Group B Streptococcus induces macrophage apoptosis by calpain activation

Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, alpha-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mphi apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mphi apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.     

Group B Streptococcus (GBS) disrupts by calpain activation the actin and microtubule cytoskeleton of macrophages

Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS β-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress β-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (β-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas μ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through β-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.     

Mechanisms of group B streptococcal-induced apoptosis of murine macrophages

Apoptosis of murine and human macrophages induced by group B Streptococcus agalactiae (GBS) is likely an important virulence mechanism that is used by the bacteria to suppress the host immune response and to persist at sites of infection. The mechanisms by which GBS induces apoptosis are, however, largely unknown. In this study, we report that in murine macrophages GBS induces unique changes in the regulation and localization of the apoptotic regulators Bad, 14-3-3, and Omi/high-temperature requirement A2 and leads to the release of cytochrome c and the activation of caspase-9 and caspase-3. Furthermore, inhibition of caspase-3 impaired GBS-induced apoptosis of macrophages. The ability to modulate the activity of effector caspases may therefore represent an unexploited avenue for therapeutic intervention in GBS infections.     

Vesicular stomatitis viruses expressing wild-type or mutant M proteins activate apoptosis through distinct pathways

Vesicular stomatitis virus (VSV) induces apoptosis by at least two mechanisms. The viral matrix (M) protein induces apoptosis via the mitochondrial pathway due to the inhibition of host gene expression. However, in some cell types, the inhibition of host gene expression by VSV expressing wild-type (wt) M protein delays VSV-induced apoptosis, indicating that another mechanism is involved. In support of this, the recombinant M51R-M (rM51R-M) virus, expressing a mutant M protein that is defective in its ability to inhibit host gene expression, induces apoptosis much more rapidly in L929 cells than do viruses expressing wt M protein. Here, we determine the caspase pathways by which the rM51R-M virus induces apoptosis. An analysis of caspase activity, using fluorometric caspase assays and Western blots, indicated that each of the main initiator caspases, caspase-8, caspase-9, and caspase-12, were activated during infection with the rM51R-M virus. The overexpression of Bcl-2, an inhibitor of the mitochondrial pathway, or MAGE-3, an inhibitor of caspase-12 activation, did not delay apoptosis induction in rM51R-M virus-infected L929 cells. However, an inhibitor of caspase-8 activity significantly delayed apoptosis induction. Furthermore, the inhibition of caspase-8 activity prevented the activation of caspase-9, suggesting that caspase-9 is activated by cross talk with caspase-8. These data indicate that VSV expressing the mutant M protein induces apoptosis via the death receptor apoptotic pathway, a mechanism distinct from that induced by VSV expressing the wt M protein.     

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Helicobacter pylori infects the human stomach by escaping the host immune response. One mechanism of bacterial survival and mucosal damage is induction of macrophage apoptosis, which we have reported to be dependent on polyamine synthesis by arginase and ornithine decarboxylase. During metabolic back-conversion, polyamines are oxidized and release H(2)O(2), which can cause apoptosis by mitochondrial membrane depolarization. We hypothesized that this mechanism is induced by H. pylori in macrophages. Polyamine oxidation can occur by acetylation of spermine or spermidine by spermidine/spermine N(1)-acetyltransferase prior to back-conversion by acetylpolyamine oxidase, but recently direct conversion of spermine to spermidine by the human polyamine oxidase h1, also called spermine oxidase, has been demonstrated. H. pylori induced expression and activity of the mouse homologue of this enzyme (polyamine oxidase 1 (PAO1)) by 6 h in parallel with ornithine decarboxylase, consistent with the onset of apoptosis, while spermidine/spermine N(1)-acetyltransferase activity was delayed until 18 h when late stage apoptosis had already peaked. Inhibition of PAO1 by MDL 72527 or by PAO1 small interfering RNA significantly attenuated H. pylori-induced apoptosis. Inhibition of PAO1 also significantly reduced H(2)O(2) generation, mitochondrial membrane depolarization, cytochrome c release, and caspase-3 activation. Overexpression of PAO1 by transient transfection induced macrophage apoptosis. The importance of H(2)O(2) was confirmed by inhibition of apoptosis with catalase. These studies demonstrate a new mechanism for pathogen-induced oxidative stress in macrophages in which activation of PAO1 leads to H(2)O(2) release and apoptosis by a mitochondrial-dependent cell death pathway, contributing to deficiencies in host defense in diseases such as H. pylori infection.     

Activation of PKC but not of ERK is required for vitamin E-succinate-induced apoptosis of HL-60 cells.

Vitamin E-succinate (VES) induced HL-60 human leukemia cells to undergo apoptosis. Treatment with VES induced membrane translocation of Fas; cleavages of caspase-3, PARP, and lamin B; hypophosphorylation of retinoblastoma protein; and increase of p21(WAF1) protein level. During the induction of apoptosis, activity of PKC was gradually increased with downregulation of VES-induced ERK activity and accompanied by activation of caspase-3. Inhibition of PKC by GF109203X blocked VES-mediated membrane translocation of PKC-alpha and cleavage of caspase-3 cascade, resulting in prevention of VES-induced apoptosis. On the contrary, PKC activation by cotreatment with LPC or thapsigargin and VES synergistically increased VES-mediated apoptosis. However, inhibition of ERK activity by PD98059 showed no significant effect on VES-induced PKC activity and apoptosis. Taken together, our data suggest that VES induces activation of PKC and PKC-dependent hypophosphorylation of retinoblastoma protein, which results in induction of apoptosis, and that VES-induced early activation of ERK and ERK-dependent induction of p21(WAF1) are not required for apoptosis.     

Ethanol induced NF-{kappa}B activation protects against cell injury in cultured rat gastric mucosal epithelium

Ethanol is a well-established irritant inducing inflammation in gastric mucosa, but the effects at the cellular level remain unclear. This study investigates NF-kappaB activation in gastric mucosal cells by ethanol and assesses the effects of heat shock pretreatment in this ulcerogenic situation. Rat gastric mucosal epithelia were exposed to ethanol for different time periods. Heat shock was induced by incubating the cells at 42 degrees C for 1 h prior to the experiments. For evaluation of NF-kappaB activation, the nuclear fraction of the cell lysates was analyzed with an EMSA or an ELISA-based assay. Caspase-3 (a promoter of apoptosis) activity was measured with a time-resolved fluorescence based assay, cell viability with a tetrazolium assay, and cell membrane integrity with a LDH assay. Ethanol (1-5%) induced NF-kappaB activation, reaching a maximum after 3 h, and also led to moderately increased COX-2 expression. Heat shock pretreatment and the intracellular calcium chelator BAPTA were able to inhibit ethanol-induced NF-kappaB activation. Heat shock pretreatment decreased ethanol-induced caspase-3 activation, decreased cell membrane damage, and retained cellular viability. Inhibition of NF-kappaB activation by NEMO-binding peptide, by decreasing RelA expression, or by inhibiting COX-2 activity by CAY-14040 promoted the effects of ethanol, such as increased caspase-3 activity and decreased cell viability. In conclusion, ethanol induces NF-kappaB activation via a calcium-dependent pathway and induces COX-2 expression. Inhibition of the NF-kappaB activation or COX-2 activity potentiates apoptosis and cell damage induced by ethanol, suggesting a protective role for NF-kappaB activation and COX-2 expression.     

PKC inhibition is involved in trichosanthin-induced apoptosis in human chronic myeloid leukemia cell line K562

Trichosanthin (TCS), a type I ribosome-inactivating protein, induces cell death in various cell types including several tumor cell lines. However, the mechanism remains largely uncharacterized. In this study, we investigated the possible mechanism underlying its cytotoxicity by using human chronic myeloid leukemia cell line K562. We found that TCS induced apoptosis in K562 cells in a time- and concentration-dependent manner and can be blocked by caspase-3 inhibitors. Interestingly, TCS treatment induced a transient elevation in intracellular calcium concentration and a slow increase in reactive oxygen species production, while calcium chelators and antioxidants had no obvious effect on TCS-induced apoptosis, suggesting that calcium changes and reactive oxygen species may not be involved in TCS-mediated apoptosis in K562 cells. Instead we found that TCS partly inhibited PKC activity. Indeed, the PKC activator, PMA, inhibited while the PKC inhibitor, calphostin c, enhanced TCS-induced apoptosis. These PKC modulators had similar effects on TCS-induced cleavage of caspase-3, and caspase-3 inhibitors prevented calphostin c-enhanced apoptosis induced by TCS. In summary, we conclude that TCS induces apoptosis in K562 cells partly via PKC inhibition and caspase-3 activation.     

Cholesterol is required to trigger caspase-1 activation and macrophage apoptosis after phagosomal escape of Shigella

Pro-inflammatory macrophage apoptosis is pivotal in the aetiology of bacillary dysentery, an acute inflammatory diarrhoea caused by Shigella spp. S. flexneri triggers its uptake by macrophages, escapes the phagosome and kills the host cell by a cytotoxic pathway, which activates and requires caspase-1 [interleukin (IL)-1beta-converting enzyme] and releases mature IL-1beta. The bacterial type III-secreted translocator/effector protein IpaB triggers cell death and directly binds to caspase-1. Here, we demonstrate that in S. flexneri-infected macrophages, activated caspase-1 is present in the cytoplasm, the nucleus and on vesicular membranes. IpaB partitions with membrane and cytoplasmic fractions and colocalizes with activated caspase-1 on the surface of bacteria, in the macrophage cytoplasm and on vesicular membranes. Macrophages treated with the cholesterol-sequestering compound methyl-beta-cyclodextrin (MCD) were depleted from cholesterol within minutes and were impaired for phagocytosis of S. flexneri. Consequently, cytotoxicity as determined by lactate dehydrogenase release was blocked. Interestingly, if MCD was added 15-30 min post infection, cytotoxicity, activation of caspase-1, and apoptosis were inhibited, while phagocytosis of the bacteria, escape from the phagosome and type III secretion of IpaB was not affected. Inhibition of Shigella cytotoxicity by MCD coincided with a reduced association of IpaB to host cell membranes. Contrarily, the activation of caspase-1 and cytotoxicity triggered by the K+/H+ antiport ionophore nigericin or by ATP was not affected or even increased by MCD. These results indicate that cholesterol is specifically required for caspase-1 activation and apoptosis triggered by Shigella after the escape from phagosomes, and suggest that membrane association of IpaB contributes to the activation of caspase-1.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse)(159.3 ± 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse)(906 ± 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Okadaic acid stimulates caspase-like activities and induces apoptosis of cultured rat mesangial cells

Glomerular mesangial cells play an important role in the development of glomerulosclerosis. Mesangial cell apoptosis has been shown to be involved in different stages of development of glomerulonephritis. The aim of the present study was to evaluate the effect of inhibition of serine/threonine phosphatases by okadaic acid, a shell fish toxin, on rat mesangial cell apoptosis and to examine the molecular mechanisms particularly the role of caspases. Okadaic acid significantly induced mesangial cell apoptosis, as measured by an increase in cytoplasmic nucleosome-associated DNA fragmentation. The induction of apoptosis was dependent on protein synthesis, because cyclohexamide, a protein synthesis inhibitor, blocked okadaic acid-induced apoptosis. In addition, okadaic acid stimulated caspase activities (as measured by caspase substrate peptide hydrolysis) in cultured rat mesangial cells at different time points. After 12 h treatment, okadaic acid caused a modest increase in caspase-8 (IETD-pNAse) (159.3 +/- 6.7%) activity, while after 18 h treatment, okadaic acid caused a significant increase in caspase-3 (DEVD-pNAse) (906 +/- 245%) activity. Okadaic acid-stimulated caspase-3 activity was inhibited by Z-IETD-FMK (caspase-8 inhibitor) suggesting that the caspase-3 activity is downstream of caspase-8 activity. Both caspase-3 and caspase-8 inhibitors blocked okadaic acid-stimulated apoptosis. These data suggest that inhibition of protein phosphatases by okadaic acid induces apoptosis in rat mesangial cells by activating caspase-3- and -8-like activities and that caspase-3-like activity is downstream of caspase-8-like activity.     

Type I IFN signaling is crucial for host resistance against different species of pathogenic bacteria

It is known that host cells can produce type I IFNs (IFN-alphabeta) after exposure to conserved bacterial products, but the functional consequences of such responses on the outcome of bacterial infections are incompletely understood. We show in this study that IFN-alphabeta signaling is crucial for host defenses against different bacteria, including group B streptococci (GBS), pneumococci, and Escherichia coli. In response to GBS challenge, most mice lacking either the IFN-alphabetaR or IFN-beta died from unrestrained bacteremia, whereas all wild-type controls survived. The effect of IFN-alphabetaR deficiency was marked, with mortality surpassing that seen in IFN-gammaR-deficient mice. Animals lacking both IFN-alphabetaR and IFN-gammaR displayed additive lethality, suggesting that the two IFN types have complementary and nonredundant roles in host defenses. Increased production of IFN-alphabeta was detected in macrophages after exposure to GBS. Moreover, in the absence of IFN-alphabeta signaling, a marked reduction in macrophage production of IFN-gamma, NO, and TNF-alpha was observed after stimulation with live bacteria or with purified LPS. Collectively, our data document a novel, fundamental function of IFN-alphabeta in boosting macrophage responses and host resistance against bacterial pathogens. These data may be useful to devise alternative strategies to treat bacterial infections.     

PS-341 (bortezomib) induces lysosomal cathepsin B release and a caspase-2-dependent mitochondrial permeabilization and apoptosis in human pancreatic cancer cells

PS-341 (bortezomib) is a potent and reversible proteosome inhibitor that functions to degrade intracellular polyubiquitinated proteins. PS-341 induces apoptosis and has shown broad antitumor activity with selectivity for transformed cells. We studied the effect of PS-341 on lysosomal and mitochondrial permeabilization, including the role of caspase-2 activation in apoptosis induction in the BxPC-3 human pancreatic carcinoma cell line. PS-341 induced a dose-dependent apoptosis in association with reactive oxygen species generation and cleavage of caspase-2 to its 33- and 14-kDa fragments. PS-341 disrupted lysosomes with redistribution of cathepsin B to the cytosol, as shown using fluorescence confocal microscopy, that was blocked by the free radical scavenger tiron but not by a caspase-2 inhibitor (benzyloxycarbonyl (Z)-VDVAD-fluoromethyl ketone (FMK)). PS-341-induced caspase-2 activation was attenuated by a selective pharmacological inhibitor of cathepsin B (R-3032), suggesting that cathepsin B release occurs upstream of caspase-2. PS-341-induced mitochondrial depolarization was attenuated by Z-VDVAD-FMK, tiron, and an inhibitor of the mitochondrial permeability transition pore (bongkrekic acid). Regulation of mitochondrial permeability by caspase-2 was confirmed using caspase-2 small interfering RNA. PS-341-induced cytochrome c release and phosphatidylserine externalization were attenuated by Z-VDVAD-FMK and partially by R-3032. PS-341 activated the BH3-only proteins Bik and Bim and down-regulated Bcl-2 and Bcl-xL mRNA and protein expression. Taken together, PS-341 induces lysosomal cathepsin B redistribution upstream of caspase-2. Caspase-2 activation regulates PS-341-induced mitochondrial depolarization and apoptosis, suggesting that caspase-2 can serve as a link between lysosomal and mitochondrial permeabilization.     

Ceramide-induced apoptosis: role of catalase and hepatocyte growth factor

The aim of this study was to elucidate cellular mechanisms involved in ceramide-induced apoptosis and its attenuation by hepatocyte growth factor (HGF). Human retinal pigmented epithelial cells (RPE) incubated with C2 ceramide accumulated reactive oxygen species (ROS) in mitochondria and underwent apoptosis in a dose-dependent manner. Ceramide-treated cells showed increased caspase-3 activation and an increase in mitochondrial membrane permeability transition (MPT). Low doses of H2O2 (100 microM) alone induced negligible apoptosis; however, ceramide-induced apoptosis was significantly enhanced by co-incubation with H2O2 (100 microM). Furthermore, ceramide treatment significantly decreased catalase enzymatic activity and protein expression. HGF pretreatment (20 ng/ml) significantly inhibited ceramide-induced apoptosis and reduced the accumulation of ROS, the activation of caspase-3, and the increase in MPT and prevented the reduction in catalase activity and expression. Together, the data suggest that ceramide induces apoptosis in RPE cells by increasing ROS production, MPT, and caspase-3 activation. The ceramide effect is potentiated by H2O2 and associated with a reduction in catalase activity, suggesting that catalase plays a central role in regulating this apoptotic response. The ability of HGF to attenuate these effects demonstrates its effectiveness as an antioxidant growth factor.     

结核分枝杆菌对宿主巨噬细胞死亡方式的调控

结核分枝杆菌（Mycobacterium tuberculosis,Mtb）感染后能抑制宿主巨噬细胞（M西）的免疫反应,并在其中生存、复制。研究表明Mtb减毒株感染主要诱导宿主Mφ凋亡,凋亡能抑制胞内Mtb的活力;而Mtb毒力株感染能抑制凋亡的完成,诱导Mφ坏死,最终导致Mtb扩散、感染临近细胞。通过对Mtb感染诱导宿主Mφ不同死亡方式的讨论,进一步认识Mtb的致病机制。     

Inflammasome-associated cell death: Pyroptosis,apoptosis, and physiological implications

Inflammasomes are innate immune mechanisms that promote inflammation by activating the protease caspase-1. Active caspase-1 induces pyroptosis, a necrotic form of regulated cell death, which facilitates the release of intracellular proinflammatory molecules, including IL-1 family cytokines. Recent studies identified mediators of inflammasome-associated cell death and suggested that inflammasomes induce not only pyroptosis, but also apoptosis. Caspase-1 has the potential to induce pyroptosis and apoptosis in a manner that is dependent on the expression of the pyroptosis mediator gasdermin D. Caspase-1-induced apoptosis is mediated by Bid and caspase-7. Caspase-8 is also activated following the formation of inflammasomes and may induce apoptosis. Because inflammasomes contribute to the pathogenesis of inflammatory disorders and host defenses against microbial pathogens, a more detailed understanding of the mechanisms underlying inflammasome-associated cell death may contribute to the development of novel therapeutic strategies for inflammasome-related diseases. Pyroptosis has been implicated in inflammasome-related diseases, and compounds that inhibit this process have been reported. The molecular mechanisms of inflammasome-associated cell death and its physiological implications are discussed herein.     

Galectin-9 induces apoptosis through the calcium-calpain-caspase-1 pathway

Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.