Interferon alpha augments activation-induced T cell death by upregulation of Fas (CD95/APO-1) and Fas ligand expression.

Interferon alpha (IFN-alpha) plays a prominent role in the therapy of a variety of diseases. The Fas/FasL system is crucial for the cytotoxic function and the peripheral elimination of activated T lymphocytes (ATC) by a mechanism referred to as activation-induced cell death (AICD). Recent studies suggest a link between IFN-alpha, the 2', 5'- oligoadenylate system and apoptosis. We therefore asked whether IFN-alpha is able to regulate the Fas/FasL pathway and thereby affects AICD. Peripheral blood mononuclear cells (PBMC), purified T cells and ATC of healthy volunteers were stimulated with various agents and the influence of IFN-alpha on Fas/FasL was assessed by mRNA and protein studies. The proportion of ATC undergoing AICD or anti-Fas-induced apoptosis was determined by FITC-annexin V staining and propidium iodide uptake. IFN-alpha upregulated mRNA expression of Fas and FasL in activated PBMC. Furthermore the concentration of the soluble form of FasL (sFasL) was increased in PBMC and T cells co-stimulated with IFN-alpha and various agents, whereas Fas surface expression was enhanced by IFN-alpha alone. IFN-alpha enhanced apoptosis induced by anti-Fas antibody and augmented AICD via the Fas/FasL pathway. IFN-alpha-regulated AICD may contribute to lymphopenia observed during IFN-alpha therapy. Our data further support that IFN-alpha is a multifunctional cytokine with profound effects on the immune cascades.     

Induction of human monocyte IL-1 mRNA and secretion during anti-CD3 mitogenesis requires two distinct T cell-derived signals.

The T cell signals that regulate the induction of human monocyte IL-1 during primary immune activation were investigated by using anti-CD3 mitogenesis. The induction of monocyte IL-1 alpha and beta mRNA during anti-CD3 mitogenesis was rapid (less than or equal to 1 h) and required the presence of both T cells and anti-CD3. The addition of T cells plus a nonmitogenic anti-CD5 antibody failed to induce IL-1 alpha or beta mRNA, indicating that IL-1 mRNA induction by anti-CD3 required T cell activation. Experiments using double chamber culture wells revealed that the major initial phase of IL-1 alpha and beta mRNA induction (1 to 12 h) required direct cell contact between monocytes and T cells. A subsequent minor late phase (greater than or equal to 12 h) of IL-1 mRNA was induced independently of cell contact in monocytes that received only soluble factors generated during anti-CD3 mitogenesis and was temporally associated with the appearance in culture supernatants of the late phase IL-1-inducing cytokines, IL-2, IFN-gamma, and TNF-alpha. Metabolic inactivation of T cells using paraformaldehyde demonstrated that the ability of T cells to induce IL-1 mRNA via cell contact was acquired only after activation of T cells via solid phase anti-CD3. Furthermore, pretreatment of T cells with the protein synthesis inhibitor emetine had no effect on T cell-mediated induction of monocyte IL-1 mRNA or cell-associated IL-1 alpha and beta, indicating that the expression of the IL-1 inductive signal did not require protein synthesis. Despite their ability to induce monocyte IL-1 alpha and beta mRNA, activated T cells treated with paraformaldehyde or emetine were no longer able to induce monocytes to secrete IL-1 beta into culture supernatants. However, supernatants from purified T cells that were activated with solid-phase anti-CD3 restored the ability of paraformaldehyde or emetine-treated T cells to induce IL-1 secretion. These studies provide evidence that supports a two-signal model of monocyte IL-1 production during primary immune activation. The first signal leads to the induction of monocyte IL-1 mRNA and is mediated by direct contact with activated T cells, and the second signal is provided by soluble T cell factors and results in IL-1 secretion.     

Infection of human macrophages and dendritic cells with Mycobacterium tuberculosis induces a differential cytokine gene expression that modulates T cell response

Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-alpha, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-gamma-inducing cytokines, IL-12 and IFN-alpha, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-gamma production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-alpha, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-alpha and IL-18 play a significant role in enhancing IFN-gamma synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.     

Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.     

The critical role of protein kinase C-theta in Fas/Fas ligand-mediated apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-theta (PKC-theta), superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) CD4(+) T cells was defective in PKC-theta(-/-) mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-theta(-/-) mice. PKC-theta is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-theta-mediated activation of NF-AT pathway. In addition, PKC-theta(-/-) T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-theta, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-theta(-/-) T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-theta is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.     

Different induction patterns of mRNA for IFN-alpha and -beta in human mononuclear leukocytes after in vitro stimulation with herpes simplex virus-infected fibroblasts and Sendai virus

Human peripheral blood mononuclear leukocytes (PBL) were stimulated in vitro by HSV type 1-infected glutaraldehyde-fixed fibroblasts, or Sendai virus (SV). The PBL containing mRNA for IFN-alpha 2 or -beta 1 were clearly identified by RNA-RNA in situ hybridization by using 35S-labeled alpha 2- and beta 1-probes. Although the two inducers gave similar levels of IFN in the culture medium (about 20 U/10(4) PBL), the patterns of expression of mRNA at the cellular level differed. The HSV induced only IFN-alpha mRNA in the PBL, with a lag of 1 to 2 h, and with a peak frequency of about 10 labeled cells/10(4) PBL at 6 h. Grain counts were high, the majority of cells having more than 50 grains. They were morphologically medium to large lymphocytes. The HSV-infected glutaraldehyde-fixed fibroblasts therefore induce IFN-alpha 2 mRNA in infrequent but highly efficient PBL, each cell capable of producing as much as 2 antiviral units of IFN-alpha. In contrast, SV induced both IFN-alpha 2 and -beta 1 mRNA in PBL, and without clear lags. IFN-beta 1 mRNA-positive PBL peaked somewhat earlier (4 h) than cells containing IFN-alpha 2 mRNA (6 h), and their mean frequencies were approximately 80 and 60/10(4) PBL, respectively, in a panel of PBL from six blood donors. Grain counts were lower than with the HSV inducer, the majority of cells having less than 50 grains, and most labeled cells were morphologically monocytes. The frequency of labeled PBL rapidly decreased with increasing culture time with both the SV and HSV inducers, was low at 12 h and almost absent at 24 h.     

Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIP(S) expression

The nature of inflammatory signals determines the outcome of T cell responses. However, little is known about how inflammatory cytokines provided to human CD8 T cells during activation affects their susceptibility to post-activation cell death. We have examined and compared the effects of the inflammatory cytokine IL-12, as well as the combination of IL-1, IL-6, and IL-23 (IL-1/6/23) on the susceptibility of primary human CD8 T cells to post-activation cell death. Human CD8 T cells activated in the presence of IL-1/6/23 underwent significantly less cell death after activation as compared with those activated in IL-12. This was due to reduced susceptibility to Fas-mediated activation-induced cell death (AICD). Mechanistically, the reduced level of cell death in CD8 T cells activated in IL-1/6/23 was a result of a low level of FasL expression and high level of c-FLIP(S) expression. When the effect of IL-1, IL-6, and IL-23 individually was examined, IL-1 or IL-6 alone was sufficient to inhibit CD8 T cell death that occurs after activation in IL-12. IL-1, but not IL-6, inhibited expression of FasL, whereas IL-6, but not IL-1, increased c-FLIP(S) expression. Our findings show that the presence of IL-1 and/or IL-6 during activation of human CD8 T cells attenuates Fas-mediated AICD, whereas IL-12 increases the susceptibility of activated CD8 T cells to this form of cell death.     

IL-12 decreases activation-induced cell death in human naive Th cells costimulated by intercellular adhesion molecule-1. I. IL-12 alters caspase processing and inhibits enzyme function.

Th cells can receive costimulatory signals through the LFA-1/ICAM-1 accessory pathway that are sufficient to induce early Th cell proliferation, but not subsequent cell expansion and maintenance of cell viability. To investigate the regulatory role for IL-12 in ICAM-1-mediated costimulation, human naive Th cells were stimulated with coimmobilized anti-CD3 mAb and ICAM-1 Ig in the presence or absence of IL-12. The ICAM-1-mediated costimulatory signals in this model resulted in early Th cell proliferation followed by cell death that was partially mediated by Fas and involved loss of mitochondrial membrane potential, processing of procaspase-9 and -3, and activation of caspase-3. Addition of IL-12 prevented activation-induced cell death and promoted late proliferation. ICAM-1 + IL-12-costimulated Th cells were resistant to Fas-mediated cell death through a mechanism that did not appear to involve a decrease in either Fas or Fas ligand expression. IL-12 did not inhibit the loss of mitochondrial membrane potential induced by ICAM-1-mediated costimulation, and this finding was consistent with the inability of IL-12 to increase expression of the antiapoptotic Bcl-2 family members, Bcl-2 and Bcl-x(L). Interestingly, IL-12 promoted an altered processing of procaspase-9 and -3 and a decrease in the percentage of cells displaying caspase-3 catalytic function. In conclusion, we now describe a novel regulatory function for IL-12 in preventing Th cell death and, as a result, in greatly increasing Th cell viability and expansion. Together, our findings indicate that IL-12 may perform this regulatory role by preventing Fas-mediated activation-induced cell death through inhibition of caspase-3 enzyme activity.     

Apoptosis-associated speck-like protein containing a caspase recruitment domain is a regulator of procaspase-1 activation

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)/target of methylation-induced silencing/PYCARD represents one of only two proteins encoded in the human genome that contains a caspase recruitment domain (CARD) together with a pyrin, AIM, ASC, and death domain-like (PAAD)/PYRIN/DAPIN domain. CARDs regulate caspase family proteases. We show here that ASC binds by its CARD to procaspase-1 and to adapter proteins involved in caspase-1 activation, thereby regulating cytokine pro-IL-1beta activation by this protease in THP-1 monocytes. ASC enhances IL-1beta secretion into the cell culture supernatants, at low concentrations, while suppressing at high concentrations. When expressed in HEK293 cells, ASC interferes with Cardiak/Rip2/Rick-mediated oligomerization of procaspase-1 and suppresses activation this protease, as measured by protease activity assays. Moreover, ASC also recruits procaspase-1 into ASC-formed cytosolic specks, separating it from Cardiak. We also show that expression of the PAAD/PYRIN family proteins pyrin or cryopyrin/PYPAF1/NALP3 individually inhibits IL-1beta secretion but that coexpression of ASC with these proteins results in enhanced IL-1beta secretion. However, expression of ASC uniformly interferes with caspase-1 activation and IL-1beta secretion induced by proinflammatory stimuli such as LPS and TNF, suggesting pathway competition. Moreover, LPS and TNF induce increases in ASC mRNA and protein expression in cells of myeloid/monocytic origin, revealing another level of cross-talk of cytokine-signaling pathways with the ASC-controlled pathway. Thus, our results suggest a complex interplay of the bipartite adapter protein ASC with PAAD/PYRIN family proteins, LPS (Toll family receptors), and TNF in the regulation of procaspase-1 activation, cytokine production, and control of inflammatory responses.     

Chronic morphine treatment promotes specific Th2 cytokine production by murine T cells in vitro via a Fas/Fas ligand-dependent mechanism

Improper homeostasis of Th1 and Th2 cell differentiation can promote pathological immune responses such as autoimmunity and asthma. A number of factors govern the development of these cells including TCR ligation, costimulation, death effector expression, and activation-induced cell death (AICD). Although chronic morphine administration has been shown to selectively promote Th2 development in unpurified T cell populations, the direct effects of chronic morphine on Th cell skewing and cytokine production by CD4(+) T cells have not been elucidated. We previously showed that morphine enhances Fas death receptor expression in a T cell hybridoma and human PBL. In addition, we have demonstrated a role for Fas, Fas ligand (FasL), and TRAIL in promoting Th2 development via killing of Th1 cells. Therefore, we analyzed whether the ability of morphine to affect Th2 cytokine production was mediated by regulation of Fas, FasL, and TRAIL expression and AICD directly in purified Th cells. We found that morphine significantly promoted IL-4 and IL-13 production but did not alter IL-5 or IFN-gamma. Furthermore, morphine enhanced the mRNA expression of Fas, FasL and TRAIL and promoted Fas-mediated AICD of CD4(+) T cells. Additionally, blockade of Fas/FasL interaction by anti-FasL inhibited the morphine-induced production of IL-4 and IL-13 and AICD of CD4(+) T cells. These results suggest that morphine preferentially enhances Th2 cell differentiation via killing of Th1 cells in a Fas/FasL-dependent manner.     

Proinflammatory cytokines induce proteinase 3 as membrane-bound and secretory forms in human oral epithelial cells and antibodies to proteinase 3 activate the cells through protease-activated receptor-2

Anti-neutrophil cytoplasmic Abs targeting proteinase 3 (PR3) have been detected in relation to a wide range of inflammatory conditions such as periodontitis, and interaction of anti-PR3 Abs with endothelial and epithelial cells provokes cell activation, although the underlying mechanism has been unclear. The present study showed that human oral epithelial cells expressed PR3 mRNA after treatment with proinflammatory cytokines such as IL-1alpha, TNF-alpha, IFN-alpha, IFN-beta, and IFN-gamma. A 29-kDa PR3 was expressed on the cell surface and released into culture supernatants by the cells upon stimulation with these cytokines. The membrane and supernatant fractions of oral epithelial cells exhibited enzymatic activity, which was inhibited by serine proteinase inhibitors, but not by a cysteine proteinase inhibitor or secretory leukocyte protease inhibitor. Addition of anti-PR3 Abs to cytokine-primed oral epithelial cells in culture induced remarkable secretion of IL-8 and monocyte chemoattractant protein 1 and aggregation of PR3 on the cells. RNA interference targeted to protease-activated receptor-2 mRNA and intracellular Ca2+ mobilization assays revealed that anti-PR3 Abs activated the epithelial cells through protease-activated receptor-2, a family of G protein-coupled receptors. The anti-PR3 Ab-mediated cell activation was completely abolished by RNA interference targeted to PR3 mRNA and by inhibition of phospholipase C and NF-kappaB. Immunohistochemistry showed that inflamed oral epithelium actually expresses PR3 protein. These results suggest that oral epithelial cells express functional PR3 in the inflamed sites and respond to anti-PR3 Abs detected in diseased sera, and that these mechanisms may actively participate in the inflammatory process, including periodontitis.     

YopJ-induced caspase-1 activation in Yersinia-infected macrophages: independent of apoptosis, linked to necrosis, dispensable for innate host defense

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity.     

Fas-mediated apoptosis in a rat acinar cell line is dependent on caspase-1 activity

Apoptosis plays an important role in the dysfunction of exocrine glands. Fas is a death-inducing receptor found on many types of cells including epithelial acinar cells. To elucidate the intracellular mechanism of Fas-mediated cell death in exocrine glands, an epithelial acinar cell line, SMG-C6, was studied. Caspase-1, -3, -8, and -9 activities were elevated in SMG-C6 cells after the induction of apoptosis by soluble Fas ligand (FasL). The activation of caspase-1 and -8 occurred prior to caspase-3 and -9 activation. The caspase-1 inhibitor, zYVAD-fmk, was effective in preventing cell death, whereas the caspase-3 and -8 inhibitors (ac-DEVD-CHO and ac-IETD-CHO, respectively) were not. zYVAD-fmk was able to inhibit caspase-3 activation indicating that caspase-1 is upstream to caspase-3. Furthermore, kinetic studies show that caspase-1 is an early event in the Fas apoptotic pathway. This study shows that caspase-1 participates in Fas-mediated apoptosis of epithelial cells by initiating the caspase cascade.     

IL-15 maintains T-cell survival via S-nitrosylation-mediated inhibition of caspase-3

Caspase activity is critical for both T-cell survival and death. However, little is known regarding what determines caspase activity in cycling T cells. Interleukin (IL)-2 and IL-15 confer very different susceptibilities to T-cell death. We therefore considered that IL-2 and IL-15 differentially regulate caspase activity to influence T-cell survival. We observed that IL-2-cultured primary murine effector T cells manifested elevated levels of caspase-3 activity compared with IL-15-cultured T cells. T cell receptor (TCR) restimulation further increased caspase activity and induced considerable cell death in IL-2-cultured T cells, but provoked only a minimal increase of caspase activity and cell death in IL-15-cultured T cells. IL-2 sensitization to cell death was caspase-3 mediated. Interestingly, increased active caspase-3 levels with IL-2 were independent of active initiator caspase-8 and caspase-9 that were similar with IL-2 and IL-15. Rather, caspase-3 activity was inhibited by posttranslational S-nitrosylation in IL-15-cultured T cells, but not in the presence of IL-2. This paralleled increased reactive nitrogen and oxygen species with IL-15 and reduced glycolysis. Taken together, these data suggest that the metabolic state conferred by IL-15 inhibits T-cell apoptosis in part by maintaining low levels of active caspase-3 via S-nitrosylation.     

Interaction between cytokines and 8-mercaptoguanosine in humoral immunity: synergy with interferon

In previous studies it has been demonstrated that a T cell-like differentiation signal is transmitted by C8-substituted guanine ribonucleosides such as 8-mercaptoguanosine (8MGuo) to antigen-stimulated B cells. A large subset of potentially reactive B cells remains unresponsive to antigen even in the presence of signals provided by these nucleosides except when this signal is preceded by a soluble activity present in mixed lymphocyte culture supernatants. Studies with purified preparations of interleukin (IL)-1, IL-2, IL-3, granulocyte-macrophage colony stimulating factor, B cell stimulatory factor 1 (IL-4), and B cell growth factor II (IL-5) indicated that none of these activities is capable of synergizing with 8MGuo to augment B cell responsiveness to antigen. Therefore, supernatants from a number of cloned cell lines were examined for activity that could synergize with 8MGuo, in order to determine the cellular source of this activity. Soluble products secreted by cloned 24/G1 T cells act synergistically with 8MGuo to evoke enhanced antibody responses to specific antigen in populations of purified B cells. Because concanavalin (Con) A-activated 24/G1 cells produce large quantities of interferon-gamma (IFN-gamma), the possibility that interferons might mediate synergy with 8MGuo was investigated. Purified murine IFN-gamma is unable to interact synergistically with 8MGuo; moreover, treatment of active 24/G1 supernatants with monoclonal anti-IFN-gamma antibodies or at pH 2 fails to abrogate their ability to synergize. In contrast to IFN-gamma, when B cells were supplemented with either IFN-alpha or IFN-beta, antigen-dependent synergy with 8MGuo was observed. However, abrogation of IFN-alpha and IFN-beta activity with specific antibodies fails to interfere with synergy between 8MGuo and mixed lymphocyte culture or Con A supernatants. Therefore, it appears that although IFN-alpha and IFN-beta are not responsible for the synergizing activity present in activated T cell supernatants, they nonetheless represent a previously unrecognized source of synergizing activity.     

Engagement of the Fc epsilon RI stimulates the production of IL-16 in Langerhans cell-like dendritic cells.

Preferential uptake and presentation of IgE-bound allergens by epidermal Langerhans cells (LC) via the high affinity IgE receptor, FcepsilonRI, is regarded as an important mechanism in the induction of cutaneous inflammation in atopic dermatitis. Here, we show that activation of monocyte-derived LC-like dendritic cells (LLDC) through engagement of FcepsilonRI induces the expression of IL-16, a chemoattractant factor for dendritic cells, CD4+ T cells, and eosinophils. We found that ligation of FcepsilonRI on LLDC derived from atopic dermatitis patients that express high levels of FcepsilonRI increases IL-16 mRNA expression and storage of intracellular IL-16 protein and enhances the secretion of mature IL-16 in a biphasic manner. An early release of IL-16 (peak at 4 h) is independent of protein synthesis, while a more delayed release (peak at 12 h) requires protein synthesis and occurs subsequent to the induction of IL-16 mRNA and intracellular accumulation of pro-IL-16. There was evidence that LLDC use caspase-1 to process IL-16, as inhibition of caspase-1, but not of caspase-3, partially prevented the release of IL-16 in response to ligation of FcepsilonRI. In an in vivo model of IgE-dependent LC activation, the atopy patch test, positive skin reactions were also associated with the induction of IL-16 in epidermal dendritic cells. These data indicate that IL-16 released from LC after allergen-mediated activation through FcepsilonRI may link IgE-driven and cellular inflammatory responses in diseases such as atopic dermatitis.     

IL-12 plays a significant role in the apoptosis of human T cells in the absence of antigenic stimulation

Interleukin-12 (IL-12) is an immunoregulatory cytokine that plays an essential role in cell-mediated immunity. It is known to induce T cell apoptosis in in vivo systems such as graft-versus-host disease (GVHD) and experimental autoimmune uveitis (EAU). However, the role of IL-12 in T cell apoptosis in the absence of antigenic stimulation has not been clearly defined. This study was conducted to investigate whether IL-12, in the absence of an antigen, is able to induce T cell apoptosis, and also, which signalling pathways utilized by IL-12 are involved in this process. Our data clearly showed that IL-12 in the absence of an antigen induces apoptosis in T cells. Flow cytometry and ELISA showed FasL up-regulation and increased IFN-gamma synthesis in IL-12 treated T cells, while Fas and TNF-R1 showed little change. Semi-quantitative RT-PCR demonstrated that IL-12 was able to up-regulate TNF-alpha and FasL mRNA expression. Furthermore, IL-12 induced apoptosis was associated with caspase-3, caspase-2, caspase-7, DNA fragmentation factor 45 (DFF45) and Fas associated death domain (FADD) whereas TNF receptor associated death domain (TRADD) and receptor interacting protein (RIP) were not. Inhibition of Janus tyrosine kinase (JAK) was able to suppress IL-12 induced T cell apoptosis. Anti-FasL antibody was able to block IL-12 induced T cell apoptosis. In conclusion, our findings suggest that IL-12 is able to induce T cell apoptosis in the absence of an antigen. In addition, the present data suggest that this process is FasL mediated and caspase-3 dependent. Furthermore, JAK was shown to be involved in this process. These results may have significant implications in the understanding of IL-12 mediated T cell apoptosis.     

Intestinal fibroblast-derived IL-10 increases survival of mucosal T cells by inhibiting growth factor deprivation- and Fas-mediated apoptosis

Mucosal T cells are essential to immune tolerance in the intestine, an organ constantly exposed to large amounts of dietary and bacterial Ags. We investigated whether local fibroblasts affect mucosal T cell survival, which is critical for maintenance of immune tolerance. Coculture with autologous fibroblasts significantly increased viability of mucosal T cells by inhibiting IL-2 deprivation- and Fas-mediated apoptosis, an effect that was both contact- and secreted product-dependent. Investigation of anti-apoptotic factors in the fibroblast-conditioned medium (FCM) revealed the presence of IL-10 and PGE2, but not IFN-beta, IL-2, or IL-15. Although recombinant IFN-beta, but not PGE2, effectively prevented T cell apoptosis, neutralizing Ab studies showed that only IL-10 blockade significantly increased T cells apoptosis, whereas neutralizing IFN-beta or IFN-alpha failed to inhibit the anti-apoptotic effect of FCM. To confirm that fibroblast-derived IL-10 was responsible for preserving mucosal T cell viability, IL-10 mRNA was demonstrated in fibroblasts by Southern blotting and RT-PCR. When FCM was submitted to HPLC fractionation, only the peak matching rIL-10 contained the anti-apoptotic activity, and this was eliminated by treatment with an IL-10-neutralizing Ab. Finally, when fibroblasts were transiently transfected with IL-10 antisense oligonucleotides, the conditioned medium lost its T cell anti-apoptotic effect, whereas medium from fibroblasts transfected with IFN-beta antisense oligonucleotides displayed the same anti-apoptotic activity of medium from untransfected fibroblasts. These results indicate that local fibroblast-derived IL-10 is critically involved in the survival of mucosal T cells, underscoring the crucial importance of studying organ-specific cells and products to define the mechanisms of immune homeostasis in specialized tissue microenvironments like the intestinal mucosa.