Immunoreactivity For Cathepsin Research Articles

Cathepsin K (Clone EPR19992) Demonstrates Uniformly Positive Immunoreactivity in Renal Oncocytoma, Chromophobe Renal Cell Carcinoma, and Distal Tubules.

Introduction. Cathepsin K is overexpressed in several tumors associated with microphthalmia transcription factor (MiTF) family or mechanistic target of rapamycin (mTOR) upregulation. Among renal neoplasms, MiTF translocation renal cell carcinoma (RCC), perivascular epithelioid cell neoplasms (PEComa), and eosinophilic solid and cystic RCC have demonstrated Cathepsin K immunoreactivity. In this study, we demonstrate a uniform Cathepsin K expression in oncocytoma, chromophobe RCC (CHRCC), and distal tubules. Design. We stained 13 oncocytomas, 13 CHRCC, 14 clear cell RCC (CCRCC), 9 papillary RCC (PRCC), 9 PEComas, and 5 MiTF RCC. Additionally, we assessed immunoreactivity for Cathepsin K in non-neoplastic renal parenchyma. Immunolabeling was performed on regularly charged slides from formalin-fixed paraffin-embedded tissue with monoclonal anti-rabbit antibodies to human Cathepsin K (clone EPR19992, Abcam). Results. All oncocytomas demonstrated diffuse strong cytoplasmic immunolabeling. CHRCC demonstrated uniform less intense immunolabeling in all cases with membranous accentuation. The assessment of the non-neoplastic renal parenchyma in all cases showed strong cytoplasmic immunoreaction in distal tubules and proximal tubules stained faintly. Mesangial cells were not immunoreactive. All MiTF RCC and PEComas were immunoreactive for Cathepsin K, whereas CCRCC and PRCC were negative in all cases. Conclusions. In this study, we expand the spectrum of renal neoplasms reactive with a particular clone of Cathepsin K (EPR19992). Distal tubules are strongly immunoreactive for Cathepsin K. Our conclusions need to be taken into consideration when differential diagnosis includes MiTF RCC or PEComa and this Cathepsin K clone is included in the immunohistochemical panel. This newer antibody clone was not tested in prior publications, potentially explaining the difference in conclusions.

Immunoreactivity of cytotoxic T-lymphocyte antigen 2 alpha in mouse pancreatic islet cells.

Cells of the pancreatic islets produce several molecules including insulin (beta cells), glucagon (alpha cells), somatostatin (delta cells), pancreatic polypeptide (PP cells), ghrelin (epsilon cells), serotonin (enterochromaffin cells), gastrin (G cells) and small granules of unknown content secreted by the P/D1 cells. Secretion mechanism of some of these molecules is still poorly understood. However, Cathepsin L is shown to regulate insulin exocytosis in beta cells and activate the trypsinogen produced by the pancreatic serous acini cells into trypsin. The structure of the propeptide region of Cathepsin L is homologous to Cytotoxic T-lymphocyte antigen-2 alpha (CTLA-2 alpha) which is also shown to exhibit selective inhibitory activities against Cathepsin L. It was thought that if CTLA-2 alpha was expressed in the pancreas; then, it would be an important regulator of protease activation and insulin secretion. The purpose of this study was, therefore, to examine by immunohistochemistry the cellular localization and distribution pattern of CTLA-2 alpha in the pancreas. Results showed that strong immunoreactivity was specifically detected in the pancreatic islets (endocrine pancreas) but not in the exocrine pancreas and pancreatic stroma. Immunostaining was further performed to investigate more on localization of Cathepsin L in the pancreas. Strong immunoreactivity for Cathepsin L was detected in the pancreatic islets, serous cells and the pancreas duct system. These findings suggest that CTLA-2 alpha may be involved in the proteolytic processing and secretion of insulin through regulation of Cathepsin L and that the regulated inhibition of Cathepsin L may have therapeutic potential for type 1 diabetes.

Cathepsin K expression in clear cell "sugar" tumor (PEComa) of the lung.

Clear cell "sugar" tumor is a rare benign neoplasm arising in the lung, considered as a part of the PEComa family. As PEComas of other sites, this tumor expresses melanocytic markers such as HMB45 and Melan-A. Despite cathepsin K, MITF and CD68 staining are known to be positive in a large number of PEComas and TFE3 rearrangement has been reported in a subset of PEComas, no data is available regarding the expression of these markers and the occurrence of TFE3 and TFEB rearrangement in clear cell "sugar" tumor of the lung. We have investigated the immunolabeling of cathepsin K, MITF, and CD68 in five cases of clear cell "sugar" tumor. Moreover, we have also sought the presence of TFE3 and TFEB rearrangement by fluorescence in situ hybridization (FISH) assay. In all tumors, strong immunoreactivity of cathepsin K and CD68 (PG-M1 and KP1 clone) was demonstrated, whereas none of them labeled for MITF staining and showed TFE3 or TFEB rearrangement. These findings widen the immunohistochemical profile of clear cell "sugar" tumor providing useful new markers for challenging cases. The expression of lysosomal markers, such as cathepsin K and CD68, strengthens the hypothesis that this tumor is part of the PEComa family.

Participation of osteoclastogenic factors in immunopathogenesis of human chronic periapical lesions.

Chronic periapical lesions (CPLs) are common lesions of the oral cavity and are the result of caries, tooth fracture, iatrogenic causes, or factors causing contamination and pulp necrosis. Inflammatory cells participate in the expansion of CPLs by releasing factors that stimulate or inhibit osteolytic activity. The objective of this study was to investigate the participation of RANKL, TNF-α, cathepsin K, IL-33, and OPG in the development of radicular cysts (RCs) and periapical granulomas (PGs). Paraffin-embedded sections of 30 RCs and 22 PGs were submitted to immunohistochemistry. Immunoexpression of the proteins studied was observed in the epithelium and capsule of RCs, as well as in connective tissue of PGs. The expression of the osteoclastogenic factors studied differed significantly in RCs and PGs (P<.001), with lower expression of OPG in RCs. In PGs, the lowest expression was observed for cathepsin K. Comparison of the 2 lesions showed a similar participation of RANKL and IL33, while a significant difference was observed for OPG (P<.001), TNF-α (P=.002), and cathepsin K (P=.016). No association of the expression of the proteins with lesions size was observed. This study demonstrated the participation of RANKL, TNF-α, IL-33, cathepsin K, and OPG in the development of RCs and PGs, with emphasis on the highest immunoreactivity of cathepsin in RCs and TNF-α and OPG in PGs. OPG possibly determines the slower growth of PGs compared to RCs.

Increased immunoreactivity of cathepsins in the rat esophagus under chronic acid reflux esophagitis.

We have designed a stable rat chronic acid reflux esophagitis (RE) model. In gastrointestinal lesions, several lysosomal cathepsins are known to participate in epithelial permeability in cell-cell connections, such as tight junctions in ulcerative colitis. However, very few studies have focused on the distribution of cathepsins in the esophageal multilayer squamous epithelium. Therefore to clarify the role of cathepsins in RE, we investigated their immunohistological localization in the esophageal epithelium under normal conditions and after RE. Of the cathepsins examined (cathepsins B, C, D, F, H, L, S, and X), granular immunoreactivity for cathepsins B, C, D and L was observed in the control esophageal epithelia; although, their distribution differed depending on the enzyme examined. In the RE model, immunoreactivity of these cathepsins was increased in esophageal epithelial cells and activated macrophages. The immunoreactivity for cathepsins F, H, S and X was barely detectable in the control esophageal epithelium. However, in the RE model, we noticed a slight increase in the expression of cathepsins H and X in the epithelial cells. Furthermore, activated macrophages of the RE model possessed intense immunoreactivity for these cathepsins, which may have been related to esophageal inflammatory mechanisms.

Expression patterns of cathepsins B, H, K, L and S in the human endometrium

Cathepsins B, H, K, L and S belong to the family of lysosomal cysteine proteinases and participate in a variety of proteolytic processes, including degradation of the extracellular matrix (ECM). In the present study, we used Northern hybridization to demonstrate the presence of mRNAs for cathepsins B, H, K, L and S in human endometrium during both the proliferative and secretory phases of menstrual cycle. The mRNA levels for cathepsins H and K were significantly lower in secretory phase endometrium in comparison with proliferative phase endometrium. Immunohistochemical localization of the different cathepsins revealed widespread distribution of all cathepsins in both stroma and epithelial cells. The immunoreactivity for cathepsins B, H and K exhibited changes related to endometrial location and/or to the phase of the cycle. The strongest immunoreactivity for cathepsins B, H, L and S was observed in the surface epithelium of the endometrium. The staining for cathepsins was predominantly intracellular, but immunoreactivity was also detected on the surface of small lymphoid cells in the stroma. The findings of the present study suggest that cysteine cathepsins are needed for normal development and function of human endometrium during both the proliferative and secretory phases.

Specific localization of lysosomal aminopeptidases in type II alveolar epithelial cells of the rat lung.

We previously demonstrated that lysosomal cysteine proteinases, cathepsins B, H, and L were localized in lysosomes of alveolar macrophages and bronchial epithelial cells in the rat lung, while cathepsin H, a typical aminopeptidase, was additionally distributed in lamellar bodies containing surfactant in type II alveolar epithelial cells (ISHII et al., 1991). The present immunohistochemical study further examined the localization of lysosomal aminopeptidases, cathepsin C, and tripeptidyl peptidase I (TPP-I) in the rat lung. Western blotting confirmed the presence of cathepsin C and TPP-I as active forms in the pulmonary tissue, showing 25 kD and 47 kD, respectively. Immunohisto/cytochemical observations demonstrated that positive staining for cathepsin C and TPP-I was more intensely localized in alveolar epithelial regions than in bronchial or bronchiolar epithelial cells. By double immunostaining using confocal laser microscopy, immunoreactivity for cathepsin H was found to be co-localized with that for cathepsin C or TPP-I in both type II cells and macrophages. Moreover, when doubly stained with anti-cathepsin C and ED2, single-positive type II cells could be clearly distinguished from double-positive macrophages in the alveolar region. Immunoelectron microscopy revealed the gold labeling of cathepsin C or TPP-I in multivesicular and composite bodies, and lamellar bodies of Type II cells. These results showing that lysosomal aminopeptidases such as cathepsin H, cathepsin C and TPP-I are localized in lamellar bodies of type II alveolar epithelial cells strongly argue for the participation of lysosomal aminopeptidases in the formation process of surfactant containing specific proteins.

Cytochemical and ultrastructural examination of apoptotic odontoclasts induced by bisphosphonate administration.

Since odontoclasts share similar characteristics with osteoclasts, this study has examined whether odontoclasts exhibit cytological alteration after treatment with bisphosphonate, which induces apoptosis of osteoclasts. After the administration of bisphosphonate to 6-day-old rabbits, many odontoclasts detached from the dentine surface of the deciduous teeth, resulting in the reduction of tartrate-resistant acid phosphatase (TRAP-ase) and immunoreactivity for cathepsin K. Transmission electron microscopy revealed a number of odontoclasts showing poorly developed or a lack of ruffled borders, a Golgi apparatus markedly reduced in size, and numerous cytoplasmic vesicles. The bisphosphonate-treated odontoclasts displayed fragmented DNA in the pyknotic nuclei evidenced by terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick-end labeling, indicating that bisphosphonate can induce apoptosis of the odontoclasts. Ultrastructural observations of the apoptotic odontoclasts revealed condensed heterochromatin at the margin of the nuclear envelope, assembled arrays of rough endoplasmic reticulum, and many vacuoles and vesicles. Some apoptotic odontoclasts showed ladder-like structures between the adjacent nuclear envelopes, enlargement of the nuclear envelopes, and the formation of a ribosome-like granular structure in the nuclei. Thus, odontoclasts are able to undergo apoptosis after bisphosphonate treatment; this results in cytological alterations, including reduced resorption activity and the inhibition of protein synthesis/transport as indicated by the diminished TRAPase and cathepsin K and the poorly developed Golgi apparatus, respectively. Nuclear alteration as evidenced by the appearance of ladder-like and ribosome-like structures was characteristic of apoptotic odontoclasts.

Distribution of Cathepsin D in Human Eyes with or without Age-related Maculopathy

Cathepsin D is a ubiquitous enzyme which plays an important role in the catabolism of proteins. Enzymatic studies showed that cathepsin D is the most important lysosomal enzyme in the proteolysis of opsin. The importance of cathepsin D in the lysosomal digestion of phagocytosed photoreceptor outer segments by the retinal pigment epithelium suggests that a decrease in cathepsin D activity might contribute to the development of hyalinized drusen and to the development of age-related maculopathy. The aim of this project was to study the immunohistochemical localization of cathepsin D in human eyes and particularly to compare the immunoreactivity of cathepsin D normal retinal pigment epithelial cells and in cells surrounding hyalinized drusen or lesions of age-related maculopathy. Following clinicopathological examinations the eyes were fixed, paraffin embedded and individual sections were subjected to Picro-Mallory staining for histopathological examination. Bleaching was performed then immunohistochemistry was carried out using a monoclonal mouse anti-human cathepsin D antibody. On the basis of the appearance of basal laminar deposit the eyes were divided into five groups corresponding to levels of progression in age-related maculopathy development. Following optimization of bleaching cathepsin D immunostaining was clearly visible in the iris epithelium, ciliary body and the retinal pigment epithelial layer of all eyes with the highest immunoreactivity present in the RPE cells. Within the neural retina the ganglion cells demonstrated a weak signal. Retinal pigment epithelial cathepsin D immunoreactivity was not impaired by age, geographical location or by age-related maculopathy status. There was a small increase in cathepsin D immunoreactivity around hyalinized drusen. The maintenance of cathepsin D immunoreactivity in eyes with hyalinized drusen or in samples with age-related maculopathy suggest that down-regulation of cathepsin D expression in the affected locations does not precede the development of these conditions. However, further studies are required to establish if the immunoreactive cathepsin D represents the fully processed biologically active enzyme.

Prognostic significance of angiogenesis and immunoreactivity of cathepsin d and type IV collagen in high-grade stage T1 primary bladder cancer

Objectives. To assess the prognostic significance of biologic parameters such as angiogenesis, expression of cathepsin D (a lysosomal protease), and degradation of type IV collagen (a basement membrane protein), we studied 20 patients with primary grade III Stage T1 transitional cell carcinoma of the bladder. Methods. Endothelial cells were labeled immunohistochemically using factor VIII–related antigen. The vascular surface density (VSD) and the microvessel number (NVES) were assessed by means of stereology. The tumor tissues were also analyzed by immunohistochemical methods for the expression of cathepsin D and the staining pattern of type IV collagen. Results. Eight patients (40%) having either recurrence or progressive disease showed greater NVES and VSD values ( P = 0.002 and P = 0.01, respectively) than patients without. The significance of vascular parameters was found to be statistically independent from coexisting carcinoma in situ, bacille Calmette-Guérin (BCG) treatment, tumor size, and number. Additionally, these parameters did not show statistical significance between progressive and recurrent tumors. However, tumors with solid morphologic features had higher VSD values and a significantly greater rate of recurrence or progression ( P = 0.01 and P = 0.07, respectively). Tissue from 17 (85%) of 20 tumors showed absent or patchy basement membrane staining for type IV collagen, and 12 (60%) showed strong immunoreactivity for cathepsin D antibody. There were no differences for either molecule with either BCG treatment or other parameters related to prognosis. Conclusions. Angiogenesis may have an independent role in predicting prognosis in grade III Stage T1 bladder carcinoma. Grade III Stage pT1 tumors with solid morphologic features have higher angiogenetic activity and a worse prognosis. Cathepsin D and type IV collagen do not seem to play a role in predicting biologic behavior.

Postictal Blockade of Ischemic Hippocampal Neuronal Death in Primates Using Selective Cathepsin Inhibitors

This paper is to study the participation of cathepsin in ischemic neuronal death of the monkey hippocampal cornu ammonis (CA) 1 sector and also to clarify whether its selective inhibitor epoxysuccinyl peptides such as CA-074 and E-64c can inhibit the neuronal death or not. In the preceding reports, we demonstrated μ-calpain activation and subsequent rupturing of the lysosomal membrane of postischemic CA1 neurons and also increase of enzyme activity of cathepsins B and L in monkeys undergoing a complete 20-min whole brain ischemia. Here, morphological, immunohistochemical and enzymatical analyses were performed to examine the efficacy of two selective cathepsin inhibitors in the postictal blockade of delayed neuronal death in the monkey hippocampus. Both inhibitors could significantly decrease enzyme activities of cathepsins B and L in all hippocampal sectors. When CA-074 was intravenously administered immediately after the ischemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 after ischemia. In contrast, when E-64c was similarly administered, approximately 84% of CA1 neurons were saved from delayed neuronal death on day 5. The surviving neurons showed mild central chromatolysis and negligible immunoreactivity for cathepsins B and L. These observations indicate that the use of cathepsin inhibitors may become novel strategy for prevention of ischemic delayed neuronal death in the primate hippocampus.

Overexpression of Cation-Dependent Mannose 6-Phosphate Receptor Prevents Cell Death Induced by Serum Deprivation in PC12 Cells

PC12 cells express well cation-independent mannose 6-phosphate receptors (CI-MPR), but not cation-dependent (CD)-MPR as much. To examine CD-MPR dependency of transport of cathepsins B and D to lysosomes in PC12 cells, we prepared the cells overexpressing CD-MPR. Immunoreactivity for cathepsin B became more distinct and larger in size in the transfected cells than in wild-type cells. No difference in the distribution of cathepsin D was seen between these two cells. The viability of the cells following serum deprivation was significantly higher in the transfected cells than in wild-type cells. This increased viability of the transfected cells was blocked by CA074, a specific inhibitor of cathepsin B, while pepstatin A suppressed the action of CA074. The results suggest that CD-MPR preferentially transport cathepsin B in PC12 cells, and cathepsins B and D participate in the regulation of PC12 cell apoptosis.

Inhibition of ischaemic hippocampal neuronal death in primates with cathepsin B inhibitor CA-074: a novel strategy for neuroprotection based on 'calpain-cathepsin hypothesis'.

Although Cornu Ammonis (CA) 1 neurons of the hippocampus are known to be vulnerable to transient ischaemia, the mechanism of ischaemic neuronal death is still unknown, and there are very few strategies to prevent neuronal death at present. In a previous report we demonstrated micro-calpain activation at the disrupted lysosomal membrane of postischaemic CA1 neurons in the monkey undergoing a complete 20 min whole brain ischaemia. Using the same experimental paradigm, we observed that the enzyme activity of the lysosomal protease cathepsin B increased throughout the hippocampus on days 3-5 after the transient ischaemia. Furthermore, by immunocytochemistry cathepsin B showed presence of extralysosomal immunoreactivity with specific localization to the cytoplasm of CA1 neurons and the neuropil of the vulnerable CA1 sector. When a specific inhibitor of cathepsin B, the epoxysuccinyl peptide CA-074 (C18H29N3O6) was intravenously administered immediately after the ischaemic insult, approximately 67% of CA1 neurons were saved from delayed neuronal death on day 5 in eight monkeys undergoing 20 min brain ischaemia: the extent of inhibition was excellent in three of eight and good in five of eight monkeys. The surviving neurons rescued by blockade of lysosomal activity, showed mild central chromatolysis and were associated with the decreased immunoreactivity for cathepsin B. These observations indicate that calpain-induced cathepsin B release is crucial for the development of the ischaemic neuronal death, and that a specific inhibitor of cathepsin B is of potential therapeutic utility in ischaemic injuries to the human CNS.

An ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special reference to autophagy and lysosomal cathepsins.

In addition to the caspase family of proteinases, cathepsin D, a lysosomal aspartic proteinase, has been suggested to act as a proapoptotic mediator in mammalian cells. To further understand the roles of cathepsins B and D in apoptosis of the cells, we examined the precise alteration processes of ultrastructures and immunoreactivity for these enzymes in PC12 cells cultured under serum deprivation. Laser scanning microscopy showed immunoreactivity for cathepsins B and D to be finely distributed in the cytoplasm of PC12 cells at the onset of culture under serum deprivation. At 3 h after the onset of culture, the immunoreactivity for cathepsin B slightly decreased in the cells, while immunodeposits for cathepsin D in the cells became more intense and larger in size than those at 0 h. Positive staining for TUNEL in nuclei of the cells appeared at 6 h, though fewer in number. Corresponding to the increase in the number of TUNEL-positive cells at 12 h and 24 h, the immunoreactivity for cathepsin B was drastically diminished in the cells, whereas that for cathepsin D was significantly augmented, especially in TUNEL-positive cells. Electron microscopically, autophagic vacuoles/autolysosomes appeared in the cytoplasm of the cells 3 h after the onset of culture. A distinct nuclear change showing relatively condensed chromatin first appeared in the peripheral part of the nuclei at 6 h. The number of PC12 cells having nuclei with chromatin condensation increased especially at 24 h, while these cells showed shrinkage of both their cytoplasm and nuclei. Dense bodies and autophagic vacuoles with limiting membranes were seen in these cells. These results showing the occurrence of autophagy and imbalance of protein amounts between cathepsins B and D during apoptosis may argue for our hypothesis that these enzymes are, in part, involved in the cell death cascade for PC12 cells following serum deprivation.

Phospholipase C-? Immunostaining in Human Breast Carcinoma: Clinical Significance and Correlations with Protease and Growth-Factor Receptor Species

: Cryostat sections of 73 invasive breast carcinomas were immunostained with a rabbit polyclonal antibody to phosphoinositide-specific phospholipase C-γ (PLC-γ), an enzyme that mediates signal transduction in tyrosine kinase growth-factor pathways. Degree of immunoreactivity was then correlated with clinicopathologic data (stage, ER status, recurrence) and immunostaining for tyrosine kinase growth-factor receptors (EGFR, ERBB-2) as well as selected “invasion-associated” proteases (cathepsin D, urokinase plasminogen activator, matrix metalloproteinases 2 and 9 [MMP-2, MMP-9]). Neoplastic epithelial populations were PLC-γ immunoreactive in 95% of tumors although staining was focally distributed (in <50% of cells) in 51% of positive cases. Forty-four percent also exhibited immunostaining of peritumoral, spindle-shaped cells (i.e., fibroblasts, endothelium, inflammatory cells). The degree of PLC-γ immunoreactivity in neoplastic epithelium was not significantly correlated with clinicopathologic features, growth factor receptor overexpression, or protease immunostaining. Stromal cell PLC-γ staining, however, was significantly associated with stromal cell immunoreactivity for cathepsin D (p = 0.03), urokinase plasminogen activator (p = 0.01), and MMP-2 (p = 0.04). Disease recurrences were also more frequent in tumors with stroma/spindle cell PLC-γ immunoreactivity (66% vs. 41%, p = 0.04). We conclude that PLC-γ immunostaining, compatible with increased tyrosine kinase pathway signaling activity, is observed not only in a high proportion of neoplastic breast epithelial populations but also in accompanying stromal cell populations in a significant number of cases. Concordance with protease immunoreactivity among peritumoral stromal cells suggests that tyrosine kinase signaling may participate in protease elaboration in vivo, possibly conferring aggressive clinical behavior.

Lysosomal generation of amyloid β protein species in transgenic mice

Soluble amyloid β protein (A β)1–40 and highly amyloidogenic A β1–42/43 were immunocytochemically labeled in lysosomes of acinar cells and macrophages in the pancreas of transgenic mice systemically expressing a C-terminal fragment of the A β precursor. A β1–42/43 and long A β species extending their C-termini were detected in the detergent-insoluble fraction. Immunoreactivity of cathepsin D was markedly increased in lysosomes filled with A β fibrils. These findings indicated that A β1–40, A β1–42, A β1–43 and longer A β species were generated in the lysosomes of the transgenic pancreas, and suggested that the activation of cathepsin D, a candidate γ-secretase, leads to acceleration of A β amyloid formation.

Immunocytochemical study of cathepsin L and rat salivary cystatin-3 in rat osteoclasts treated with E-64 in vivo

The localization of cathepsin L and rat salivary cystatin-3 (RSC-3) in rat osteoclasts (rat femoral and alveolar bones) treated with or without E-64 (control) was examined immunocytochemically. In osteoclasts pretreated with E-64, immunoreactivity for cathepsin L was very weak extracellularly compared to that in the control osteoclasts. However, it was strong intracellularly. The localization of RSC-3 was unclear in the control osteoclasts, while in E-64 treated osteoclasts, both the clear zone and ruffled border areas showed a very strong immunoreaction. At the electron-microscopic level, in normal osteoclasts, numerous immunoreaction products for cathepsin L were found extracellularly in the bone matrix under the ruffled border, while few intracellular products were observed. In contrast, in the 1E-64-treated osteoclasts, only a few immunoreaction products were found extracellularly, while intracellularly cathepsin L was found in numerous endosome-lysosomel vacuoles. In the immunoreaction for RSC-3, the cytoplasm of the ruffled border was positive, and the tips of the RSC3-positive ruffled border appeared to enter deeply into the bone matrix. Intracellularly, the granular reaction products of RSC-3 were found in the vacuoles (probably autophagolysosomes). Thus, in E-64-treated osteoclasts, inhibition of the extracellular release of cathepsin L was demonstrated. In addition, intralysosomal accumulation of RSC-3 and deep penetration of the RSC-3-positive ruffled border into the bone matrix were found. These findings suggest that RSC-3 is associated with the inhibition of cathepsin L in both the lysosomes (in the osteoclasts) and bone matrix.

Immunocytochemical localization of lysosomal cysteine and aspartic proteinases, and ubiquitin in rat epidermis.

To analyze the degradation system in epidermal cells during their generation, differentiation, and cell death, immunocytochemical localization of lysosomal cysteine and aspartic proteinases, an endogenous cysteine proteinase inhibitor, cystatin beta, and ubiquitin were examined using rat sole skin. By confocal laser microscopy, granular immunodeposits for lysosomal proteinases were well demonstrated in epidermal cells; immunoreactivity for cathepsins B and C was prominent in the lower spinous and basal layers, while that for cathepsins L and D was intense in the upper spinous and granular layers, although immunoreactivity for cathepsin D was also detected in the lower epidermal layers. Immunoreactivity for cathepsin H was weakly detected only in the spinous layer, where there were some intensely immunopositive cells with processes which were also immunopositive for S-100 alpha, indicating that these cells were Langerhans cells. Diffuse immunoreactivity for cystatin beta was intense in the spinous and granular layers and weak in the basal layer. In addition, we also examined the localization of ubiquitin, which is a signal peptide for cytosolic proteolysis; clear-cut granular immunodeposits for ubiquitin were detected in spinous and granular cells, and some were co-localized with cathepsin B immunoreactivity. In the basal layer, mitotic cells were strongly immunopositive for ubiquitin. These results suggest that cysteine and aspartic proteinases are involved in the lysosomal system of the epidermis, showing different distributions in the epidermal layers depending on the enzymes examined. Moreover, ubiquitin may be associated with the cell cycle-dependent degradation in basal cells while it also participates in the non-lysosomal proteolysis and probably, lysosomal proteolysis in the spinous and granular cells.

Immunoelectron Microscopical Characterization of the Epithelioid Type of Smooth Muscle Cells in Human Glomus Organs

The wall structure of arterio-venous anastomoses in human glomus organs was studied by immunohistochemistry and immunoelectron microscopy. Smooth muscle cells of the epithelioid type I and of the dense type II could be found in the media. The immunohistochemical study confirmed the immunopositivity of both smooth muscle cell types for α-smooth muscle actin, vimentin, and smooth muscle myosin. All smooth muscle cells also stained positively for caveolin, a recently described structural protein of microvesicles present in selected epithelial and nonepithelial cells. The immunoreactivity for cathepsin D, however, was much higher in the type I cells than in the type II cells. Immunoelectron microscopy revealed that type I cells contain loose arrays of alpha smooth muscle actin positive microfilaments, sometimes arranged in small bundles, whereas the dense medial smooth muscle cells of the type II have tightly packed actin filaments. Only type I cells contained cathepsin D positive lysosomes. The data suggest that two types of phenotypic variants of vascular smooth muscle cells in the human arterio-venous anastomosis exist: a more “synthetic” type I cell and a more contractile type II cell.

Lysosomal cysteine and aspartic proteinases and ubiquitin in rat and human urinary bladder epithelium.

To examine localization of cysteine and aspartic proteinases, and ubiquitin in rat and human urinary bladders, immunocytochemistry was applied to the tissues. In semi-thin sections, immunoreactivity for cathepsins B and D was densely localized throughout epithelial layers of rats and humans, while that for cathepsins H and L was mainly localized in rat superficial and human intermediate cells. Immunoreactivity for cathepsin C was relatively high in rat and human epithelia, especially in humans. Immunoreactivity for ubiquitin was detected in rat and human epithelial cells. By electron microscopy, vesicular or heterogeneously dense lysosomes labeled with immunogold particles indicating cathepsin B were seen in rat and human epithelial cells; particularly, they often appeared near fusiform vesicles in rat superficial cells and in human intermediate and superficial cells. By double immunostaining, lysosomes with or without vesicular structures were co-labeled with immunogold particles showing both cathepsin B and ubiquitin. The results suggest that cathepsins B, C, H, and L, and cathepsin D are involved in the lysosomal system of rat and human bladder epithelia. Moreover, considering that ubiquitin is a cofactor in the soluble ATP-dependent proteolysis, the results may also indicate that epithelial cells actively form autophagolysosomes.