Direct comparison of the sensitivity of enzyme histochemical and immunohistochemical methods: cathepsin B expression in human colorectal mucosa

Immunohistochemical localization of the proteinase cathepsin B has been compared directly with localization of cathepsin B activity with a catalytic (enzyme) histochemical method. The 2 approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is active or not whereas the catalytic method visualizes the functionally active enzyme only. Sensitivity of both approaches to localize low amounts of enzyme protein or activity has never been compared. In the present study, we show that cathepsin B protein has a wider distribution pattern than cathepsin B activity in human colorectal mucosa, which means that inactive cathepsin B protein is present. With respect to sensitivity of the methods, it is shown that cathepsin B protein could only be demonstrated properly when strong signal amplification was applied by using Nanogold with silver enhancement, whereas activity could be demonstrated with a simple and direct fluorogenic histochemical assay. It is concluded that catalytic histochemical methods are relatively simple methods for the localization of activity of enzymes in tissues and cells and that their sensitivity is high in comparison with immunohistochemical methods.     

Cathepsin B expression in colorectal carcinomas correlates with tumor progression and shortened patient survival.

Cathepsin B is a lysosomal cysteine proteinase that has the ability to degrade several extracellular matrix components at both neutral and acidic pH and has been implicated in the progression of several human and rodent tumors. We have studied the expression of cathepsin B in human colorectal tissues using a monospecific polyclonal rabbit antibody raised against human liver cathepsin B. In immunoblots of normal and neoplastic colorectal tissues this antibody specifically recognized only cathepsin B. We studied 101 cases of formalin-fixed, paraffin-embedded tissue (15 normal mucosa, 17 adenomas, and 69 carcinomas). Epithelial cells of normal mucosa and adenomas were either negative or showed a weak granular reactivity located in the paranuclear and apical cytoplasm of superficial cells. Small clusters of histiocytes were also positive in the region of the superficial area of the lamina propria. In carcinomas, increased expression of cathepsin B correlated with advanced stage of the disease. Increased immunoreactivity of cathepsin B in malignant cells was associated with either a diffuse cytoplasmic staining or was polarized to the basal pole of the cells. This is in contrast to the punctate paranuclear staining pattern observed in normal colonic mucosal cells. In tumor stromal cells, increased expression of the enzyme correlated with neoplastic progression. Expression of high levels of cathepsin B in the tumor epithelial cells was associated with a significantly shorter survival of the patients. In conclusion, our results indicate that cathepsin B expression is up-regulated in human colorectal carcinomas compared with normal mucosa and adenomas and correlates with tumor progression.     

Immunolocalization of cathepsin D in normal and neoplastic human tissues.

The aspartic proteinase cathepsin D was purified from human spleen and localised in various formalin fixed paraffin embedded human tissues using the peroxidase-antiperoxidase (PAP) technique. Cathepsin D was shown not only in macrophages but also in other connective tissue cells, and in epithelium. It was present in spleen (littoral cells and cells within Malpighian bodies), liver (hepatocytes and Kupffer cells), lung (alveolar macrophages and bronchial epithelium), brain (neurones), lymph nodes (histiocytes in germinal centres, sinusoid lining cells) and stomach (parietal and mucous neck cells). Cathepsin D was also found in carcinomas of bronchus, stomach, colon, kidney, breast, ovary, bladder and pancreas, both in neoplastic epithelium and in stromal cells, but was seldom present in connective tissue neoplasms. A group of malignant lymphomas also contained the enzyme within scattered cells. The distribution of cathepsin D seems to be much wider than that of the structurally related aspartic proteinases pepsin, gastricsin, and renin.     

Immunohistochemical demonstration of cathepsin B in the macrophages of benign and malignant lymphoid tissues

Cathepsin B has been demonstrated by immunohistochemical means in the macrophages of palatine tonsils, reactive lymph nodes and in specimens of Hodgkin's disease (HD) and non-Hodgkin's lymphomas (NHL). Two cases of genuine histiocytic lymphoma showed strong staining for the enzyme in most cells. In Hodgkin's disease, many Reed–Sternberg cells and Hodgkin cells were positive. Branching and ‘tingible body’ macrophages (histiocytic reticulum cells. HRCs) were strongly positive in all of the specimens. In reactive lymph nodes, the sinus-lining cells and intrasinusoidal macrophages were positive for cathepsin B. True dendritic reticulum cells (DRCs) appeared to be negative. Unlike muramidase (lysozyme), cathepsin B is not seen in neutrophil polymorph leucocytes.     

Activity and concentration of cathepsin B as prognostic criteria for the development of mouse LS lymphosarcoma and Lewis lung adenocarcinoma

We measured activity and content of cathepsin B in tumor tissues, liver, and spleen in mice with Lewis adenocarcinoma and LS-lymphosarcoma. Cathepsin B activity in Lewis adenocarcinoma cells was lower than in LS-lymphosarcoma cells, which was probably related to differences in their metastatic properties. Antitumor therapy increased activity and content of cathepsin B in tumor tissues. Changes in the content and activity of cathepsin B in tumor tissues can serve as a prognostic criterion for tumor regression during therapy. Cathepsin B is probably involved in apoptosis of tumor cells during chemotherapy of lymphosarcoma-LS with cyclophosphamide.     

Progestin increases cathepsin D synthesis in uterine luminal epithelial cells

Early in blastocyst implantation, cells of the uterine luminal epithelium deteriorate and die in response to the presence of the blastocyst. Destruction of the epithelial cells appears to depend on control of the autophagic activity and enzyme content of lysosomes in these cells. Concentrations of the lysosomal proteinase, cathepsin D, have been identified in luminal epithelial cells, and these studies examined changes in epithelial cathepsin D activity and their hormonal control during early pseudopregnancy in the rat. Cathepsin D activity in luminal epithelial cells increases during early pseudopregnancy to maximal levels at the time of sensitivity to deciduogenic stimuli. Rates of cathepsin D synthesis in luminal epithelial cells also increase during early pseudopregnancy, but neither enzyme activity nor rates of synthesis increase in stromal-myometrial tissues. In ovariectomized rats, progestins rather than estradiol increase cathepsin D activity and rates of synthesis in luminal epithelial cells. These studies suggest that cell death in the luminal epithelium during blastocyst implantation may depend in part on the accumulation of lysosomal cathepsin D in these cells in response to progesterone secretion during early pregnancy.     

Localization of cathepsin B in normal and hyperplastic human prostate by immunoperoxidase and protein A-gold techniques

Cathepsin B, a lysosomal cysteine protease, was localized in normal prostate and benign prostatic hyperplasia (BPH) using immunoperoxidase and protein A-gold techniques. Our objective was to determine whether cathepsin B was involved in the prostatic epithelium affected by nodular hyperplasia. All samples were collected immediately after prostatectomy. Immunohistochemical studies showed that the enzyme was expressed in the supranuclear cytoplasm of columnar cells and in numerous basal cells of normal and BPH acini. The strongest localization of cathepsin B occurred in acinar basal cells; hence, it is possible that cathepsin B could be useful as a marker for such cellular elements. Stromal macrophages showed reaction products, but lymphocytes and neutrophils did not. In both normal and hyperplastic glands, the enzyme was localized by gold particles in lysosomes, secretory granules, and vacuoles of columnar epithelial acinar cells. Immunoelectron microscopic study also showed the presence of cathepsin B in the heterochromatin (condensed chromatin) and nuclear membranes of columnar and basal cells, but not in euchromatin or nucleoli. At present, the function of cathepsin B in the nuclei of basal and columnar cells remains unknown. However, the cathepsin B in the cytoplasmic compartment might be associated with the lysosomal function of the cells. The role of cathepsin B as a marker for basal cell participation in the development of prostatic lesions should be studied further.     

Differential expression patterns of cathepsins B, H, K, L and S in the mouse ovary

Cathepsins B, H, K, L and S belong to a family of lysosomal cysteine proteinases which participate in a variety of proteolytic processes, including degradation of extracellular matrix. Although the presence of cathepsin mRNAs in the ovary has been reported earlier, very little information is available on their temporospatial expression. In the present study, Northern analysis revealed cyclic changes in the mRNA levels for cathepsins B, H, K, L and S during the 4-day oestrous cycle in the mouse ovary. Immunohistochemical localization revealed distinct expression patterns suggesting different functions for the cathepsins studied. Cathepsin B was predominantly seen in the germinal epithelium throughout the oestrous cycle. Upon follicular maturation, an increasing number of granulosa cells became positive for all cathepsins. Strong cathepsin H staining was sharply defined in theca externa which also stained for cathepsins K and S. Corpus luteum was the predominant location of cathepsin L. The distribution of cathepsin S resembled that of cathepsin L. The developing oocyte stained positive for all cathepsins. In-situ hybridization confirmed the differential production of cathepsin mRNAs by granulosa, thecal and luteal cells. These complex temporal and spatial expression patterns at different stages of the oestrous cycle and follicular development suggest divergent functions for specific cathepsins in follicular development, growth and rupture.     

Turnover of intestinal brush-border proteins during postnatal development in rat

Protein turnover in brush-border membranes of rats during postnatal development has been studied by the double isotope technique. Unlike adult animals where only large proteins (mol wt > 140,000) show relatively rapid turnover, most brush-border proteins in 12-day-old rats show high 3H-to-14C ratios of leucine incorporation, consistent with rapid turnover. Lysosomal proteases, including cathepsin B, are partly responsible for this rapid turnover. This conclusion is based on the following findings: 1) In vivo treatment of 12-day-old animals with leupeptin, an inhibitor of cathepsin B, alters relative turnover rates, enzyme activity, and content of many brush-border proteins. Activities of maltase and trehalase rise while lactase falls. 2) Cathepsin B activity falls rapidly in intestine after the animals are 16 days of age, at a time when luminal pancreatic proteases are rising. Moreover, cathepsin B activity shows less latency in distal intestine at 12 and 16 days than at later ages or in proximal intestine. It is suggested that during postnatal development lysosomal enzymes, e.g., cathepsin B, play an important role in the turnover of intestinal brush-border proteins.     

Cathepsin K – a marker of macrophage differentiation?

Cathepsin K is a cysteine protease with high matrix-degrading activity. Initially, cathepsin K was described as being expressed exclusively by osteoclasts. It was suggested that cathepsin K expression is a specific feature of cells involved in bone remodelling. The aim of this study was to investigate the hypothesis that cathepsin K is expressed not only in bone-resorbing macrophages, but also more generally in specifically differentiated macrophages, such as epithelioid cells and multinucleated giant cells in soft tissues. Specimens obtained from different organs and anatomical locations of patients suffering from sarcoidosis, tuberculosis, granulomas caused by foreign materials, and sarcoid-like lesions were investigated for the expression of cathepsins B, K, and L. Immunohistochemistry and in situ hybridization showed cathepsin K in epithelioid cells and multinucleated giant cells irrespective of the pathological condition and anatomical location, but not in normal resident macrophages. By immunoelectron microscopy, cathepsin K was discovered in cytoplasmic granules of multinucleated giant cells. In contrast, cathepsin B and cathepsin L were expressed ubiquitously in CD68-positive tissue macrophages, epithelioid cells, and multinucleated giant cells. The results demonstrate that cathepsin K, but not cathepsin B or cathepsin L, differentiates specific phenotypes of macrophages independently of the anatomical site. Its enzymatic characteristics, particularly its high matrix-degrading activity, suggest that cathepsin K-positive epithelioid cells and multinucleated giant cells are characterized by an enhanced specific proteolytic capability.     

Cathepsin B in gingival crevicular fluid of adult periodontitis patients: Identification by immunological and enzymological methods

Cathepsin B (EC 3.4.22.1), a typical lysosomal cysteine proteinase was identified immunologically with anti-human cathepsin B antibody in inflammatory exudate, gingival crevicular fluid (GCF) of adult periodontitis patients. The sensitive enzyme immunoassay (EIA) system initially developed, was rarely influenced by the presence of endogenous, cysteine proteinase inhibitors, cystatin(s), indicating that it is possible to quantify the gross amount of cathepsin B including free enzyme forms and enzyme-inhibitor complex forms using this EIA system. The cathepsin B levels in, GCF as determined by EIA and the activity measured with Z-Arg-Arg-MCA showed positive and significant correlation with various clinical parameters. Immunoblotting analysis revealed that the molecular form was a 29 kDa mature enzyme. More than 95% of Z-Arg-Arg-MCA hydrolytic activity in each GCF sample was inhibited by CA-074, specific inhibitor of cathepsin B. These results strongly suggested that the gross amount of cathepsin B in GCF as well as its activity level is closely associated with the severity of the disease and that cathepsins B play an important role in the pathogenesis of periodontitis.accepted by W. B. van den Berg     

Immunocytochemical localization of cathepsin D in rat ventral prostate: evidence for castration-induced expression of cathepsin D in basal cells

Cathepsin D (EC3.4.23.5) is an aspartyl endopeptidase involved in lysosomal proteolysis. Its functional role is uncertain. This study was undertaken to determine the cellular and subcellular distribution of cathepsin D in the normal rat ventral prostate and its possible role in the castration-induced atrophy of the gland. Cathepsin D was localized immunohistochemically to perinuclear lysosomes in secretory cells, in capillary endothelial cells, and, occasionally, in stromal cells of the untreated animal. Castration resulted in an increased number of cathepsin D-positive cells in the stroma within 24 hr. By 48 hr after castration autophagolysosomes formed in secretory cells and apoptotic bodies appeared in the epithelium. Although apoptotic bodies generally contained immunoreactive cathepsin D, a subpopulation of larger apoptotic bodies, which commonly rested on the basement membrane and contained multiple inclusions, were more variable in cathepsin D expression. The induction of cathepsin D in dendritic cells basally oriented in the epithelium was noted at 4 days of castration. These cells had a phagocytic phenotype, were distributed periodically along the basement membrane, and were not found in ductal epithelia. Treatment with actinomycin D or hydrocortisone to reduce the rate of regression of the ventral prostate blocked the appearance of these cathepsin D-positive, basally oriented epithelial cells. Our data indicate that this cathepsin D-positive, phagocytic cell differentiates from a cell resident in the prostatic epithelium. We suggest that it differentiates from basal cells in the secretory tubuloalveolar portion of the gland and that it is involved in the destruction of regressed secretory cells.     

Cathepsin D-like activity in neutrophils and monocytes.

Monocytes-macrophages and polymorphonuclear leukocytes contain an acid proteolytic enzyme that cleaves tritiated hemoglobin. The monocyte-macrophage-derived enzymatic activity was completely inhibited by pepstatin A, a property of cathepsin D. Monocyte-derived macrophages developed detectable cathepsin D-like activity after 5 days in culture, and this activity coincided with the appearance of other known indicators of macrophage maturation. The cathepsin D activity further increased significantly with time after day 5 of culture. The proteinase activity extracted from neutrophils was only partially inhibitable by pepstatin A, which indicates that this activity is contributed by more than one proteolytic enzyme, including cathepsin D. Cathepsin D activity demonstrated in neutrophils and macrophages may be an important marker of phagocyte function.     

Clinicopathologic significance of cystatin C expression in gliomas

Cathepsin B, one of the lysosomal cysteine proteases, has been related to tumor invasiveness. Cystatin C is the strongest inhibitor of cathepsin B. Knowledge of its participation in the progression of gliomas is limited. We investigated the expression of cystatin C and its association with the clinicopathologic features of 57 gliomas. Cystatin C and cathepsin B expressions were evaluated by immunohistochemical methods and by semiquantitative real-time polymerase chain reaction analysis for the corresponding messenger RNA. Disease-free survival was analyzed by the Kaplan-Meier method. Tumors with low cystatin C protein expression and high cathepsin B protein expression were significantly more likely to be of high grade, and this pattern was significantly correlated with high Ki-67 LI and tumor recurrence. Depressed expression of cystatin C messenger RNA in glioblastomas compared with low-grade astrocytomas was demonstrated. Multivariate analysis demonstrated high tumor grade, high Ki-67 labeling index, high cathepsin B expression, and low cystatin C expression correlated significantly with shorter disease-free survival. These results suggest that gliomas in patients with an unfavorable clinical outcome are characterized by depressed expression of cystatin C. Evaluation of cystatin C expression in gliomas provides useful clinical information, especially as a prognostic indicator.     

The Role of Cathepsin D in the Pathogenesis of Tuberculosis: A Histochemical Study Employing Unlabeled Antibodies and the Peroxidase-Antiperoxidase Complex

Cathepsin D was visualized in free pulmonary alveolar macrophages (AM), in oil-induced peritoneal macrophages (MN) and in rabbit pulmonary and dermal BCG lesions with unlabeled antibodies and the peroxidase-antiperoxidase (PAP) complex. Large amounts of cathepsin D were present in AM and lower amounts in MN. In the lung this enzyme was richest in the alveolar macrophages that accumulated around the BCG lesions. In the dermal lesions, cathepsin D was in highest concentration in macrophages at the border of the necrotic (liquefying) centers. It was also found in high concentration in keratinizing cells of the dermal epithelium and hair follicles. It did not, however, increase appreciably in many of the activated macrophages that stained intensely for the lysosomal enzyme β-galactosidase. In fact, many epithelioid cells with high β-galactosidase activity contained no visible cathepsin D. This proteinase does not, therefore, seem to be primarily involved in the lymphocyte-mediated macrophage activation associated with acquired cellular resistance to tubercle bacilli. It is probably more involved with cell autolysis, with the digestion of ingested necrotic debris and, in all likelihood, with the process of liquefaction, the most adverse event in the pathogenesis of tuberculosis in man.     

Anti-immunoglobulin E antibody treatment blocks histamine release and tissue contraction in sensitized mice.

Cathepsin G is a serine protease located in the azurophil granules of neutrophils. In this study, we investigated the effect of cathepsin G on the functions of human natural killer (NK) cells in vitro. Cathepsin G enhanced NK cytotoxicity rapidly in a dose-dependent fashion. The ability to augment NK cytotoxicity was markedly reduced in the presence of the inhibitor, phenylmethanesulphonyl fluoride (PMSF) or chymostatin, demonstrating that the proteolytic activity of cathepsin G is essential for the induction of NK cytotoxicity. Granulocyte exocytosis is required for NK cell-dependent target killing. Cathepsin G induced the release of the granule enzyme, N-acetyl-beta-D-glucosaminidase, from human NK cells. Moreover, an increase in the cytosolic-free Ca2+ concentration was observed in NK cells after stimulation with cathepsin G. When human granulocytes were stimulated with cytochalasin B and N-formyl-methionyl-leucyl-phenylalanine (fMLP), cathepsin G was released. The cathepsin G released from granulocytes also caused enhancement of NK cytotoxicity. In the presence of serine protease inhibitor the supernatant including cathepsin G obtained from stimulated granulocytes did not enhance NK cytotoxicity, but the stimulated granulocytes did. Highly purified human NK cells treated with cathepsin G enhanced NK cytotoxicity, but NK-depleted lymphocytes did not, demonstrating that cathepsin G regulates NK cytotoxicity independently of other factors. We have shown recently that human cathepsin G binds to human NK cells. These combined data indicate that cathepsin G released from granulocytes binds to NK cells and augments NK cytotoxicity through its protease activity.     

Cathepsin D as a potential prognostic marker for lung adenocarcinoma

We previously identified cathepsin D as a possible marker for lung adenocarcinoma (AD). The purpose of the present study is to evaluate the correlation between cathepsin D expression and clinicopathological findings or prognosis. We conducted immunohistochemistry (IHC) to assess 150 AD tissues. For these 150 tumors, TTF-1 expression, EGFR and KRAS gene mutations, and ALK rearrangements had already been examined. Cathepsin D expression was detected in 44% (66 of 150, IHC score ≥1+) and 27.3% (41 of 150, IHC score ≥2+). Cathepsin D-positive (IHC score ≥2+) tumors were more poorly differentiated than cathepsin D-negative ones, while all lepidic predominant invasive adenocarcinomas showed no cathepsin D expression. Univariate analysis revealed a poor prognosis for cathepsin D-positive lung AD patients with an IHC score ≥2+ (P=0.044). Cathepsin D expression was more frequent in TTF-1-negative than in TTF-1-positive ADs (P=0.034), and more frequent in ADs with EGFR wild genotype than mutant EGFR (P<0.001). Regarding AD patients with ALK rearrangements, 4 were positive for Cathepsin D, while 2 were negative. Cathepsin D expression is indicated to be a possible prognostic marker for lung AD and to correlate with a more poorly differentiated form.     

The effect of interleukin-1β and transforming growth factor β on cathepsin B activity in human articular chondrocytes

Cathepsin B is a lysosomal cysteine proteinase, thought to be involved in the degradation of connective tissue breakdown products internalized by endocytosis. It has also been implicated in the extracellular matrix degradation of collagens and proteoglycans in disease states such as tumour invasion, rheumatoid arthritis and osteoarthritis. To date it is still unclear which factors can potentially modulate the release and/or activation of this enzyme.

The aim of this study was to investigate the effect of interleukin-1β and transforming growth factor β on cathepsin B activity in both cell lysates and cell supernatants, using first passage cultures of human articular chondrocytes. Enzyme activity was determined using a specific synthetic substrate for cathepsin B, in a fluorimetric assay.

After 24 h incubation, IL-1β (10–100 U/ml) significantly stimulated cathepsin B activity dose dependently in cell lysates whereas no activity could be detected in the media. Production of cathepsin B activity was unaffected by the presence of 1.4 μM indomethacin, which suggests that prostaglandins are not regulators of cathepsin B activity. TGFβ had no significant effect on basal intracellular cathepsin B levels and variable effects on IL-1β stimulated levels. In three of the five experiments carried out, TGFβ showed a down-regulation of IL-1β-induced enzyme activity. The other two experiments showed an additive effect when TGFβ and IL-1β were co-incubated. Basal and IL-1β-induced levels of cathepsin B activity in cell lysates were both abolished after E-64 (5 μM) was incorporated in the assay incubation medium, which established the specificity of the reaction.

These results suggest that IL-1β and TGFβ may be involved in the regulation of cathepsin B activity and thus cartilage breakdown associated with rheumatic diseases.