Although it has been well established that murine immature thymocytes are sensitive to apoptosis when exposed to various apoptotic stimuli, these cells as well as mature T lymphocytes were resistant to jasplakinolide-induced apoptosis. The results suggest that jasplakinolide induces apoptotic cell death through a caspase-3-like protease-dependent pathway. Another important outcome is that transformed cell lines were more susceptible to jasplakinolide-induced apoptosis than normal nontransformed cells.

Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-α, and addition of IFN-γ inhibited SLPI but augmented TNF-α production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-α production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.

Captopril is an orally active inhibitor of angiotensin‐converting enzyme (ACE) which is widely used as an anti‐hypertensive agent. In addition to its ability to reduce blood pressure, captopril has a number of other biological activities. Recently the drug was shown to inhibit Fas‐induced apoptosis in human activated peripheral T cells and human lung epithelial cells. In this study, we investigated whether captopril blocks activation‐induced apoptosis in murine T cell hybridomas, and found that captopril inhibited IL‐2 synthesis and apoptotic cell death upon activation with anti‐CD3 antibody. In addition, captopril inhibited an inducible caspase‐3‐like activity during activation‐induced apoptosis. On the other hand, captopril did not interfere with Fas signalling, since anti‐Fas antibody‐induced apoptosis in Fas+ Jurkat cells was unaffected by the drug. Furthermore, we examined whether captopril blocks activation‐induced apoptosis by interfering with expression of Fas, Fas ligand (FasL), or both on T cell hybridomas. FasL expression on activated T cells was significantly inhibited by captopril, whereas up‐expression of Fas was partially inhibited, as assessed by cell surface staining. Taking all data together, we conclude that captopril prevents activation‐induced apoptosis in T cell hybridomas by interfering with T cell activation signals. Captopril has been reported to induce systemic lupus erythematosus syndrome, and our findings may be useful for elucidating the mechanism of captopril‐induced autoimmunity.

Stimulation of murine thymocytes with phorbol ester or calcium ionophore for 18-24 h resulted in 70%-80% fragmentation of DNA into 180-200-bp multiples, followed by cell death. Experiments with fractionated subpopulations by panning or flow cytometry revealed that DNA fragmentation was selectively observed in CD4+CD8+ cells and in a portion of CD4-CD8+ cells. To investigate whether DNA cleavage is also inducible via antigen-specific receptors, thymocytes were incubated in wells precoated with anti-CD3 antibody. An approximately 20% increase of DNA fragmentation was constantly observed when unseparated thymocytes were stimulated with anti-CD3 antibody. In this anti-CD3-induced DNA degradation, CD4+CD8+ cells are probably the target cells, since (a) fetal thymocytes at day 18 of gestation were found vulnerable to anti-CD3-induced DNA cleavage and (b) flow cytometry analysis of viable cells recovered after cultivation in the anti-CD3-coated wells revealed that CD4+CD8+ cells were preferentially decreased. Further experiments with purified CD4+CD8+ cells, however, could not define a clear-cut increase of DNA fragmentation when isolated CD4+CD8+ cells were stimulated with anti-CD3 antibody. Addition of interleukin (IL) 1, IL 2, IL 3, IL 4 or interferon-gamma to the CD4+CD8+ cell cultures failed to yield a DNA cleavage similar to that of unseparated thymocytes.

S U M M A R Y Thymic mesenchymal cells are known to be important for the development of the early fetal thymus into a functionally mature organ supporting T cell differentiation. We examined the expression of mesenchymal markers: pan-mesenchymal marker ER-TR7, desmin, a-smooth muscle actin (a-SMA), and a-and b-chain of platelet-derived growth factor receptor (PDGFRa, PDGFRb) in thymi of normal adult mice. Desmin and ER-TR7 revealed specific staining in the capsule, septa, and perivascular cells. Most perivascular cells highly expressed PDGFRb at the same levels as desmin. Low expression of PDGFRa was detected in the capsule, intralobular septa, and some perivascular cells of normal adult thymi. a-SMA, used to identify vascular smooth muscle cells, was detectable on arterioles and some large venules but not on capillaries. Thus, desmin, PDGFRa, and PDGFRb were localized in the capsule, septa, and perivascular cells in thymus of adult mouse, although there were differences in the expression level among these markers. On the other hand, the expression of mesenchymal markers was detectable in the region of the thymic medullary epithelium of lymphotoxin b receptor-deficient mice and plt/plt mice, indicating that mesenchymal cells were abnormally localized in the region. These results suggest that disorganization of the medullary epithelium may be accompanied by aberrant distribution of mesenchyme in adult mouse thymus. (J Histochem Cytochem 57:373-382, 2009)