it is expressed in dendritic cells as well as osteoclasts and macrophages, as previously shown. (J Histochem Cytochem 48:219-227, 2000)     

The spectrum of human kallikrein 6 (zyme/protease M/neurosin) expression in human tissues as assessed by immunohistochemistry.

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 avidin-biotin 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.     

The synthetic analogue of mycoplasmal lipoprotein FSL-1 induces dendritic cell maturation through Toll-like receptor 2

Granulocyte-macrophage colony-stimulating factor-differentiated bone marrow-derived dendritic cells were stimulated with the synthetic lipopeptide S-(2,3-bispalmitoyloxypropyl)-CGDPKHSPKSF (FSL-1) or the Escherichia coli lipopolysaccharide. FSL-1 induced the production of TNF-alpha and IL-12 by C57BL/6-derived bone marrow-derived dendritic cells but not by bone marrow-derived dendritic cells from Toll-like receptor 2-deficient (TLR2(-/-)) mice. Lipopolysaccharide induced the production of TNF-alpha and IL-12 by bone marrow-derived dendritic cells derived from either type of mice. FSL-1 did not induce production of IL-10 by bone marrow-derived dendritic cells from either type of mice, whereas lipopolysaccharide induced small amounts of IL-10 by bone marrow-derived dendritic cells from both types of mice. The upregulation by FSL-1 of the expression of CD80, CD86 and the MHC class II molecule IA(b) was dose- and time-dependent on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells but not on the surface of TLR2(-/-)-derived bone marrow-derived dendritic cells. Lipopolysaccharide upregulated the expression of these molecules on the surfaces of bone marrow-derived dendritic cells from both types of mice. The expression of CD11c on the surfaces of C57BL/6-derived bone marrow-derived dendritic cells was upregulated by stimulation with both FSL-1 and lipopolysaccharide up to 12 h; thereafter, the expression was downregulated. The results suggest that FSL-1 can accelerate maturation of bone marrow-derived dendritic cells and this FSL-1 activity is mediated by TLR2.     

Human breast cancer cell lines and tissues express tartrate-resistant acid phosphatase (TRAP)

Tartrate-resistant acid phosphatase (TRAP) is expressed by osteoclasts, macrophages and dendritic cells. TRAP has been identified in a wide variety of tissues, however, its biological function is not fully understood. Serum TRAP is a marker of diseases involving excessive bone resorption including metastatic bone disease in breast cancer patients and can be used to monitor responses to treatment. Our aim in this study was to determine whether TRAP is expressed by human breast tumours. Four breast cancer cell lines were assayed for TRAP activity. MDA-MB-435, the most tumourigenic line, had an activity twofold higher than the other cell lines. Immunohistochemistry using a TRAP specific antibody confirmed that both cell lines and human breast tumours express TRAP. Expression was absent in benign tissues and abundant in more aggressive tumours. This work suggests that tumour derived TRAP contributes to the raised enzyme activity found in the serum of breast cancer patients.     

A monoclonal antibody directed against high Mr salivary mucins recognizes the SO3-3Gal{beta}1-3GlcNAc moiety of sulfo-Lewisa: a histochemical survey of human and rat tissue

Using a panel of synthetic oligosaccharides attached to a polyacrylamidecarrier, the epitope of monoclonal antibody F2, evoked to highM_r salivary mucins, was mapped to the SO₃-3Galß1-3GlcNAc-moiety of the sulfo-Le^a antigen. Using immunochemical techniques,the expression of the F2-epitope was investigated in a numberof different isolated human mucin species, as well as in humanand rat tissue specimens. The mAb F2 bound to high M_r salivarymucins, cervical mucins, colon mucins and gallbladder mucins,but not to low M_r salivary mucins nor to gastric mucins. Immunohistochemicalscreening of human tissues with mAb F2 revealed a positive reactionwith a number of epithelia, including the (sero)mucous salivaryglands, the goblet cells of the colon, submucosal glands ofthe lung, the lining epithelium of cervical and esophageal glands,the suprabasal skin keratinocytes, and Hassall's corpusclesof the thymus. No staining was found in normal breast, pancreas,small intestine, spleen, and lymph nodes. Normal gastric glandswere negative, but gastric intestinal metaplastic glands stronglystained with the antibody. In rat tissues, mAb F2 labeled epithelialcells of salivary glands, colon and stomach. In addition toepithelial cells, extracellular matrix components in rat thymusand skin were labeled by mAb F2. No labeling of erythrocytes,granulocytes, lymphocytes or bone marrow cells was found byFACScan analysis. The present data shows a tissue specific distributionof the F2-epitope in cells from the epithelial lineage in humanand rat. epithelial tissue sulfo-Lewis^a mucins mAbs immunohistochemistry     

The major source of endogenous prostaglandin D2 production is likely antigen-presenting cells. Localization of glutathione-requiring prostaglandin D synthetase in histiocytes, dendritic, and Kupffer cells in various rat tissues

The cellular localization of glutathione-requiring PGD synthetase, which catalyzes the predominant formation of PGD2 in various peripheral tissues, was investigated in adult rats by immunoperoxidase-staining with a polyclonal antibody specific for this enzyme. Although the 25 N-terminal amino acid residues of synthetase are 56% identical and 76% similar to those of several rat glutathione S-transferase subunits, the antibody cross-reacted only with synthetase in dot blotting and was nearly completely inactive with all transferase isozymes thus far purified. In Western blotting after SDS-PAGE of crude extracts of rat spleen, the antibody showed a single positive band at the same position as that of the purified enzyme (Mr = 26,000). The positive immunocytochemical stain was found in a number of histiocytes and/or dendritic cells in spleen, thymus, and Peyer's patch of intestine. The immunostain was also observed in such cells in lamina propria of the villus in small intestine and colon, in submucosal layer of stomach, and in Kupffer cells in liver. Immunoelectron microscopy confirmed that immunoreactivity of this enzyme was distributed in cytoplasm of those cells. Such immunoreactive cells were not observed in brain, spinal cord, kidney, heart, testis, and skeletal muscle. These observations suggest that PGD2 is produced by glutathione-requiring PGD synthetase localized in these types of APC in various tissues and may play a critical role in dictating the progression of immune responses.     

Nuclear RelB+ cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions

Differentiated dendritic cells (DC) have been identified by the presence of nuclear RelB (nRelB) and HLA-DR, and the absence of CD20 or high levels of CD68, in lymph nodes and active rheumatoid arthritis synovial tissue. The current studies aimed to identify conditions in which nRelB is expressed in human tissues, by single and double immunohistochemistry of formalin-fixed peripheral and lymphoid tissue. Normal peripheral tissue did not contain nRelB+ cells. nRelB+ DC were located only in T- or B-cell areas of lymphoid tissue associated with normal organs or peripheral tissues, including tonsil, colon, spleen and thymus, or in association with T cells in inflamed peripheral tissue. Inflamed sites included skin delayed-type hypersensitivity reaction, and a wide range of tissues affected by autoimmune disease. Nuclear RelB+-HLA-DR- follicular DC were located in B-cell follicles in lymphoid organs and in lymphoid-like follicles of some tissues affected by autoimmune disease. Lymphoid tissue T-cell areas also contained nRelB(-)-HLA-DR+ cells,some of which expressed CD123 and/or CD68. Nuclear RelB+ cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions.     

The enhancer domain of the human cytomegalovirus major immediate-early promoter determines cell type-specific expression in transgenic mice.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides -670 to +54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.     

A monoclonal antibody that defines basal cells of stratified epithelia in various human and rabbit tissues

Monoclonal antibodies were produced against monkey lung lavage fluid by using a mouse hybridoma technique. One monoclonal antibody, KP8D4, specifically reacted with basal cells in human bronchial epithelia by immunohistological staining of acetone-fixed, frozen sections and it recognized a protein with an apparent molecular weight of 84000, as determined by gel immunoblotting. The distribution of this protein was immunohistochemically examined in various human tissues (lung, tongue, esophagus, stomach, intestine, liver, pancreas, salivary gland, spleen, thymus, heart, aorta, vena cava, prostate, breast, kidney, urinary bladder, thyroid, brain, skin, striated muscle) and various tissues of rats, rabbits and pigs. The results showed a specific affinity of KP8D4 to basal cells of stratified epithelia in the various human and rabbit tissues. This antibody may be a useful tool for studies of normal development and diverse pathological disorders.     

Role of Shiga toxin versus H7 flagellin in enterohaemorrhagic Escherichia coli signalling of human colon epithelium in vivo

Enterohaemorrhagic Escherichia coli O157:H7 (EHEC) is a clinically important foodborne pathogen that colonizes human colon epithelium and induces acute colonic inflammation, but does not invade the epithelial cells. Whereas Shiga toxin (Stx) and bacterial flagellin have been studied for their ability to upregulate the production of proinflammatory chemokines by cultured human colon cancer cell lines, the relevance of studies in colon cancer cell lines to the production of proinflammatory signals by normal epithelial cells in EHEC-infected human colon is not known. We show herein that Stx does not bind to human colon epithelium in vivo. Moreover, globotriaosylceramide (Gb3/CD77) synthase, the enzyme required for synthesis of the Gb3/CD77 receptor for Stx, was not expressed by normal or inflamed human colon epithelium in vivo. In contrast, Toll-like receptor (TLR) 5, the receptor for bacterial flagellin, was expressed by normal human colon epithelium and by colon epithelium in human intestinal xenografts. EHEC H7 flagellin instilled in the lumen of human colon xenografts that contain an intact human epithelium upregulated the expression of epithelial cell proinflammatory chemokines, which was accompanied by a subepithelial influx of neutrophils. Isogenic mutants of EHEC that lacked flagellin did not significantly upregulate prototypic neutrophil and dendritic cell chemoattractants by model human colon epithelia, irrespective of Stx production. We conclude that EHEC H7 flagellin and not Stx is the major EHEC factor that directly upregulates proinflammatory chemokine production by human colon epithelium in vivo.     

Fascin expression in human embryonic, fetal, and normal adult tissue.

This study investigates the distribution of fascin in human embryonic, fetal, and normal adult tissues. Tissue microarray technology was used to perform immunohistochemical experiments on human embryos and fetuses at 4-22 weeks of gestation and adult specimens. Fascin was widely expressed in the nervous system. At 4 weeks of gestation, fascin was present in the neural tube. At 8-12 weeks of gestation, homogenous gene expression was seen in cells of the cerebellum and gastrointestinal tract. In later developmental stages and in adults, Purkinje cells of the cerebellum and glandular epithelium of the gastrointestinal tract showed no expression. Fascin was expressed in the cortex and medulla of the adrenal gland at 8-12 weeks of gestation, whereas immunoreactivity decreased from the zona glomerulosa through the zona reticularis and was essentially negative in the adrenal medulla of adults. Significant expression of fascin was seen throughout development in neurons, follicular dendritic cells of lymphoid tissue, basal layer cells of stratified squamous epithelia, mesenchyme, and vascular endothelial cells. Simple columnar epithelia of the biliary duct, colon, ovary, pancreas, and stomach were all negative for fascin expression. These results show that expression of fascin is time specific and highly tissue specific. Parallels between fascin expression in embryogenesis and carcinogenesis are discussed.     

Expression of the lymphotoxin beta receptor on follicular stromal cells in human lymphoid tissues

The lymphotoxin beta receptor (LTbetaR), and its ligand, LTalpha1beta2, have been proposed to play a key role in the development and organization of lymphoid tissues. The LTbetaR is expressed on a variety of human primary and transformed cells, but strikingly absent on T or B lymphocytes and primary monocytes or peripheral dendritic cells, although LTbetaR is detected on some myeloid leukemic lines. In the developing thymus LTbetaR is prominent along the trabeculae and into the medulla upto corticomedullary junction. In the spleen, LTbetaR is prominently expressed by cells in the red pulp and along the borders of red and white pulp which colocalizes with reticular stromal cells. The LTbetaR is expressed on a human follicular dendritic cell line, FDC-1, and signals expression of CD54 when ligated with the LTalpha1beta2 complex. These results support the concept that directional interactions between LTalpha1beta2 bearing lymphocytes and LTbetaR bearing stromal cells are involved in the organization of lymphoid tissue.     

TACC3 expression is tightly regulated during early differentiation

Transforming acidic coiled-coil (TACC) proteins are hypothesized to play a role in normal cellular growth and differentiation and to be involved in centrosomal microtubule stabilization. Our current studies aim to delineate the expression pattern of TACC3 protein during cellular differentiation and in a variety of normal human tissues. TACC3 is known to be upregulated in differentiating erythroid progenitor cells following treatment with erythropoietin and is required for replication of hematopoietic stem cells. However, we demonstrate that a dramatic upregulation of TACC3 also occurs during the early differentiation of NIH 3T3-L1 cells into adipocytes and PC12 cells into neurons, indicating that TACC3 mediates cellular differentiation in several cell types. Using real-time PCR, we quantitated the mRNA levels of TACC3 compared to TACC1 and TACC2 in various human adult tissues. We observed the highest expression of TACC3 mRNA in testis, spleen, thymus and peripheral blood leukocytes, all tissues undergoing high rates of differentiation, and a lower level of expression in ovary, prostate, pancreas, colon, small intestine, liver and kidney. In contrast, TACC1 and TACC2 mRNA levels are more widespread. By immunohistochemistry, we confirm that the TACC3 protein localizes to differentiating cell types, including spermatocytes, oocytes, epithelial cells, bone marrow cells and lymphocytes. Thus, these observations are concordant with a basic role for TACC3 during early stages of differentiation in normal tissues.     

Differential expression of HSPA1 and HSPA2 proteins in human tissues; tissue microarray-based immunohistochemical study

In the present study we determined the expression pattern of HSPA1 and HSPA2 proteins in various normal human tissues by tissue-microarray based immunohistochemical analysis. Both proteins belong to the HSPA (HSP70) family of heat shock proteins. The HSPA2 is encoded by the gene originally defined as testis-specific, while HSPA1 is encoded by the stress-inducible genes (HSPA1A and HSPA1B). Our study revealed that both proteins are expressed only in some tissues from the 24 ones examined. HSPA2 was detected in adrenal gland, bronchus, cerebellum, cerebrum, colon, esophagus, kidney, skin, small intestine, stomach and testis, but not in adipose tissue, bladder, breast, cardiac muscle, diaphragm, liver, lung, lymph node, pancreas, prostate, skeletal muscle, spleen, thyroid. Expression of HSPA1 was detected in adrenal gland, bladder, breast, bronchus, cardiac muscle, esophagus, kidney, prostate, skin, but not in other tissues examined. Moreover, HSPA2 and HSPA1 proteins were found to be expressed in a cell-type-specific manner. The most pronounced cell-type expression pattern was found for HSPA2 protein. In the case of stratified squamous epithelia of the skin and esophagus, as well as in ciliated pseudostratified columnar epithelium lining respiratory tract, the HSPA2 positive cells were located in the basal layer. In the colon, small intestine and bronchus epithelia HSPA2 was detected in goblet cells. In adrenal gland cortex HSPA2 expression was limited to cells of zona reticularis. The presented results clearly show that certain human tissues constitutively express varying levels of HSPA1 and HSPA2 proteins in a highly differentiated way. Thus, our study can help designing experimental models suitable for cell- and tissue-type-specific functional differences between HSPA2 and HSPA1 proteins in human tissues.     

9 alpha,11 beta-prostaglandin F2 formation in various bovine tissues. Different isozymes of prostaglandin D2 11-ketoreductase, contribution of prostaglandin F synthetase and its cellular localization

9 alpha,11 beta-prostaglandin F2 was formed from prostaglandin D2 by its 11-ketoreductases in 100,000 x g supernatants of various bovine tissues in the presence of an NADPH-generating system. The reductase activities were high in liver (51.09 nmol/h/mg of protein), lung (24.99), and spleen (14.20); moderate in heart and pancreas (3.09-3.61); weak in stomach, intestine, colon, kidney, uterus, adrenal gland, and thymus (0.11-2.63); and undetectable in brain, retina, carotid artery, and blood (less than 0.10). No formation of prostaglandin F2 alpha from prostaglandin D2 was detected in all tissues. In immunotitration analyses with a polyclonal antibody specific for prostaglandin F synthetase, the reductase activities in lung and spleen showed identical titration curves to that of the purified synthetase and decreased to less than 15% of the initial activity under the condition of antibody excess. Prostaglandin F synthetase-immunoreactive protein in these two tissues showed peptide fingerprints identical to that of the purified enzyme after partial digestion with Staphylococcus aureus V8 protease. The antibody was partially cross-reactive to the reductase in liver (about 20% of that to the synthetase) but not to the reductase(s) in other tissues. The Km value for prostaglandin D2 of the reductase activity was the same in lung and spleen as that of the purified prostaglandin F synthetase (120 microM) but differed in liver (6 microM), heart, and pancreas (15 microM). The predominant distribution of prostaglandin F synthetase in lung and spleen was confirmed by radioimmunoassay (2.8 and 1.0 micrograms/mg protein, respectively) and Northern blot analyses. In immunoperoxidase staining, this enzyme was localized in alveolar interstitial cells and nonciliated epithelial cells in lung, histiocytes and/or dendritic cells in spleen, and a few interstitial cells in kidney and adrenal cortex.     

A human homologue of the rat metastasis-associated variant of CD44 is expressed in colorectal carcinomas and adenomatous polyps

A recently described splice variant of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to a non-metastasizing rat pancreatic carcinoma cell line and to non- metastasizing sarcoma cells. Homologues of this variant as well as several other CD44 splice variants are also expressed at the RNA level in human carcinoma cell lines from lung, breast, and colon, and in immortalized keratinocytes. Using antibodies raised against a bacterial fusion protein encoded by variant CD44 sequences, we studied the expression of variant CD44 glycoproteins in normal human tissues and in colorectal neoplasia. Expression of CD44 variant proteins in normal human tissues was readily found on several epithelial tissues including the squamous epithelia of the epidermis, tonsils, and pharynx, and the glandular epithelium of the pancreatic ducts, but was largely absent from other epithelia and from most non-epithelial cells and tissues. In human colorectal neoplasia CD44 variant proteins, including homologues of those which confer metastatic ability to rat tumors, were found on all invasive carcinomas and carcinoma metastases. Interestingly, focal expression was also observed in adenomatous polyps, expression being related to areas of dysplasia. The distribution of the CD44 variants in human tissues suggests that they play a role in a few restricted differentiation pathways and that in colorectal tumors one of these pathways has been reactivated. The finding that metastasis-related variants are already expressed at a relatively early stage in colorectal carcinogenesis and tumor progression, i.e., in adenomatous polyps, suggests the existence of a yet unknown selective advantage linked to CD44 variant expression. The continued expression in metastases would be compatible with a role in the metastatic process.     

Long bone osteoclasts display an augmented osteoclast phenotype compared to calvarial osteoclasts

Osteoclasts are multinucleated cells specialized in degrading bone and characterized by high expression of the enzymes tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CtsK). Recent studies show that osteoclasts exhibit phenotypic differences depending on their anatomical site of action.Using immunohistochemistry, RT-qPCR, FPLC chromatography and immunoblotting, we compared TRAP expression in calvaria and long bone. TRAP protein and enzyme activity levels were higher in long bones compared to calvaria. In addition, proteolytic processing of TRAP was more extensive in long bones than calvaria which correlated with higher cysteine proteinase activity and protein expression of CtsK. These two types of bones also exhibited a differential expression of monomeric TRAP and CtsK isoforms. Analysis of CtsK^−/− mice revealed that CtsK is involved in proteolytic processing of TRAP in calvaria. Moreover, long bone osteoclasts exhibited higher expression of not only TRAP and CtsK but also of the membrane markers CD68 and CD163.The results suggest that long bone osteoclasts display an augmented osteoclastic phenotype with stronger expression of both membranous and secreted osteoclast proteins.     

Quantitation and immunohistochemical localization of cathepsins E and D in rat tissues and blood cells

The distribution of cathepsins E and D in various rat tissues and blood cells was determined by immunoprecipitation and by immunohistochemistry with discriminative antibodies specific for each enzyme. While cathepsin D was detected in all of the tissues and blood cells tested (except for erythrocytes), cathepsin E had a relatively limited distribution. The cathepsin E content was highest in the stomach and was succeeded in the following order by the urinary bladder, thymus, spleen, cervical lymph node and bone marrow. Significant amounts of cathepsin E were also found in the colon, rectum, jejunum, skin, lung, kidney and submandibular gland. The other tissues tested had little or no detectable cathepsin E content. Of the blood cells tested, lymphocytes and peritoneal neutrophils contained high levels of cathepsin E. Erythrocytes had cathepsin E only as aspartic proteinases. When the subcellular localization of cathepsin E in the neutrophils was investigated by fractionation of the postnuclear supernatants, the enzyme behaved as a soluble cytosolic enzyme. In contrast, cathepsin D was mainly associated with the granular fraction. The immunohistochemical localization of cathepsins E and D was clearly different in the stomach, large intestines, kidney and urinary bladder, but was similar in the lymph node and spleen. The tissue-fixed macrophages, which were notable in the skeletal and cardiac muscle tissues, submucosal layers of the gastrointestinal tracts, salivary gland, lung and trachea, also exhibited similar intense immunoreactivities demonstrative of both cathepsins E and D.     

A tumor-associated glycoprotein that blocks MHC class II-dependent antigen presentation by dendritic cells

Tumors evade immune surveillance despite the frequent expression of tumor-associated Ags (TAA). Tumor cells escape recognition by CD8(+) T cells through several mechanisms, including down-regulation of MHC class I molecules and associated Ag-processing machinery. However, although it is well accepted that optimal anti-tumor immune responses require tumor-reactive CD4(+) T cells, few studies have addressed how tumor cells evade CD4(+) T cell recognition. In this study, we show that a common TAA, GA733-2, and its murine orthologue, mouse epithelial glycoprotein (mEGP), function in blocking MHC class II-restricted Ag presentation by dendritic cells. GA733-2 is a common TAA that is expressed normally at low levels by some epithelial tissues and a subset of dendritic cells, but at high levels on colon, breast, lung, and some nonepithelial tumors. We show that ectopic expression of mEGP or GA733-2, respectively, in dendritic cells derived from murine bone marrow or human monocytes results in a dose-dependent inability to stimulate proliferation of Ag-specific or alloreactive CD4(+) T cells. Dendritic cells exposed to cell debris from tumors expressing mEGP are similarly compromised. Furthermore, mice immunized with dendritic cells expressing mEGP from a recombinant adenovirus vector exhibited a muted anti-adenovirus immune response. The inhibitory effect of mEGP was not due to down-regulation of functional MHC class II molecules or active suppression of T cells, and did not extend to T cell responses to superantigen. These results demonstrate a novel mechanism by which tumors may evade CD4(+) T cell-dependent immune responses through expression of a TAA.