Critical role for cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin

The potassium ionophore nigericin induces cell death and promotes the maturation and release of IL-1beta in lipopolysaccharide (LPS)-primed monocytes and macrophages, the latter depending on caspase-1 activation by an unknown mechanism. Here, we investigate the pathway that triggers cell death and activates caspase-1. We show that without LPS priming, nigericin alone triggered caspase-1 activation and IL-18 generation in THP-1 monocytic cells. Simultaneously, nigericin induced caspase-1-independent necrotic cell death, which was blocked by the cathepsin B inhibitor CA-074-Me and other cathepsin inhibitors. Cathepsin B activation after nigericin treatment was determined biochemically and corroborated by rapid lysosomal leakage and translocation of cathepsin B to the cytoplasm. IL-18 maturation was prevented by both caspase-1 and cathepsin B inhibitors in THP-1 cells, primary mouse macrophages and human blood monocytes. Moreover, IL-18 generation was reduced in THP-1 cells stably transformed either with cystatin A (an endogenous cathepsin inhibitor) or antisense cathepsin B cDNA. Collectively, our study establishes a critical role for cathepsin B in nigericin-induced caspase-1-dependent IL-18 maturation and caspase-1-independent necrosis.     

Cathepsin-mediated Necrosis Controls the Adaptive Immune Response by Th2 (T helper type 2)-associated Adjuvants

Immunologic adjuvants are critical components of vaccines, but it remains unclear how prototypical adjuvants enhance the adaptive immune response. Recent studies have shown that necrotic cells could trigger an immune response. Although most adjuvants have been shown to be cytotoxic, this activity has traditionally been considered a side effect. We set out to test the role of adjuvant-mediated cell death in immunity and found that alum, the most commonly used adjuvant worldwide, triggers a novel form of cell death in myeloid leukocytes characterized by cathepsin-dependent lysosome-disruption. We demonstrated that direct lysosome-permeabilization with a soluble peptide, Leu-Leu-OMe, mimics the alum-like form of necrotic cell death in terms of cathepsin dependence and cell-type specificity. Using a combination of a haploid genetic screen and cathepsin-deficient cells, we identified specific cathepsins that control lysosome-mediated necrosis. We identified cathepsin C as critical for Leu-Leu-OMe-induced cell death, whereas cathepsins B and S were required for alum-mediated necrosis. Consistent with a role of necrotic cell death in adjuvant effects, Leu-Leu-OMe replicated an alum-like immune response in vivo, characterized by dendritic cell activation, granulocyte recruitment, and production of Th2-associated antibodies. Strikingly, cathepsin C deficiency not only blocked Leu-Leu-OMe-mediated necrosis but also impaired Leu-Leu-OMe-enhanced immunity. Together our findings suggest that necrotic cell death is a powerful mediator of a Th2-associated immune response.     

Insights into the roles of cathepsins in antigen processing and presentation revealed by specific inhibitors

Eleven human cathepsins have been identified, however, the in vivo roles of individual cathepsins are still largely unknown. In this brief review we will summarize the functions of individual cathepsins in antigen processing and presentation, which are the initial steps of the immune response. Two general inhibitors of papain-like cysteine proteases, E-64 and pyridoxal phosphate, can completely suppress antigen presentation in vivo. To evaluate the contribution of individual cathepsins, specific inhibitors have been developed based on cathepsin tertiary structures: CA-074 for cathepsin B, CLIK-148 and -195 for cathepsin L, CLIK-60 for cathepsin S. Administration of CA-074, a cathepsin B inhibitor, suppresses the response to exogenous antigens, such as hepatitis B virus antigen, ovalbumin and Leishmania major antigen, and induces switching of the helper T cell responses from Th-2 to Th-1 of CD4+ T cells, thereby downregulating the production of IgE and IgG1. Administration of the cathepsin S inhibitor CLIK-60 impairs presentation of an autoantigen, alpha-fodrin, in Sjogren's syndrome and suppresses the Th-1 response and autoantibody production.     

Endosomal-lysosomal proteolysis mediates death signalling by TNFalpha,not by etoposide,in L929 fibrosarcoma cells: evidence for an active role of cathepsin D

In several 'in vitro' models of apoptosis, lysosomal proteolysis has been shown to play an active role in mediating the death signal by cytokines or antiblastic drugs. Depending on the experimental cell model and the cytotoxic stimulus applied, an increased expression and the cytosolic translocation of either cathepsin D or B have been reported in apoptotic cells. We have analysed the involvement of these lysosomal proteases in a canonical apoptotic cell model, namely L929 fibroblasts, in which apoptosis was induced by cytotoxic agents acting through different mechanisms: (i) the cytokine TNFalpha, which triggers the cell suicide via interaction with its membrane receptor, and (ii) the topoisomerase II-inhibitor etoposide (VP16), which directly causes DNA damage. In both cases the activity of cathepsins B and D increased in apoptosing cultures. CA074-Me, a specific inhibitor of cathepsin B, and Leupeptin, a broad inhibitor of serine and cysteine proteases (among which is cathepsin B), did not exert any protection from TNFalpha. In contrast, pre-loading the cells with pepstatin A, a specific inhibitor of cathepsin D, protected L929 cells from TNFalpha cytotoxicity by more than 50%. However, no protection was observed if pepstatin A was added concomitantly with the cytokine. Inhibition of either cathepsin B or D did not impede apoptosis induced by etoposide. Lysosomal integrity was preserved and cathepsin D remained still confined in vesicular structures in apoptotic cells treated with either TNFalpha or etoposide. It follows that proteolysis by cathepsin D is likely to represent an early event in the death pathway triggered by TNFalpha and occurs within the endosomal-lysosomal compartment.     

Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo

New derivatives of E-64 (compound CA-030 and CA-074) were tested in vitro and in vivo for selective inhibition of cathepsin B. They exhibited 10000–30000 times greater inhibitory effects on purified rat cathepsin B than on cathepsin H and L; their initial K₁ values for cathepsin B were about 2–5 nM, like that of E-64-c, whereas their initial K₁ values for cathepsins H and L were about 40–200 μM. In in vivo conditions, such us intraperitoneal injection of compound CA-030 or CA-074 into rats, compound CA-074 is an especially potent selective inhibitor of cathepsin B, whereas compound CA-030 does not show selectivity for cathepsin B, although both compounds CA-030 and CA-074 show complete selectivity for cathepsin B in vitro.     

Inhibition of phosphatidylinositol 3-kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin-dependent apoptosis

In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.     

Host cell cathepsins potentiate Moloney murine leukemia virus infection

The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B-/- cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.     

Cathepsin D mediates cytochrome c release and caspase activation in human fibroblast apoptosis induced by staurosporine

There is increasing evidence that proteases other than caspases, for example, the lysosomal cathepsins B, D and L, are involved in apoptotic cell death. In the present study, we present data that suggest a role for cathepsin D in staurosporine-induced apoptosis in human foreskin fibroblasts. Cathepsin D and cytochrome c were detected partially released to the cytosol after exposure to 0.1 muM staurosporine for 1 h. After 4 h, activation of caspase-9 and -3 was initiated and later caspase-8 activation and a decrease in full-length Bid were detected. Pretreatment of cells with the cathepsin D inhibitor, pepstatin A, prevented cytochrome c release and caspase activation, and delayed cell death. These results imply that cytosolic cathepsin D is a key mediator in staurosporine-induced apoptosis. Analysis of the relative sequence of apoptotic events indicates that, in this cell type, cathepsin D acts upstream of cytochrome c release and caspase activation.     

The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins.

A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis.     

Hypoxia Enhances the Antiglioma Cytotoxicity of B10, a Glycosylated Derivative of Betulinic Acid

B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma.     

Identification and discrimination of extracellularly active cathepsins B and L in high-invasive melanoma cells

We established a novel protocol for lithium dodecyl sulfate (LDS) gelatin zymography, which operates under reducing conditions and at a slightly acidic pH value (6.5). This zymographic assay is based on polyacrylamide gel electrophoresis and facilitates the electrophoretic separation of human cathepsins in an active state. By this technique, activity of purified human liver cathepsin B was detected at a concentration as low as 50 ng and was blocked only in the presence of the cysteine protease inhibitor E-64 and the specific cathepsin B inhibitor CA-074 but not by aspartate, serine, or matrix metalloprotease inhibitors. The method was applied to analyze cathepsin activities in cell culture supernatants of the high-invasive melanoma cell line MV3. Interestingly, LDS zymography of MV3 cell supernatants in combination with specific inhibitors of cathepsins B and L identified three forms of extracellularly active cathepsin B and two forms of proteolytically active cathepsin L. We herein describe the generation and biochemical significance of acidic LDS zymography. This novel method permits not only the enzymatic analysis of purified cysteine proteases but also the identification and discrimination of different cathepsin activities in biological fluids, cell lysates, or supernatants, especially of cathepsins B and L, which are closely linked to major inflammatory and malignant processes.     

Hypoxia inhibits TRAIL-induced tumor cell apoptosis: Involvement of lysosomal cathepsins

Tumor hypoxia interferes with the efficacy of chemotherapy, radiotherapy, and tumor necrosis factor-α. TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a potent apoptosis inducer that limits tumor growth without damaging normal cells and tissues in vivo. We present evidence for a central role of lysosomal cathepsins in hypoxia and/or TRAIL-induced cell death in oral squamous cell carcinoma (OSCC) cells. Hypoxia or TRAIL-induced activation of cathepsins (B, D and L), caspases (-3 and -9), Bid cleavage, release of Bax and cytochrome c, and DNA fragmentation were blocked independently by zVAD-fmk, CA074Me or pepstatin A, consistent with the involvement of lysosomal cathepsin B and D in cell death. Lysosome stability and mitochondrial membrane potential were reduced in hypoxia and TRAIL-induced apoptosis. However, TRAIL treatment under hypoxic condition resulted in diminished apoptosis rates compared to treatment under normoxia. This inhibitory effect of hypoxia on TRAIL-induced apoptosis may be based on preventing Bax activation and thus protecting mitochondria stability. Our data show that TRAIL or hypoxia independently triggered activation of cathepsin B and D leading to apoptosis through Bid and Bax, and suggest that hypoxic tissue regions provide a selective environment for highly apoptosis-resistant clonal cells. Molecular therapy approaches based on cathepsin inhibitors need to address this novel tumor-preventing function of cathepsins in OSCC.     

6-Hydroxydopamine induces cystatin C-mediated cysteine protease suppression and cathepsin D activation

Alteration in the lysosomal system (LS) may represent a central mechanism in neurodegeneration. 6-Hydroxydopamine (6-OHDA) induces oxidative stress and cell death in catecholaminergic cells. The LS and caspases participate in apoptosis, although the mechanism(s) that is involved is not completely understood. Here, we show that Pheochromocytoma (PC12) cells exposed to 6-OHDA results in lysosomal dysregulation, caspase activation and cell death. Cells exposed to 6-OHDA increased expression and release of cystatin C (CC) and suppressed cathepsin B (CB). CB activity significantly declined 24h following exposure to 6-OHDA, however neutralization of CC restored CB activity. Cathepsin D (CD) and caspase-3 activity also increased following exposure to 6-OHDA. Inhibition of CD and caspase-3 with pepstatin A (PA) and DEVD-Cho, respectively, attenuated the 6-OHDA induced cell death at 48 and 72 h. However, the CB inhibitor CA-074 Me failed to protect cells. Additionally, poly-ADP-ribose polymerase (PARP) cleavage was evaluated after exposure to 6-OHDA and PA, CA-074 Me, and DEVD-Cho. Only DEVD-Cho significantly decreased PARP cleavage following exposure to 6-OHDA. Hence, caspase-3 mediated PARP cleavage following exposure to 6-OHDA appears independent of CB and CD alterations. These studies suggest alternate pathways and potential therapeutic targets of cell death associated with oxidative stress, CC, and lysosomal dysregulation.     

CA-074, but not its methyl ester CA-074Me,is a selective inhibitor of cathepsin B within living cells

Studies using inhibitors that reportedly discriminate between cathepsin B and related lysosomal cysteine proteinases have implicated the enzyme in a wide range of physiological and pathological processes. The most popular substance to selectively inhibit cathepsin B in vivo is CA-074Me, the methyl ester of the E-64 derivative CA-074. However, we now have found that CA-074Me inactivates both cathepsin B and cathepsin L within murine fibroblasts. In contrast, exposure of these cells to the parental compound CA-074 leads to the selective inhibition of endogenous cathepsin B, while intracellular cathepsin L remains unaffected. These results indicate that CA-074 rather than CA-074Me should be used to specifically inactivate cathepsin B within living cells.     

Anthrax Lethal Toxin Induced Lysosomal Membrane Permeabilization and Cytosolic Cathepsin Release Is Nlrp1b/Nalp1b-Dependent

NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or ‘danger signals’. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.     

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.     

Caspase-1 protein induces apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-mediated necrosis independently of its catalytic activity

The adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), connects pathogen/danger sensors such as NLRP3 and NLRC4 with caspases and is involved in inflammation and cell death. We have found that ASC activation induced caspase-8-dependent apoptosis or CA-074Me (cathepsin B inhibitor)-inhibitable necrosis depending on the cell type. Unlike necroptosis, another necrotic cell death, ASC-mediated necrosis, was neither RIP3-dependent nor necrostatin-1-inhibitable. Although acetyl-YVAD-chloromethylketone (Ac-YVAD-CMK) (caspase-1 inhibitor) did not inhibit ASC-mediated necrosis, comprehensive gene expression analyses indicated that caspase-1 expression coincided with the necrosis type. Furthermore, caspase-1 knockdown converted necrosis-type cells to apoptosis-type cells, whereas exogenous expression of either wild-type or catalytically inactive caspase-1 did the opposite. Knockdown of caspase-1, but not Ac-YVAD-CMK, suppressed the monocyte necrosis induced by Staphylococcus and Pseudomonas infection. Thus, the catalytic activity of caspase-1 is dispensable for necrosis induction. Intriguingly, a short period of caspase-1 knockdown inhibited IL-1β production but not necrosis, although longer knockdown suppressed both responses. Possible explanations of this phenomenon are discussed.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Proteases of the nematode Caenorhabditis elegans

Crude homogenates of the soil nematode Caenorhabditis elegans exhibit strong proteolytic activity at acid pH. Several kinds of enzyme account for much of this activity: cathepsin D, a carboxyl protease which is inhibited by pepstatin and optimally active toward hemoglobin at pH 3; at least two isoelectrically distinct thiol proteases (cathepsins Ce1 and Ce2) which are inhibited by leupeptin and optimally active toward Z-Phe-Arg-7-amino-4-methylcoumarin amide at pH 5; and a thiol-independent leupeptin-insensitive protease (cathepsin Ce3) with optimal activity toward casein at pH 5.5. Cathepsin D is quantitatively most significant for digestion of macromolecular substrates in vitro, since proteolysis is inhibited greater than 95% by pepstatin. Cathepsin D and the leupeptin-sensitive proteases act synergistically, but the relative contribution of the leupeptin-sensitive proteases depends upon the protein substrate.     

Significance of cathepsin B accumulation in synovial fluid of rheumatoid arthritis

We measured and compared the activities of various kinds of proteinases, such as cysteine, serine, aspartic, and metalloproteinases, in synovial fluids of 16 patients with rheumatoid arthritis (RA) and 18 patients with osteoarthritis (OA). More than 19-fold higher activity of cathepsin B and about 6-fold higher activity of prolylendopeptidase, compared to those of OA, were accumulated in RA fluid. Moreover, levels of cathepsins B and S using the corresponding sandwich enzyme immunoassays were statistically higher in RA fluid than those in OA. Significant amounts of 41-kDa and 35-kDa procathepsin L were detected in RA fluid using gelatin zymography, while 41-kDa enzyme alone was detected in OA. Cathepsin B in RA fluid could degrade collagen, and this degradation was suppressed by the addition of CA-074, a specific inhibitor of cathepsin B. Therefore, cathepsin B may participate in joint destruction of RA, and its inhibitor may be effective for RA care.