Induction of apoptosis in bacillus Calmette-Guérin-activated T cells by transforming growth factor-beta

In view of the critical role played by bacillus Calmette-Guérin (BCG) in the development and functional activation of protective T cells against tuberculosis, it has become important to understand the mechanisms by which cytokines regulate BCG-mediated immune responses. There is evidence that cytokine-mediated suppression of T cell function by mechanisms, including apoptosis, may reduce host resistance in tuberculosis. However, it is unclear whether cytokine-mediated suppression of antigen-responsive T cells through apoptotic mechanisms may be operating during human cellular activation induced by BCG. Here we present evidence, for the first time, that treatment of BCG-activated T cells with transforming growth factor-beta (TGF-beta) induces cellular apoptosis. These results were further supported by the fact that treatment of cells with a blocking mAb directed to TGF-beta significantly inhibited the percentage of apoptosis induced by TGF-beta. Interestingly, TGF-beta-mediated death of BCG-activated T cells in cultures containing interleukin (IL)-12 was observed. Moreover, our results demonstrated the induction of apoptosis by TGF-beta in BCG-activated T cells cultured in the presence of exogenous IL-12. In addition, our data indicated that TGF-beta significantly inhibited both BCG-induced cell growth determined by thymidine uptake and BCG-induced IFN-gamma secretion. Finally, TGF-beta-induced apoptosis in BCG-activated T cells correlated inversely with BCG-induced IFN-gamma secretion. Taken together, these findings indicate that TGF-beta induces apoptosis in human T cells activated with BCG and at the same time suggest that loss of BCG-reactive T cells through apoptotic mechanisms could contribute to an increased susceptibility to Mycobacterium tuberculosis infection.     

Increased production of TGF-beta and apoptosis of T lymphocytes isolated from peripheral blood in COPD

Chronic obstructive pulmonary disease (COPD) is associated with inflammation of airway epithelium, including an increase in the number of intraepithelial T cells. Increased apoptosis of these T cells has been reported in the airways in COPD, and although this process is critical for clearing excess activated T cells, excessive rates of apoptosis may result in unbalanced cellular homeostasis, defective clearance of apoptotic material by monocytes/macrophages, secondary necrosis, and prolongation of the inflammatory response. Lymphocytes are known to traffic between the airway and the peripheral circulation, thus we hypothesized that in COPD, circulating T cells may show an increased propensity to undergo apoptosis. We analyzed phytohemagglutinin (PHA)-stimulated peripheral blood T cells from COPD patients and controls for apoptosis using flow cytometry and staining with annexin V and 7-aminoactinomycin D. As several pathways are involved in induction of apoptosis of T cells, including transforming growth factor (TGF)-beta/TGF receptor (TGFR), TNF-alpha/TNFR1, and Fas/Fas ligand, these mediators were also investigated in peripheral blood samples from these subject groups. Significantly increased apoptosis of PHA-stimulated T cells was observed in COPD (annexin positive 75.0 +/- 14.7% SD vs. control 50.2 +/- 21.8% SD, P = 0.006), along with upregulation of TNF-alpha/TNFR1, Fas, and TGFR. Monocyte production of TGF-beta was also increased. In conclusion we have demonstrated the novel finding of increased apoptosis of stimulated T cells in COPD and have also shown that the increased T-cell death may be associated with upregulation of apoptotic pathways, TGF-beta, TNF-alpha, and Fas in the peripheral blood in COPD.     

TGF-beta1-mediated control of central nervous system inflammation and autoimmunity through the inhibitory receptor CD26

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-gamma and TNF-alpha by pathogenic T cells in response to myelin Ag was enhanced in CD26(-/-) mice, production of the immunosuppressive cytokine TGF-beta1 was diminished in vivo and in vitro. In contrast to the reduction in TGF-beta1 production, responsiveness to external TGF-beta1 was normal in T cells from CD26(-/-) mice, excluding alterations in TGF-beta1 sensitivity as a mechanism causing the loss of immune regulation. Natural ligands of CD26/DP IV induced TGF-beta1 production in T cells from wild-type mice. However, natural ligands of CD26/DP IV failed to elicit TGF-beta1 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.     

Microglial phagocytosis of apoptotic inflammatory T cells leads to down-regulation of microglial immune activation.

Apoptotic cell death is an established mechanism to terminate an inflammatory response in rodent or human brains. Microglia, as the resident phagocyte, is a strong candidate for the clearance of apoptotic lymphocytes. Apoptosis was induced in cultured autologous thymocytes and in myelin basic protein (MBP)-specific, encephalitogenic T cells from Lewis rats by the addition of 0.1 microg/ml methylprednisolone. The amount of phagocytosis of apoptotic cells was assessed using an in vitro phagocytosis assay. Supernatants were collected to measure microglial cytokine secretion. The state of immune activation in microglia was investigated by a T cell proliferation assay and by flow cytometric analysis of microglial surface expression of immune molecules. Microglia ingested specifically apoptotic cells (apoptotic thymocytes as well as MBP-specific T cells) in contrast to nonapoptotic control cells (p < 0.0001). Subsequent secretion of the proinflammatory cytokines TNF-alpha and IL-12 was significantly decreased, while the secretion of IL-10 and TGF-beta was not affected. Furthermore, ingestion of apoptotic cells led to increased microglial MHC class II expression without concomitant increase in MHC class I, costimulatory molecules, and ICAM expression. The Ag-specific activation of MBP-specific T cells in cocultures with microglia that had ingested apoptotic cells was significantly less than that of identical T cells that interacted with nonphagocytosing microglia. Together with negative results obtained in a trans-well system, this is in support of a cell contact-mediated effect. Microglia might play an important role in the clearance of apoptotic cells. The uptake of apoptotic cells by microglia is tolerogenic and results in a reduced proinflammatory cytokine production and a reduced activation of encephalitogenic T cells. This might help to restrict an autoimmune inflammation and minimize damage in the inflamed brain.     

TGF-beta regulates airway responses via T cells

Allergic asthma is characterized by airway hyperreactivity, inflammation, and a Th2-type cytokine profile favoring IgE production. Beneficial effects of TGF-beta and conflicting results regarding the role of Th1 cytokines have been reported from murine asthma models. In this study, we examined the T cell as a target cell of TGF-beta-mediated immune regulation in a mouse model of asthma. We demonstrate that impairment of TGF-beta signaling in T cells of transgenic mice expressing a dominant-negative TGF-beta type II receptor leads to a decrease in airway reactivity in a non-Ag-dependent model. Increased serum levels of IFN-gamma can be detected in these animals. In contrast, after injection of OVA adsorbed to alum and challenge with OVA aerosol, transgenic animals show an increased airway reactivity and inflammation compared with those of wild-type animals. IL-13 levels in bronchoalveolar lavage fluid and serum as well as the number of inducible NO synthase-expressing cells in lung infiltrates were increased in transgenic animals. These results demonstrate an important role for TGF-beta signaling in T cells in the regulation of airway responses and suggest that the beneficial effects observed for TGF-beta in airway hyperreactivity and inflammation may be due to its regulatory effects on T cells.     

TGF-beta1 suppresses apoptosis via differential regulation of MAP kinases and ceramide production

Serum deprivation induces apoptosis in NIH3T3 cells, which is associated with increased intracellular ceramide generation and with the activation of p38 mitogen-activated protein (MAP) kinase. Treatment of cells with transforming growth factor-beta1 (TGF-beta1) activated the extracellular signal regulated kinases 1 and 2 (ERK1/ERK2), inhibited the serum deprivation-induced p38 activation and the increase in intracellular ceramide formation, leading to the stimulation of cell proliferation and the suppression of apoptosis. Inhibition of p38 MAP kinase by SB203580 significantly reduced the serum-deprivation-induced apoptosis. Overexpression of p38 increased the cell apoptosis and reduced the antiapoptotic effect of TGF-beta1. Inhibition of ERK1/ERK2 by PD98059 completely inhibited the TGF-beta1-stimulated proliferation and partially inhibited the antiapoptotic effects of TGF-beta1. Neither SB203580 nor PD98059 has obvious effect on TGF-beta1-mediated inhibition of the increased ceramide generation. Serum-deprivation-induced apoptosis in NIH3T3 cells can also be blocked by broad-spectrum caspase inhibitor. TGF-beta1 treatment has an inhibitory effect on caspase activities. Our results indicate that ceramide, p38, and ERK1/ERK2 play critical but differential roles in cell proliferation and stress-induced apoptosis. TGF-beta1 suppresses the serum-deprivation-induced apoptosis via its distinct effects on complex signaling events involving the activation of ERK1/ERK2 and the inhibition of p38 activation and increased ceramide generation.     

Regulation of Fas ligand-induced apoptosis by TNF.

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly isolated Fas(+) lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye. TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2(-/-) T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1(-/-) T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1(-/-) 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2(-/-) 2,4,6-trinitrophenyl-coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites.     

Apoptosis of human endothelial cells is accompanied by proteolytic processing of latent TGF-beta binding proteins and activation of TGF-beta

Transforming growth factors beta (TGF-betas) are multifunctional cytokines that modulate cell growth, differentiation and apoptosis. Numerous effects initiated by TGF-betas in vitro have been described, but the role of TGF-beta targeting and activation under physiological conditions has gained very little attention and understanding. We report here that apoptosis of human umbilical vein endothelial cells (HUVECs) is accompanied by release of truncated large latent TGF-beta complexes from the pericellular matrix followed by activation of TGF-beta. The activation of TGF-beta during apoptosis was accompanied by enhanced secretion of beta1-LAP protein, and apoptotic HUVECs acquired the capacity to induce the release of latent TGF-beta-binding proteins (LTBPs) from extracellular matrices. Activated TGF-beta, in turn, attenuated apoptotic death of HUVECs. Current results indicate that the activation of TGF-beta accompanies the apoptosis of HUVECs, and may play a protective feedback role against apoptotic cell death. The results suggest a role for TGF-beta as a putative extracellular modulator of apoptosis.     

Transforming growth factor-beta 1 suppresses serum deprivation-induced death of A549 cells through differential effects on c-Jun and JNK activities

Transforming growth factor (TGF)-beta1, a pleiotropic cytokine involved in regulating growth and differentiation, can exert both pro-apoptotic and anti-apoptotic effects depending on the cell type or circumstances. We observed that TGF-beta1 blocked apoptosis resulting from serum withdrawal in A549 human lung carcinoma cells. This was associated with suppression of JNK activation that occurs concomitant with the onset of apoptosis in the absence of TGF-beta1, suggesting that JNK plays an active role in the death process and that TGF-beta1 exerts its protective influence by altering JNK activity. Overexpression of a dominant negative mutant form of SEK1, an upstream activator of JNK, likewise suppressed JNK activation and inhibited apoptosis. Investigation of early events following TGF-beta1 treatment revealed an early induction and phosphorylation of c-Jun that was absent in cells subjected to serum withdrawal alone. That TGF-beta1-induced expression of c-Jun is important for survival was supported by the finding that overexpression of non-phosphosphorylatable dominant negative mutant c-Jun, c-Jun(S73A), attenuated the protective influence of TGF-beta1. Our findings suggest that JNK activation is a late but essential event in serum deprivation-induced apoptosis in A549 cells. TGF-beta1 prevents apoptosis, in part, through the early induction and phosphorylation of c-Jun, which in turn results in attenuated JNK activation.     

Transforming growth factor-beta1 potentiates renal tubular epithelial cell death by a mechanism independent of Smad signaling

Tubular atrophy resulting from epithelial cell loss is one of the characteristic features in the development of chronic renal interstitial fibrosis. Although the trigger(s) and mechanism for tubular cell loss remain undefined, the hyperactive transforming growth factor (TGF)-beta1 signaling has long been suspected to play an active role. Here we demonstrate that although TGF-beta1 did not induce cell death per se, it dramatically potentiated renal tubular cell apoptosis initiated by other death cues in vitro. Pre-incubation of human kidney epithelial cells (HKC) with TGF-beta1 markedly promoted staurosporine-induced cell death in a time- and dose-dependent manner. TGF-beta1 dramatically accelerated the cleavage and activation of pro-caspase-9, but not pro-caspase-8, in HKC cells. This event was followed by an accelerated activation of pro-caspase-3. To elucidate the mechanism underlying TGF-beta1 promotion of tubular cell death, we investigated the signaling pathways activated by TGF-beta1. Both Smad-2 and p38 mitogen-activated protein (MAP) kinase were rapidly activated by TGF-beta1, as demonstrated by the early induction of phosphorylated Smad-2 and p38 MAP kinase, respectively. We found that overexpression of inhibitory Smad-7 completely abolished Smad-2 phosphorylation and activation induced by TGF-beta1 but did not inhibit TGF-beta1-induced apoptosis. However, suppression of p38 MAP kinase with chemical inhibitor SC68376 not only abolished p38 MAP kinase phosphorylation but also obliterated apoptosis induced by TGF-beta1. These results suggest that hyperactive TGF-beta1 signaling potentiates renal tubular epithelial cell apoptosis by a Smad-independent, p38 MAP kinase-dependent mechanism.     

Apoptosis of mink lung epithelial cells by co-treatment of low-dose staurosporine and transforming growth factor-beta1 depends on the enhanced TGF-beta signaling and requires the decreased phosphorylation of PKB/Akt

We demonstrate how co-treatment of low-dose staurosporine (STS) and TGF-beta1, which alone have little effect on cell death, markedly induces apoptosis in Mv1Lu mink lung epithelial cells, but not in its clonal variant R1B cells lacking functional TGF-beta signaling. This process was associated with mitochondria-dependent apoptosis and the enhanced TGF-beta/Smad signaling in Mv1Lu cells. When R1B cells were infected with adenovirus carrying wild-type ALK5, a functional TGF-beta type I receptor gene, the apoptotic cell death was significantly restored in these cells following co-treatment of low-dose STS and TGF-beta1. Treatment of Mv1Lu cells with both low-dose STS and TGF-beta1 decreased the activity of phospho-Akt, which is involved in cell survival signal. In addition, pre-treatments of PI3 kinase inhibitors, LY294002 and wortmannin, further increased the apoptosis of MvlLu cells induced by co-treatment of low-dose STS and TGF-beta1. And overexpression of constitutively active Akt (myr-Akt) using adenoviral expression system inhibited the apoptotic cell death of Mv1Lu cells by about 50% upon co-treatment of low-dose STS and TGF-beta1. These results suggest that co-treatment of low-dose STS and TGF-beta1 induces apoptosis of mink lung epithelial cells by enhancing TGF-beta signaling and in part suppressing cytoprotective signaling.     

Regulation of T cell apoptosis during the immune response

Apoptosis of T-lymphocytes is a fundamental process regulating antigen receptor repertoire selection during T cell maturation and homeostasis of the immune system. It also plays a key role in elimination of autoreactive lymphocytes. Resting mature T cells are activated by antigen to elicit an appropriate immune response. In contrast, preactivated T cells undergo activation-induced cell death (AICD) in response to TCR triggering alone. Thus, death by apoptosis is essential for function, growth and differentiation of T-lymphocytes. This review focuses on apoptosis mechanisms involved in T cell development and during the course of an immune response.     

TGF-beta 1 regulates lymphocyte homeostasis by preventing activation and subsequent apoptosis of peripheral lymphocytes

TGF-beta1 plays an important role in the maintenance of immune homeostasis and self-tolerance. To determine the mechanism by which TGF-beta1 prevents autoimmunity we have analyzed T cell activation in splenic lymphocytes from TGF-beta1-deficient mice. Here we demonstrate that unlike wild-type splenic lymphocytes, those from Tgfb1(-/-) mice are hyporesponsive to receptor-mediated mitogenic stimulation, as evidenced by diminished proliferation and reduced IL-2 production. However, they have elevated levels of IFN-gamma and eventually undergo apoptosis. Receptor-independent stimulation of Tgfb1(-/-) T cells by PMA plus ionomycin induces IL-2 production and mitogenic response, and it rescues them from anergy. Tgfb1(-/-) T cells display decreased CD3 expression; increased expression of the activation markers LFA-1, CD69, and CD122; and increased cell size, all of which indicate prior activation. Consistently, mutant CD4(+) T cells have elevated intracellular Ca(2+) levels. However, upon subsequent stimulation in vitro, increases in Ca(2+) levels are less than those in wild-type cells. This is also consistent with the anergic phenotype. Together, these results demonstrate that the ex vivo proliferative hyporesponsiveness of Tgfb1(-/-) splenic lymphocytes is due to prior in vivo activation of T cells resulting from deregulated intracellular Ca(2+) levels.     

Cell intrinsic TGF-beta 1 regulation of B cells

TGF-beta family cytokines play multiple roles in immune responses. TGF-beta1-null mice suffer from multi-organ infiltration that leads to their premature death. T cells play a central role in the TGF-beta1 phenotype, as deficiency of TGF-beta1 only in T cells reproduces the lethal phenotype. Although it is known that TGF-beta1 controls B cells isotype switch and homeostasis, the source responsible for this control has not been characterized. Because of the major role that T cells play in regulating B cell responses, we addressed the T cell dependency of the TGF-beta1 control of B cells. The analysis of T cell-deficient, TGF-beta1 knockout mice and the production of chimeras in which B but not T cells lacked TGF-beta1 allowed us to show that B cells are controlled in part by cell autonomous production of TGF-beta1.     

Apoptotic cells and innate immune stimuli combine to regulate macrophage cytokine secretion

Macrophage interactions with apoptotic cells can suppress inflammatory responses, but cell death by apoptosis may also trigger inflammation. We now report that murine macrophages exposed to the combination of apoptotic cells and archetypal ligands for Toll-like receptors (TLRs) 2, 4, and 9 mount cytokine responses that differ importantly from those elicited by either class of stimulus alone. TLR ligands induced early and sustained secretion of TNF-alpha, macrophage-inflammatory protein (MIP) 1alpha and MIP-2 with later secretion of IL-10, IL-12, and TGF-beta1; apoptotic cells alone stimulated late TGF-beta1 secretion only. The combination of apoptotic cells and TLR ligands enhanced early secretion of TNF-alpha, MIP-1alpha, and MIP-2 and increased late TGF-beta1 secretion, while suppressing late TNF-alpha, IL-10, and Il-12 by mechanisms which could nevertheless be overridden by IFN-gamma. We propose that this combinatorial macrophage cytokine response to apoptotic cells and TLR ligands may contribute to recruitment and activation of innate immune defense when cell death occurs at infected inflamed sites while promoting later resolution with diminished engagement of adaptive immunity.