opović P

Peripheral blood granulocyte activity following contact sensitization of rats with dinitrochlorobenzene

Contact hypersensitivity (CHS) reaction is a classic example of a cell-mediated reaction. As the afferent phase of the reaction includes inflammation, CHS is a suitable model for investigating non-specific immunity. Some aspects of granulocyte activity in the afferent phase of experimentally induced CHS to dinitrochlorobenzene (DNCB) in two genetically different rat strains, AO and DA were examined in this study. A shift in the ratio of granulocytes to lymphocytes in favour of granulocytes and an increase in granulocyte survival were noted in DA rats. Granulocytes from both strains demonstrated increased levels of NBT reduction and an increase in their adhesion to plastic. Decreased granulocyte adhesion in the presence of monoclonal antibodies to beta2 integrins (anti-CD11b/c and anti-CD18) points to the contribution of these molecules to granulocyte adhesiveness during the sensitization phase of CHS. Stimulation of adhesion in the presence of anti-CD11a antibody, points to a differential modulation of adhesion molecule activity during the afferent phase of CHS. Changes in functional activity of granulocytes demonstrated in this study might contribute to the development of CHS in rats.

Different Roles of a Rat Cortical Thymic Epithelial Cell Line In Vitro on Thymocytes and Thymocyte Hybridoma Cells: Phagocytosis, Induction of Apoptosis, Nursing and Growth Promoting Activities

In this work, the interaction between a rat cortical thymic epithelial cell (TEC) line (R-TNC.1) with nursing activity and thymocytes as well as BWRT 8 thymocyte hybridoma (TH) cells has been studied. The R-TNC.1 cell line significantly bound thymocytes and TH. Binding was stronger during the first 30 min of cell incubation and was followed by a progressive deadhesion. Among adherent thymocytes the proportion of apoptotic cells increased with culture time which was a consequence of higher capacity of the line for binding of apoptotic than viable cells and induction of apoptosis in a subset of adherent thymocytes. Emperiopolesis activity of this thymic nurse cell (TNC) line was manifested by engulfment of thymocytes as well as TH cells. A subset of viable intra-TNC thymocytes has been triggered to die by apoptosis, whereas other internalized thymocytes have been stimulated to proliferate, as measured by an increase in the percentage of cells in mitosis and higher incorporation of bromodeoxyuridine (BrdU), in comparison to thymocytes cultivated alone. A significant stimulation of proliferation of engulfed TH cells was also observed. The R-TNC.1 cell line efficiently phagocytosed both apoptotic thymocytes and TH, and the process is followed by intra-TNC destruction of ingested cells. Cumulatively, these results suggest different role of the R-TNC.1 clone: phagocytosis of apoptotic cells; induction of apoptotic cell death in a subset of both bound and internalized thymocytes and stimulation of proliferation of a subset of intra-TNC thymocytes or TH cells.

Leflunomide induces apoptosis of thymocytes and T-cell hybridoma: differences in sensitivity and signaling pathways.

LEFLUNOMIDE is a novel antiphlogistic and immunomodulating agent that has been shown to be efficacious in preventing and healing autoimmune disorders and reactions leading to organ transplantation rejection. However, the mechanisms of its action are not sufficiently elucidated. It has been postulated that the immunomodulatory effect of the drug is probably a consequence of reversible inhibition of T-cell proliferation by blocking the early stage of T-cell activation and preventing the expression of interleukin 2 receptor (IL-2R). The effect is partly dependent on inhibition of certain protein tyrosine kinases (PTK). In this work we show evidence that A77 1726, an active metabolite of leflunomide, induces apoptosis of rat thymocytes and rat thymocyte hybridoma in vitro. Experiments were done on thymocytes of AO rats (9 weeks old) and BWRT8 hybridoma cells. The following chemicals were obtained from ICN (Costa Mesa, Calif, USA); RPMI 1640 medium; fetal calf serum (FCS); Z-PheAla-CH2F, an IL-1b converting enzyme (ICE)-like inhibitor; Z-Val-Ala-Asp(Ome)-CH2F, a caspase inhibitor; and dexamethasone. Anti-rat abT-cell receptor (TCR) monoclonal antibody (mAb) was purchased from Serotec (Oxford, UK). Merocyanine (MC) 540, propidium iodide (PI), phorbol myristate acetate (PMA), genistein, and herbimycin A were obtained from Sigma (Munich, Germany). Cyclosporin A was obtained from Novartis (Basel, Switzerland), whereas A77 1726 was obtained from Hoechst, AG (Wiesbaden, Germany). Apoptosis induced by A77 1726, genistein, herbimycin A, dexamethasone, or immobilized R73 mAb was measured after cultivation of the cells for 20 hours (RPMI 1 10% FCS) using morphological criteria, MC 540, and PI staining. The percentage of specific apoptosis was determined as previously described. We found that A77 1726 induced apoptosis of both thymocytes and BWRT8 hybridoma cells, as judged by staining with MC 540 (Fig 1A), morphological analysis of nuclei (chromatin condensation, pyknosis, and kariorexis) (Fig 1B), and staining with PI (Fig 1C). All the methods gave comparable results. As shown in Fig 1A and 1B, A77 1726 at concentrations between 10 mmol/L and 100 mmol/L induced apoptosis of thymocytes in a dose-dependent manner. Approximately 10 times lower concentrations of the metabolite were necessary for apoptosis induction in BWRT8 cells. This is the first report showing direct proapoptotic effect of leflunomide on thymocytes and T-cell hybridoma. Peripheral T cells are more resistant, but the compound significantly modulates activation-induced death of these cells (Colic et al, (manuscript in preparation). It is well known that glucocorticoids and TCR crosslinking promote apoptosis of thymocytes. A similar phenomenon was published for BWRT8 cells. Therefore, in the next experiments we studied the interference of A77 1726 with dexamethasone and an mAb to rat abTCR (R73) immobilized to plastic in inducing apoptosis. As shown in Fig 1, the leflunomide metabolite increased apoptosis both of thymocytes and BWRT8 cells triggered by these agents. The finding may be relevant for selection processes in the thymus because the glucocorticoid-mediated apoptosis is supposed to be a model of “death by neglect,” whereas TCR cross-linking mimics the processes of negative selection. When we studied the effect of PMA, a PKC stimulator, and CsA, an inhibitor of calcineurin, the agents known to modulate apoptosis of thymocytes, it was found that PMA potentiated apoptosis of thymocytes but significantly inhibited apoptosis of BWRT8 cells. In contrast, CsA slightly inhibited the hybridoma cell death, but did not modulate apoptosis of thymocytes, either alone or in combination with A77 1726 (Fig 1C). PMA has been shown to either promote or inhibit apoptosis of T cells. The effect probably depends on whether this compound activates and/or subsequently depletes PKC and also on the type of cells or their activation state. In our experiments, a shortterm preincubation of the cells with PMA probably reflects activation of PKC, and the differences between the two cell types might be related to the differences in their activation state and intracellular signaling pathways. This is in agreement with the requirements for calcineurin, as shown using CsA. CsA has been found to prevent the activation-induced cell death (AICD) by inhibiting the Fas-L expression. However, it is not efficacious in modulating apoptosis of

Bidirectional Interactions between Thymocytes and Thymic Epithelial Cell Lines In Vitro

In vitro interactions of thymocytes and thymocyte hybridomas with cortical (R-TNC.1) and medullary (TE-R 2.5) rat thymic epithelial-cell (TEC) lines were studied. It was found that the cortical line had better adhesion capability. It bound exclusively immature CD4+CD8+ αβTCR10 thymocytes, induced apoptosis of a subset of these cells, and stimulhted proliferation of the BWRT (CD4-CD8- αβTCR-) hybridoma. The medullary line bound both immature and mature thymocytes, decreased their apoptosis, and induced apoptosis of the BWRT 8 (CD4+CD810 αβTCRhi) hybridoma. Thymocyte differently modulated cytokine production by TEC lines, upregulating the secretion of IL-1 by R-TNC.1 and IL-6 by TE-R 2.5 cells. Finally, coculture of thymocytes with TEC lines resulted in different patterns of protein-tyrosine phosphorylation in thymocytes. These results show the existence of mutual bidirectional interactions between thymocytes and TEC lines in vitro, but these processes differed depending on phenotypic characteristics and origin of TEC lines used.

Adhesion Molecules in the Thymic Microenvironment: Interactions between Thymocytes and Cloned Thymic Epithelial Cell Lines

Publisher Summary This chapter presents a study on the adhesion molecules, which participate in the interactions between thymocytes and cloned thymic epithelial cell (TEC) lines. Various subsets of TEC provide distinct stimuli to developing thymocytes in the thymus via direct cell–cell interactions and soluble molecules, in which various receptor–ligand pairs participate. In this study, two TEC lines (R-TNC.1 and TE-R 2.5) from long-term culture of the rat thymic epithelium were cloned. Based on detailed multimarker phenotypic analysis and electron microscopy, it was concluded that the R-TNC.1 line is a type of cortical TEC with nursing activity, whereas the TE-R 2.5 line belongs to the medullary TEC. By using an in vitro assay, it is shown that R-TNC.1 and TE-R 2.5 cell lines differently bind thymocytes and T cell hybridomas. Binding of thymocytes to both lines is mediated by LFA-1/ICAM-1, CD2, and Thy 1, but the extent of binding inhibition in the presence of specific monoclonal antibodies (mAbs) depends on TEC lines used. CD4 and CD8 as well as two novel molecules expressed on TEC, defined by 4D1 and 1D6 mAbs, are involved in adhesion of thymocytes to the medullary line.

Comparison of signaling pathways involved in apoptosis of a thymocyte hybridoma triggered by a rat thymic medullary epithelial cell line, dexamethasone or T-cell receptor cross-linking

Using an in vitro co-culture assay we found that a rat medullary thymic epithelial cell (TEC) line (TE-R2.5) induces apoptosis of the BWRT8 thymocyte hybridoma (TH) (CD4(hi)CD8(low) alphabetaTCR(hi)). TH apoptosis induced by this TEC line was predominantly mediated by direct cell-cell contacts and was potentiated by cross-linking of the T cell receptor (TCR) by R73 monoclonal antibody (mAb). Dexamethasone (Dx) also triggered TH apoptosis but inhibited death of these cells induced by TE-R2.5 cells or immobilized R73 mAb. The TEC-induced apoptosis was independent of the LFA-1/ICAM-1 interaction but partly depended on a novel 29 kDa molecule expressed on TE-R2.5 cells. All three types of TH apoptosis were followed by the cleavage of poly-(ADP-ribose)-polymerase and were blocked by a caspase inhibitor Z-Val-Ala-Asp(OMe)-CH(2)F.PKC stimulation by phorbol myristate acetate interfered with the TH apoptosis induced by TE-R2.5 and Dx, but did not modulate the effect of R73 mAb. On the contrary, inhibition of calcineurin with cyclosporine A did not influence the apoptosis induced by TE-R2.5 and Dx, but completely prevented the R73-triggered TH cell death. The TE-R2.5-mediated BWRT8 apoptosis was suppressed by Na-orthovanadate, an inhibitor of protein tyrosine phosphatases (PTP) as well as by genistein, a protein tyrosine kinase (PTK) inhibitor, while both compounds potentiated the effect of Dx. Blocking PTP, but not PTK decreased the proapoptotic effect of R73 mAb. These results, including those using a BWRT8 subclone (BWRT8-MDP.2) which is resistant to TCR-triggered apoptosis, but sensitive to apoptosis stimulated by TE-R2.5 and Dx, indicate that TE-R2.5-induced TH apoptosis in our model is different from apoptosis in other TEC co-culture models, published so far.

Immunosine (7-thia-8-oxoguanosine) acts as a cofactor for proliferation of T cells

Abstract— Immunosine (7‐thia‐8‐oxoguanosine) is a novel guanosine analogue showing immunostimulatory activity both in vivo and in vitro. This compound acts on different components of the immune system including B cells, natural killer (NK) cells and antigen‐presenting cells (APC). However, its influence on functions of T cells is poorly understood. In this work we studied the effect of immunosine on proliferation of total rat splenocytes and purified T cells triggered by different mitogens and the mechanisms involved. The results demonstrate that immunosine significantly stimulates proliferation of T cells. The effect was dose‐dependent and also depended on concentrations of specific stimulators. Maximal stimulation was seen using 250 μM immunosine. The stimulatory effect of immunosine on lymphocyte proliferation triggered by Concanavalin A (Con A) correlated with increased interleukin 2 (IL‐2) production and upregulation of the IL‐2 receptor α (IL‐2Rα) expression. The dependency of T‐cell proliferation on IL‐2/IL‐2R was confirmed using neutralizing anti‐IL‐2Rα monoclonal antibodies (mAbs). Higher concentrations of immunosine in the presence of optimal concentrations of Con A (5 μg/mL) inhibited proliferation of T cells. A similar stimulatory effect of immunosine on proliferation of purified T cells and IL‐2 production was observed using an anti‐T‐cell receptor (TCR) mAb and a combination of anti‐TCR mAb and IL‐2. However, the guanosine analogue did not significantly modulate proliferation of T cells triggered by IL‐2 alone. When the combination of phorbol myristate acetate (PMA) and ionomycin was used for T‐cell stimulation different results were obtained. Under lower cell stimulation immunosine significantly potentiated T‐cell proliferation, expression of IL‐2Rα and IL‐2 production. In the presence of suboptimal stimulation the compound stimulated T‐cell proliferation and IL‐2Rα expression, whereas under maximal stimulation an enhancing effect on IL‐2 production was seen. Since direct stimulatory effect of immunosine on T‐cell growth in culture was rather weak it can be postulated that the compound acts as a cofactor for T‐lymphocyte proliferation.

Fibrinogen kao faktor rizika za ishemijsku bolest srca

Mesto fibrinogena u razvoju ateroskleroze i arterijske tromboze je verovatno znacajno jer on ucestvuje i u procesu nastanka i rasta plaka modulise hemoreoloske osobine krvi, a cini i osnovu koaguluma tokom procesa tromboze. Koncentracija fibrinogena u krvi je dobar nezavisan prognosticki parametar za razvoj akutnog infarkta miokarda, kako kod zdravih odraslih osoba, tako i kod koronarnih bolesnika. Nivo fibrinogena u krvi je delimicno genetski determinisan, ali i brojni faktori spoljasnje sredine uticu na njegov nivo. Vrlo je bitan odnos između fibrinogena i nekih drugih važnih faktora rizika. Fibrinogen i holesterol imaju izgleda sinergisticki ucinak na razvoj akutnog koronarnog sindroma. Moguce je da je fibrinogen jedna od najznacajnijih spona između pusenja i koronarne bolesti. Veoma je mali broj lekova koji se mogu dugorocno primenjivati i smanjiti nivo fibrinogena u krvi, tako da za sada ne postoje klinicke studije o vrednosti ovakve terapije u lecenju i prevenciji akutnih koronarnih sindroma. Shodno tome sve dok se ne dokaže da se smanjenjem nivoa fibrinogena u krvi smanjuje rizik za ispoljavanje koronarne bolesti, njegova uloga kao faktora rizika ostaje nedovoljno definisana.