Novel animal models for Sjögren's syndrome: expression and transfer of salivary gland dysfunction from regulatory T cell-deficient mice.

IL-2 knockout (KO), IL-2Ralpha KO and scurfy mice lack the CD4+CD25+ regulatory T (Treg) cells and develop severe inflammation in multiple organs, although organs affected vary among these strains. We asked if salivary and lacrimal glands, the main organs affected in Sjögren's syndrome, are targeted in these strains. Severe lymphocyte and neutrophil infiltration in the salivary and lacrimal glands and a decrease in salivary secretory function were observed in IL-2 KO and IL-2Ralpha KO mice, but not in scurfy mice. Interestingly, transfer of lymph node cells from scurfy mice to RAG-1 KO recipients rapidly and effectively induced inflammation and loss of function in the salivary glands. Furthermore, we observed that daily LPS feeding in scurfy mice also induced inflammation in the salivary glands. Our study demonstrates several novel models for Sjögren's syndrome, including an adoptive transfer model that shows that scurfy mice have dormant salivary gland-specific autoreactive lymphocytes that can be activated by certain environmental factors, such as those present in RAG-1 KO mice.

Sp1 is the major fasl gene activator in abnormal CD4(-)CD8(-)B220(+) T cells of lpr and gld mice.

The abnormal CD4(-)CD8(-)TCRalpha beta(+)B220(+) double-negative (DN) T cells that accumulate in lpr and gld mice are refractory to TCR cross-linking and IL-2 stimulation, yet they have an activated phenotype and express a high level of fasl mRNA. Specific binding sites for Sp1, NFAT, Egr, and NF-kappaB have been identified in the promoter region of the fasl gene. To determine the critical factor for fasl gene activation, fasl promoter reporter and mutant constructs were transiently transfected into the abnormal DN T cells. The data demonstrate that the Sp1 binding site is the major response element that regulates fasl promoter activity. Moreover, the abnormal DN T cells contain in their nuclei a high level of Sp1, a low level of NFAT and NF-kappaB, and a very low level of Egr. Ectopic expression of Egr-3 but not Sp1 protein in the abnormal DN T cells enhanced fasl promoter activity, suggesting that the Egr but not Sp1 was limiting for fasl gene activation. Comparison between the abnormal DN T cells and the Sertoli TM4 cells showed a strong correlation between Sp1 expression and fasl mRNA level and FasL function. Our study has identified Sp1 as the major transcription factor responsible for fasl gene activation in the abnormal DN T cells that are defective in signal transduction through TCR and IL-2R, thereby, implicating a novel regulatory pathway for fasl gene activation during the physiological development and elimination of the abnormal DN T cells.

Fas ligand engagement of resident peritoneal macrophages in vivo induces apoptosis and the production of neutrophil chemotactic factors.

Fas ligand (FasL) is a potent proapoptotic type-II transmembrane protein that can cause cell death in Fas+ target populations. Despite the presumed "silent" nature of apoptotic cell death, forced expression of FasL can induce a dramatic inflammatory response. To elucidate the in vivo mechanism(s) linking FasL and inflammation, we used a membrane-bound cell-free form of FasL (mFasL-vesicle preparation (VP)). We found that i.p. injection of FasL-microvesicles led to the rapid activation and subsequent demise of Mac1(high) resident peritoneal macrophages. Apoptosis of Mac1(high) peritoneal macrophages was observed within 0.5 h of mFasL-VP injection and correlated with the detection of increased macrophage inflammatory protein (MIP)-2 levels in peritoneal lavage fluid as well as induced RNA expression of IL-1beta, MIP-2, MIP-1alpha, and MIP-1beta. In vitro culture of purified peritoneal populations identified Mac1(high) cells as the major cytokine/chemokine producers in response to mFasL-VP. Purified Mac1(high) cells exposed to FasL could restore the ability of Fas-deficient mice to mount an inflammatory response. Our data demonstrate that the FasL-mediated inflammatory response starts with the production of proinflammatory mediators by preapoptotic resident tissue macrophages and suggest a general mechanism responsible for neutrophil inflammation seen in cases of FasL-expressing allografts.

Apoptosis-inducing membrane vesicles. A novel agent with unique properties.

The CD95 ligand (FasL) transmembrane protein is found on activated T cells and cells outside the immune system. A well-known turnover process of membrane FasL is mediated by matrix metalloproteinase, which generates soluble FasL (sFasL). Here, we demonstrate that membrane FasL turnover occurs effectively through the release of membrane vesicles. Quantitative analysis indicates that this process is as effective as sFasL release for FasL-3T3 cells but somewhat less effective for FasL-expressing T cells. The apoptosis-inducing membrane vesicles display unique properties not found in FasL-expressing cells and sFasL. Unlike sFasL, vesicle-associated FasL remained bioactive, killing the same panel of targets that are susceptible to FasL-expressing cells. In contrast to FasL-expressing T cells, FasL-mediated killing by vesicles do not involve LFA-1/ICAM interaction and do not depend on de novo protein synthesis. These observations indicate that the release of FasL-bearing vesicles contributes to the turnover of cell-associated FasL, but the impact of the bioactive FasL-expressing vesicles on the function of cell-associated FasL is different from that of sFasL.

CD95 (Fas) ligand-expressing vesicles display antibody-mediated, FcR-dependent enhancement of cytotoxicity.

Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1-10 microg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1-100 ng/ml significantly enhanced cytotoxicity against the FcR(+) LB27.4, M59, and LF(+) targets, but not the FcR(-) Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR(+) A20 B lymphoma but not with the FcR(-) A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR(-) Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.

Bioactivities of Fas ligand-expressing retroviral particles.

Culture supernatants from retroviral packaging cells carrying the human Fas ligand (FasL) gene killed both human (Jurkat) and mouse (LB27.4) targets within 5 h of incubation. Cytotoxicity was found both in a fraction >/=500 kDa and a fraction between 50 and 500 kDa. Following ultracentrifugation, the activity in the >/=500-kDa fraction was concentrated in the pellet (FasL vector preparation (VP)), which was also infective when added to NIH-3T3 cells. Both Polybrene and poly-l -lysine significantly enhanced the cytotoxicity of FasL VP but not anti-Fas mAb, soluble FasL (sFasL), and cell-associated FasL. In the presence of Polybrene, FasL VP killed targets that are resistant to anti-Fas mAb and sFasL. The infectivity but not FasL cytotoxicity of FasL VP was sensitive to irradiation and heat shock. By contrast, cytotoxicity of FasL VP could be enhanced or inhibited depending on the doses of anti-FasL mAb. Interestingly, the infectivity of FasL VP was specifically enhanced by anti-FasL mAb, suggesting that a nonviral gene product could be used to regulate the behavior of the retroviral vector. Thus, in addition to expressing potent FasL cytotoxicity, the FasL VP exhibits unique properties heretofore not attributed to anti-Fas mAb, sFasL, and cell-associated FasL. Our study raises the possibility of using the retroviral gene-packaging technology to make powerful, versatile, and regulatable bioactive vesicles expressing a predetermined function of the protein encoded by the target gene.

Role of activator protein-1 in TCR-mediated regulation of the murine fasl promoter.

The present study demonstrates that transcription factor interactions are important in regulating the murine fasl promoter following TCR-mediated activation. We used DNase I-footprinting, EMSAs, and transient transfection assays to identify the minimal TCR signal-responsive region within the fasl promoter. This region contains the previously identified binding sites for NF-kappaB and Egr and the AP-1 site identified in this study. We found that TCR signaling induces AP-1 binding to this site and regulates the fasl promoter function in a fashion dependent on NF-kappaB binding. However, mutation in the AP-1 site alone did not show a significant effect on the promoter function. The data suggest that the minimal promoter required at least two transcription factors to function.

Protection of T cells from activation-induced cell death by Fas+ B cells.

Naive CD4+ T cells proliferate strongly in response to stimulation by superantigens such as staphylococcal enterotoxin B (SEB). However, when these same cells revert to a resting phenotype and are subjected to restimulation with either SEB or anti-CD3, the majority of these SEB-responsive cells undergo Fas ligand (FasL)-mediated activation-induced cell death (AICD). We investigated the impact of Fas expression on T cell AICD by utilizing B cell stimulators that lacked functional FasL and either expressed or did not express the Fas receptor. Our results indicate that B cells play an important role in modulating the level of T cell AICD via the Fas/FasL pathway. Activated B cells expressing high levels of Fas receptor can redirect the FasL expressed by T cells primed to undergo AICD away from the T cells and prevent the induction of AICD in these cells. Furthermore, B cells stimulated through both the CD40 receptor and membrane IgM appear to mediate a stronger protective effect on T cells by virtue of their resistance to FasL-mediated cytolysis. These observations suggest a mechanism by which normal B cell and T cell responses to foreign antigen are maintained, while responses to self antigen are not.

Retroviral membrane display of apoptotic effector molecules.

Retroviruses have been widely used in gene transmission studies. In this paper, we show that nonviral apoptotic proteins can be displayed on viral membrane surfaces and that the displayed proteins can execute their normal effector functions. We introduced the genes encoding the apoptosis effector proteins, human CD95 ligand (hFasL) or human tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL), into a cell line that packages Moloney murine leukemia virus vectors. Retrovirus preparations from these lines killed target cells efficiently, and target killing was prevented by Fas-Ig fusion protein or soluble TRAIL receptor (sDR5), respectively. We show that the virus preparation exhibiting Fas-specific cytotoxicity has the same density as a retrovirus, contains full-length FasL protein, and can be depleted of infectivity by immunoadsorption with anti-FasL antibody. This novel property of retroviruses-the display of functional effector proteins-may allow the custom design of reagents whose normal function requires their being embedded in a membrane.

FasL promoter activation by IL-2 through SP1 and NFAT but not Egr-2 and Egr-3.

Recently activated peripheral T cells treated with IL-2 for 4 days expressed Fas ligand (FasL)-mediated cytotoxicity. These IL-2-treated T cells had high nuclear expression of SP1 and NFAT, but lacked the Egr-2 and Egr-3 that could be induced by anti-CD3 stimulation and had been implicated in FasL gene activation. A minimal promoter region that responded to IL-2 was identified by transient transfection assays using deletion mutants. The data suggests that the GGGCGGAAA site present in the 5' end of the minimal FasL promoter is critical to IL-2-induced FasL gene activation. The GGGCGGAAA sequence contains an overlapping site used by two transcription factor families, one (GGGCGG) for the SP1 family and the other (GGAAA) for the NFAT family. FasL promoter activity was partially but statistically significantly reduced with constructs mutated at either site. More activity was lost with a construct mutated at both sites. In contrast, mutation at the Egr site had no effect on IL-2-induced FasL promoter activity. Our study identified a new FasL promoter site responding to IL-2-induced SP1 and NFAT factors. Furthermore, the nuclei of IL-2-treated cells express SP1 and NFAT, but not Egr-2 and Egr-3, for FasL gene activation.

Hypothesis: A recurrent, moderate activation fosters systemic autoimmunity--the apoptotic roles of TCR, IL-2 and Fas ligand.

Studies of several gene knockout mice suggest an interesting association of a moderate T cell response with systemic autoimmune diseases. In addition, CD95 ligand (FasL) expression in some strains of these mice is impaired. Because FasL is critically involved in regulating peripheral tolerance, there may be a link between autoimmune diseases and a moderate T cell response that cannot activate the FasL gene. Here, we propose that there are two thresholds of T cell activation. When moderately stimulated, T cells can be activated to the low (1st) threshold, which permits the induction of CD40L, IL-2, IL-4, and other components that help the immune response. The high (2nd) activation threshold can only be achieved by a strong and concurrent stimulation through TCR and IL-2R. Once the high threshold is reached, FasL is produced to induce apoptosis of the activated T and B cells. In the absence of the FasL-mediated downregulation, the activated B cells become efficient antigen-presenting cells for self-antigens and excellent responders for T cell help. Such an exacerbating condition, induced by recurrent and moderate activation, favors the development of autoreactive T cells and autoantibody production. Evidence supporting this hypothesis and some predictions that can be tested are described.

Molecular and cellular mechanisms regulating T and B cell apoptosis through Fas/FasL interaction.

Fas (CD95) and Fas ligand (FasL) are a receptor/ligand pair critically involved in lymphocyte homeostasis and peripheral tolerance such that genetic defect in either Fas or FasL results in an autoimmune lymphoproliferative syndrome. Fas is a type I transmembrane protein and a member of the tumor necrosis factor receptor (TNFR) family whereas FasL is a type II transmembrane protein and a member of TNF family. Binding of Fas by FasL induces apoptosis of the Fas-expressing cells. In the past few years, Fas/FasL interaction has been connected to a series of important phenomena previously viewed as independent immune processes. The activation-induced T cell death (AICD) and the FasL-mediated cytotoxicity by activated T cells are two critical mechanisms that can account for most of these phenomena. It is in the context of the two mechanisms that we discuss in this review the molecular and cellular events that occur during T/T and T/B interactions that account for the down-regulation of the immune response. We have also discussed recent advances in the areas of FasL gene regulation, lymphokine regulation of AICD, and regulation of B cell susceptibility to FasL. Investigation in these areas should help elucidate the role of Fas/FasL in the complex network of regulatory mechanisms that control immune response and autoimmunity.

Activation-induced apoptosis of mature T cells is dependent upon the level of surface TCR but not on the presence of the CD3 zeta ITAM.

Activation-induced cell death (AICD) occurs primarily in recently activated T cells after a second TCR triggering. Since a threshold in the activation status may be critical for AICD, it is likely that the CD3 ITAM, docking sites for tyrosine kinases, regulate AICD. A 'threshold model' for AICD was tested by using two targeted mutant mouse strains lacking either the zeta chain (CD3zeta-/-) or the ITAM of the zeta chain (CD3zeta-/-:Tgzetadelta67-150). Although the T cells from the CD3zeta-/- mice express extremely low levels of surface TCR, a subpopulation (approximately 18%) of activated T cells could be induced to express TCR/FcepsilonRI gamma by using a powerful polyclonal activation protocol. These activated TCR/FcRI gamma T cells were capable of undergoing AICD, but its induction required 10 times as much anti-CD3epsilon mAb as that required for AICD of wild-type T cells. Thus, the intensity of AICD correlated with the level of CD3 expression and was less efficient with activated, CD3zeta(-/-)-derived T cells. By contrast, AICD of T cells from the CD3zeta-/-:Tgzetadelta67-150 mice could be induced with low doses of anti-CD3epsilon mAb and the extent of AICD was comparable to T cells from wild-type mice. The AICD induced in T cells from CD3-/-, CD3zeta-/-:Tgzetadelta67-150 and normal controls was specifically inhibited by Fas-Ig fusion proteins. Our data support the 'threshold model' of AICD by demonstrating that AICD is controlled by the strength of T cell activation.

Acceleration of lpr lymphoproliferative and autoimmune disease by transgenic protein kinase CK2 alpha.

MRL-lpr/lpr mice have a Fas receptor mutation that leads to abnormalities of apoptosis, lymphoproliferation, and a lupus-like autoimmune disease associated with the production of autoantibodies. Other than Fas pathway defects, little is known about molecular abnormalities that predispose to autoimmunity. Protein kinase CK2 (also termed casein kinase II), a serine-threonine protein kinase whose targets include many critical regulators of cellular growth, is highly expressed in a lymphoproliferative disease of cattle and in many human cancers. Overexpression of the CK2alpha catalytic subunit in lymphocytes of transgenic mice leads to T cell lymphoma. We hypothesized that CK2 dysregulation and Fas mutation might cooperatively augment lymphocyte proliferation and transformation. We find that in MRL-lpr/lpr mice bearing the CK2alpha transgene, the lymphoproliferative process is dramatically exacerbated, as these mice develop massive splenomegaly and lymphadenopathy by 12 wk of age in association with increased autoantibody production and accelerated renal disease. The lymphoid organs are filled with the unusual B220+CD4-CD8- T cells typically seen in MRL-lpr/lpr mice, not the B220-CD4+CD8+ or B220-CD4-CD8+ T cells typically seen in CK2a transgenic lymphomas. The T cells do not fulfill the criteria for transformation, as they are polyclonal and not transplantable or immortal in cell culture. Thus, although the lpr lymphoproliferative and autoimmune syndrome is potentiated by the presence of the CK2a transgene, this combination of apoptotic and proliferative abnormalities appears to be insufficient to transform lymphoid cells.

Identification of two NF-kappa B sites in mouse CD95 ligand (Fas ligand) promoter: functional analysis in T cell hybridoma.

Fas ligand (FasL) gene expression is critically involved in peripheral T cell tolerance and lymphocyte homeostasis. Previous studies have suggested that nuclear translocation of NF-kappaB during T cell activation is a critical event for FasL gene activation. In the present study we have identified two NF-kappaB sites (designated FasL-kappaB1 and FasL-kappaB2) on the promoter (approximately 700 bp) of FasL. The NF-kappaB sites were identified by electrophoretic mobility shift assay. Transient transfection reporter analyses showed that the FasL promoter activity was comparable between a construct that contains both sites and a shorter construct (433 bp) that contains only the FasL-kappaB1 site. Furthermore, elimination of FasL-kappaB1 by site-directed mutagenesis significantly inhibited FasL promoter activity. These observations provide strong evidence that NF-kappaB directly binds to the FasL-kappaB1 site and up-regulates FasL gene expression.

Lymphokine regulation of activation-induced apoptosis in T cells of IL-2 and IL-2R beta knockout mice.

Recent studies using IL-2R alpha knockout mice have generated conflicting results regarding the hypothesis that IL-2/IL-2R interaction is obligatory for the development of AICD, which plays a central and pivotal role in maintaining peripheral tolerance. A relevant consequence of AICD defect is the demonstrated development of autoimmune lymphoproliferative disease in IL-2, IL-2R alpha, and IL-2R beta knockout mice, but not in IL-4, IL-7, or IL-7R knockout mice. Whether IL-4, IL-7, or IL-15 can provide the required signal for AICD development is addressed here using IL-2 and IL-2R beta knockout mice. Lymph node T cells from knockout mice were stimulated with Con A plus rIL-1 for 3 days and then maintained in high concentrations of rIL-4, rIL-7, or rIL-15 for an additional 3 days before they were subjected to AICD analysis. Our study demonstrates that IL-4, IL-7, and IL-15 can transduce signals critical for AICD development in the absence of IL-2-mediated signals. The requirement for relatively high concentrations of these lymphokines suggests their limited role in maintaining peripheral T cell tolerance, thus explaining the differential expression of autoimmune lymphoproliferative disease in the targeted mutant strains described above.

Proteasome regulation of Fas ligand cytotoxicity.

The role of NF-kappa B in regulating FasL-mediated cytotoxicity was investigated by using lactacystin. Lactacystin is a microbial metabolite known to inhibit only the protease activity of the proteasome, which is required for NF-kappa B translocation. When activated by immobilized anti-CD3 monoclonal antibody, hybridoma T cells (5D5) degraded I kappa B beta, translocated NF-kappa B into the nucleus, transcribed immediate-early genes and the Fas ligand (FasL) gene, and expressed FasL-mediated cytotoxicity. Lactacystin strongly blocked I kappa B beta degradation and the translocation of NF-kappa B (p50/RelA heterodimer), but had little effect on the expression of the transcription factors, Oct-1 and AP-1. Moreover, lactacystin did not inhibit the nuclear translocation of NF-ATp whereas cyclosporin A inhibited the translocation of both NF-kappa B and NF-ATp. The expression of c-myc and nur77, two immediate-early genes implicated in FasL gene activation, was blocked by lactacystin. Subsequently, the expression of FasL gene and FasL-mediated cytotoxicity was inhibited. LLnL, a well-known peptide aldehyde which inhibits the protease activities of the proteasome and cysteine proteases, also inhibited NF-kappa B translocation and FasL-mediated cytotoxicity. However, these events were not inhibited by the highly specific cysteine protease inhibitor E64. These observations provide further evidence that FasL cytotoxicity is regulated by the proteasome. Furthermore, lactacystin must be added early in order to efficiently inhibit the induction of FasL cytotoxicity, indicating that the early events are critical for FasL gene activation. Our study integrates the proteasome-dependent I kappa B degradation and NF-kappa B translocation into a T cell activation cascade which results in FasL gene activation and the expression of FasL-mediated cytotoxicity.

CD4+ T cells reactivated with superantigen are both more sensitive to FasL-mediated killing and express a higher level of FasL.

Naive CD4(+) T cells proliferate strongly in response to superantigens such as staphylococcal enterotoxin B (SEB). When these cells are rested and challenged a second time, they undergo activation-induced cell death (AICD). Fas/FasL interactions have been shown to mediate AICD, even though the level of Fas expression in the 2 degrees SEB responder populations is no higher than in the 1 degrees cultures. To determine whether the dissimilarity between the 1 degrees and 2 degrees cultures could be attributed to differences in FasL cytotoxic activity or in the sensitivity of the Fas apoptosis signaling pathway, we compared these parameters during the 1 degrees and 2 degrees responses of lpr and gld CD4+ T cells (which do not undergo AICD due to a lack of Fas and an inactive FasL, respectively) so that each parameter could be evaluated independently. The results demonstrate that 2 degrees responders both express a higher level of functional FasL and are more sensitive to FasL-mediated killing. These findings account for the differences between the 1 degrees and 2 degrees responses of CD4+ T cells to superantigen. In addition, we found that the apparent level of FasL-mediated cytotoxic activity in the 2 degrees lpr CD4+ T cell population is much higher than that of wild-type cells, suggesting that deficient Fas expression leads to inordinately high levels of FasL expression or subsaturation of FasL binding sites.

Proteasome regulation of activation-induced T cell death.

Lactacystin, a microbial metabolite that inhibits protease activity only in the proteasome, was used to study the role of the proteasome in the activation-induced cell death (AICD) of T cells. Lactacystin induces DNA fragmentation and apoptosis in a T cell hybridoma (DO.11. 10) in a dose-dependent manner. Between 1 and 10 microM, the mildly cytotoxic lactacystin inhibited the AICD of DO.11.10 cells cultured in anti-CD3-coated wells. Degradation of IkappaBbeta and the translocation of the NF-kappaB (p50/RelA) into the nucleus, which occurred at 1.5 hr after anti-CD3 activation, were inhibited by lactacystin. Lactacystin did not inhibit the expression of nuclear transcription factor Oct-1. The activation-induced expression of the immediate-early gene, Nur77, and the T cell death genes, CD95 (Fas) and CD95 ligand (FasL), were inhibited. Functional expression of FasL cytotoxicity and the increase of cell surface Fas were also inhibited. Lactacystin must be added within 2 hr of activation to efficiently block AICD. In addition, lactacystin failed to inhibit the killing of DO.11.10 by FasL-expressing allo-specific cytotoxic effector cells. These observations strongly suggest a direct link between the proteasome-dependent degradation of IkappaBbeta and the AICD that occurs through activation of the FasL gene and up-regulation of the Fas gene.