Programming for cytotoxic effector function occurs concomitantly with CD4 extinction during CD8(+) T cell differentiation in the thymus. (1/8)

CD4(+) T cells are generally specialized to function as helper cells and CD8(+) T cells are generally specialized to function as cytotoxic effector cells. To explain how such concordance is achieved between co-receptor expression and immune function, we considered two possibilities. In one case, immature CD4(+)CD8(+) thymocyte precursors might first down-regulate expression of one co-receptor molecule, with the remaining co-receptor molecule subsequently activating the appropriate helper or cytotoxic functional program. Alternatively, we considered that the same intrathymic signals that selectively extinguished expression of one or the other co-receptor molecule might simultaneously initiate the appropriate helper or cytotoxic functional program. In the present study, we attempted to distinguish between these alternatives by examining thymocyte precursors of CD8(+) T cells for expression of Cathepsin C and Cathepsin W, molecules important for cytotoxic effector function. We report in developing thymocytes that Cathepsin C and Cathepsin W are expressed coordinately with extinction of CD4 co-receptor expression. We conclude that CD4 extinction and initiation of the cytotoxic functional program occurs simultaneously during differentiation of CD8(+) T cells in the thymus.  (+info)

Human cathepsin W, a cysteine protease predominantly expressed in NK cells, is mainly localized in the endoplasmic reticulum. (2/8)

Human cathepsin W (also called lymphopain) is a recently described papain-like cysteine protease of unknown function whose gene expression was found to be restricted to cytotoxic cells. Here we demonstrate that cathepsin W is expressed predominantly in NK cells and, to a lesser extent, in CTLs. Quantitative RT-PCR revealed that NK cells contained approximately 21 times more cathepsin W transcript than CTLs. The predominant expression of cathepsin W in NK cells was further confirmed by Western blot analysis and immunohistochemistry. IL-2-mediated stimulation of NK cells and CTLs revealed a stronger up-regulation of the cathepsin W gene and protein expression in NK cells (7-fold) than in CTLs (2-fold). Transfection experiments of HeLa cells and biochemical analyses revealed that cathepsin W is exclusively "high mannose-type" glycosylated and is mainly targeted to the endoplasmic reticulum (ER). Interestingly, the ER localization of cathepsin W was also found in NK cells, in which colocalization studies revealed an overlapping staining of cathepsin W and Con A, an ER-specific lectin. Furthermore, subcellular fractionation of cathepsin W-expressing cells confirmed the ER localization and showed that cathepsin W is membrane associated. Based on the results of this study, cathepsin W might represent a putative component of the ER-resident proteolytic machinery. The constitutive expression in NK cells and the stronger up-regulation of cathepsin W by IL-2 in NK cells than CTLs suggest that cathepsin W is not just a marker of cytotoxic cells but is, rather, specifically expressed in NK cells.  (+info)

Functional involvement of cathepsin W in the cytotoxic activity of NK-92 cells. (3/8)

Human cathepsin W (lymphopain) is a papain-like cysteine protease of unknown function that is specifically expressed in natural killer (NK) cells and to a lesser extent in cytotoxic T cells (CTL). In order to analyze the functional importance of cathepsin W for the cytotoxic process, we investigated NK-92 cells that have an NK cell-like phenotype and express cathepsin W. NK-92 cells possess strong cytotoxic activity against Jurkat and K562 cells. The cytotoxic activity of NK-92 cells against K562 was decreased in the presence of antisense phosphorothioate oligonucleotides against the cathepsin W-cDNA. Western blot analysis showed that the impaired cytotoxic activity of NK-92 cells was accompanied by reduced amounts of cathepsin W in the antisense-treated cells. In addition, co-cultivation experiments between NK-92 and K562 cells revealed a time-dependent decrease of cathepsin W by Western blot and immunofluorescence analysis during the cytotoxic attack, whereas CD56 expression of NK-92 cells was not affected. During cytotoxic attack, cathepsin W was neither targeted to K562 cells or other subcellular compartments, as shown by immunofluorescence analysis. The decrease of cathepsin W protein was associated with stable cathepsin W transcript levels. Control experiments using HT-29 cells, which are resistant against NK-92-mediated cytotoxicity, showed no change of cathepsin W expression, implying that the decrease of cathepsin W in the NK-92/K562 assay is linked to the cytotoxic process. Although the exact function of cathepsin W with respect to its enzymatic activity and its site of action still needs to be elucidated, our data demonstrate for the first time that cathepsin W is important for cellular cytotoxicity mediated by NK cells.  (+info)

Characterization of murine cathepsin W and its role in cell-mediated cytotoxicity. (4/8)

Cathepsin W is a member of the papain-like family of cysteine proteases. In this report, we have isolated the cDNA for murine CtsW (mCtsW) from a splenocyte library. The deduced 371-amino-acid sequence shares 68% identity with human CtsW and includes the conserved catalytic triad cysteine, histidine, and asparagine found in all members of this family. In addition to the fulllength form of mCtsW, we have isolated an alternatively spliced form of the mRNA that lacks a complete catalytic triad. An S1 nuclease protection assay and a Western blot analysis showed that mCtsW is mainly restricted to the CD8(+) T cell and natural killer cell compartments. In addition, we confirmed that, like its human homologue, mCtsW is localized mainly to the endoplasmic reticulum and its expression is up-regulated upon activation. We also characterized the mCtsW locus using bacterial artificial chromosome clones. The gene consists of 10 coding exons and 9 introns spanning 3.2 kb. To elucidate the physiologic role of this protease, we generated mice deficient in mCtsW. Our data establish that mCtsW is not required for cytotoxic lymphocyte-induced target cell death in vitro. In addition, mCtsW deficiency does not alter the susceptibility of cytotoxic lymphocytes to suicide or fratricide after degranulation. Thus, mCtsW does not have a unique role in target cell apoptosis or cytotoxic cell survival in vitro.  (+info)

Upregulation of cathepsin W-expressing T cells is specific for autoimmune atrophic gastritis compared to other types of chronic gastritis. (5/8)

AIM: To investigate a pathophysiological role of cathepsin W (CatW), a putative thiol-dependent cysteine protease, which is specifically expressed in cytotoxic lymphocytes, in different types of chronic inflammation of the gastric mucosa. METHODS: Gastric and duodenal biopsies of patients with Helicobacter pylori (H pylori)-associated active gastritis (Hp, n = 19), chemically induced reactive gastritis (CG, n = 17), autoimmune atrophic gastritis (AIG, n = 20), lymphocytic corpus gastritis (LG, n = 29), celiac disease (CD, n = 10), and corresponding controls (n = 24) were analyzed by immunohistochemistry for the expression of CatW and CD45. Furthermore, immunohistochemical double staining with anti-CD3 and anti-cathepsin was performed for the samples of AIG. RESULTS: Median values of CatW-expressing cells among CD45-positive immune cells were between 2% and 6% for normal gastric mucosa, CG, and LG, whereas the corresponding value was significantly increased for AIG (24.7%, P<0.001) and significantly decreased for HP (0.7%, P<0.05). Double staining with anti-CD3 and anti-CatW antibodies revealed that >90% of CatW-expressing cells in gastric mucosa of AIG were T cells. Duodenal mucosa had significantly more CatW/CD45-positive cells than normal gastric mucosa (median: 17.8% vs 2%, P<0.01). The corresponding proportion of CatW/CD45-postive cells was decreased in CD compared to duodenal mucosa (median: 2.1% vs 17.8%, P<0.05). CONCLUSION: The opposite findings regarding the presence of CatW-positive cells in AIG (increase) and CD (decrease) reflects the different cellular composition of immune cells involved in the pathogenesis of these diseases.  (+info)

The peri-islet basement membrane, a barrier to infiltrating leukocytes in type 1 diabetes in mouse and human. (6/8)

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Human cathepsin W, a putative cysteine protease predominantly expressed in CD8+ T-lymphocytes. (7/8)

A 750-bp fragment of a novel human cysteine protease has been identified from the dbEST databank. PCR cloning and DNA sequencing yielded a 1.38-kb full-length cDNA which encodes a polypeptide of 376 amino acids. The protein consists of a putative 21-residue signal peptide, a 106-residue propeptide and a 252-residue mature protein. The deduced amino acid sequence contains the highly conserved residues of the catalytic triad of papain-like cysteine proteases: cysteine, histidine, and asparagine. Furthermore, the protein sequence possesses two potential N-glycosylation sites: one in the propeptide and one in the mature protein. Comparison of the amino acid sequence of human cathepsin W with other human thiol-dependent cathepsins revealed a relatively low degree of similarity (21-31%). In contrast to cathepsins L, S, K, B, H and O, cathepsin W contains a 21-amino acid peptide insertion between the putative active site histidine and asparagine residues and an 8-amino acid C-terminal extension. This unique sequence may indicate that cathepsin W belongs in a novel subgroup of papain-like proteases distinct from that of cathepsin L- and B-like proteases. Northern blot analysis indicates a specific expression of cathepsin W in lymphatic tissues. Further analysis revealed predominant levels of expression in T-lymphocytes, and more specifically in CD8+ cells. The expression of the protease in cytotoxic T-lymphocytes may suggest a specific function in the mechanism or regulation of T-cell cytolytic activity.  (+info)

Human cathepsin F. Molecular cloning, functional expression, tissue localization, and enzymatic characterization. (8/8)

A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a lambdagt10-skeletal muscle cDNA library. The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B. Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup. Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus. The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalytically or by incubation with pepsin. Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min. This may suggest a function in an acidic cellular compartment. Transient expression of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distribution of the gene product in the juxtanuclear region of the cells. However, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting that the protease is targeted to the lysosomal compartment via an N-terminal signal peptide-independent lysosomal targeting pathway.  (+info)