SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

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The T cell marker CD26/dipeptidyl peptidase (DP) IV is associated with an effector phenotype and markedly elevated in the human CNS disorder multiple sclerosis. However, little is known about the in vivo role of CD26/DP IV in health and disease, and the underlying mechanism of its function in CNS inflammation. To directly address the role of CD26/DP IV in vivo, we examined Th1 immune responses and susceptibility to experimental autoimmune encephalomyelitis in CD26−/− mice. We show that gene deletion of CD26 in mice leads to deregulation of Th1 immune responses. Although production of IFN-γ and TNF-α by pathogenic T cells in response to myelin Ag was enhanced in CD26−/− mice, production of the immunosuppressive cytokine TGF-β1 was diminished in vivo and in vitro. In contrast to the reduction in TGF-β1 production, responsiveness to external TGF-β1 was normal in T cells from CD26−/− mice, excluding alterations in TGF-β1 sensitivity as a mechanism causing the loss of immune regulation. Natural ligands of CD26/DP IV induced TGF-β1 production in T cells from wild-type mice. However, natural ligands of CD26/DP IV failed to elicit TGF-β1 production in T cells from CD26−/− mice. The striking functional deregulation of Th1 immunity was also seen in vivo. Thus, clinical experimental autoimmune encephalomyelitis scores were significantly increased in CD26−/− mice immunized with peptide from myelin oligodendrocyte glycoprotein. These results identify CD26/DP IV as a nonredundant inhibitory receptor controlling T cell activation and Th1-mediated autoimmunity, and may have important therapeutic implications for the treatment of autoimmune CNS disease.

Alveolar and bronchial epithelial cells have been shown to have regulatory functions in the maintenance of lung structure and function. Recent evidence supports the premise that these cells can synthesize a variety of extracellular matrix components in vitro, suggesting an active participation in connective tissue remodeling. Their possible role in extracellular matrix degradation, however, is less clear. This study addresses the question of whether alveolar and bronchial epithelial cells express the highly collagenolytic and elastinolytic cysteine proteinase cathepsin K, which has recently been newly described. We provide evidence that the epithelial cell lines A549 and BEAS-2B are capable of expressing cathepsin K messenger RNA. Furthermore, we show that cathepsin K is expressed in normal bronchial epithelial cells. Western blot analyses of human lung-tissue lysates revealed specific immunoreactivity at molecular weights of 46 and 27 kD, corresponding to the procathepsin and the mature cathepsin K. Immunohistochemical analyses showed a pronounced staining of bronchial epithelial cells and in single alveolar epithelial cells. Using a specific fluorogenic cytochemical assay, the intracellular activity of the enzyme was localized. These findings demonstrate that bronchial and alveolar epithelial cells are capable of expressing cathepsin K, which could be of considerable importance for remodeling processes of the extracellular matrix in the lung.

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.

Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL(-/-) mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the single-chain isoform of catD. Furthermore, we found that catL-deficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catL-deficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.

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Tissue remodeling is crucial in different lung diseases, in the embryonal development as well as in bronchial carcinoma. Cathepsins were proposed to be involved in the degradation of matrix proteins. Cathepsin K is one of the most potent matrix-degrading cysteine proteinases known as yet. The elastinolytic and collagenolytic activity of this papain-like protease is comparable with that of neutrophil elastase. We have investigated the cathepsin K expression in normal adult lung tissues, in embryonal lung tissue and in bronchial carcinoma. With help of specific anti-cathepsin K antibodies it could be shown that cathepsin K was expressed in bronchial epithelial cells. These data could be confirmed at mRNA level using a quantitative RT-PCR as well as by visualisation of the specific enzymatic activity in epithelial cell lines. During the embryonal development cathepsin K was expressed in the epithelial cells of the developing bronchi. The expression seemed to be upregulated in parallel with the development of the bronchial and alveolar lumen. In the later phase of lung development the cathepsin K expression was restricted to bronchial epithelial cells. Furthermore, using quantitative RT-PCR it could be shown that cathepsin K-mRNA was upregulated in lung tumor tissues in comparison to normal tissues from the same patients. These data suggest that cathepsin K may play an important role in matrix remodeling of the lung under physiological and pathological conditions.