Relationship between disease activity and type 1 interferon- and other cytokine-inducible gene expression in blood in dermatomyositis and polymyositis.

The objective of this study was to evaluate the relationship between blood mRNA, disease activity and treatment effects in a longitudinal study of patients with dermatomyositis (DM) or polymyositis (PM). In all, 24 patients with DM or PM were followed for up to 6 years (mean of 1.9 years) at 2-7 follow-up visits while receiving standard clinical care. Clinical data and blood samples collected at 80 patient visits were used for the analysis of cytokine-induced gene expression for the signaling pathways of type 1 interferon (IFN), tumor necrosis factor-α, IL-1ß, granulocyte-monocyte colony-stimulating factor, IL-10 and IL-13. A type 1 IFN signature score, but not other cytokine signature scores in the blood of patients with DM or PM, correlated highly with disease activity, decreased significantly with immunomodulatory therapies and showed concordant changes with major changes in disease activity. Type 1 IFN signature score in the blood correlates with disease activity in longitudinal follow-up of individual patients with DM or PM. The type 1 IFN-inducible gene transcripts in the blood have potential utility for monitoring disease activity in patients with DM or PM.

Immunoprophylaxis of RSV infection: advancing from RSV-IGIV to palivizumab and motavizumab.

Antibodies mediate humoral immune responses and play key roles in the defense of viral infection by the recognition, neutralization, and elimination of viruses from the circulation. For the prevention of respiratory syncytial virus (RSV) infection, the natural immune response to RSV from pooled human plasma has been harvested and successfully developed as a prophylactic polyclonal RSV hyperimmune globulin, RespiGam (RSV-IGIV; MedImmune, Gaithersburg, MD). The success of RSV-IGIV validated the immunoprophylaxis approach for RSV prevention and led to the development of Synagis (palivizumab; MedImmune, Gaithersburg, MD), a humanized monoclonal antibody (mAb) that binds to the RSV F protein. Palivizumab is a potent anti-RSV mAb that is about 50-fold more potent than RSV-IGIV, and since obtaining regulatory approval in 1998 it has been used extensively to help prevent severe RSV disease in high-risk infants and children. However, a very small number of patients receiving the drug do not appear to be adequately protected. To further improve protection against RSV, we have applied a directed evolution approach to enhance the binding of palivizumab to F protein by manipulation of both the on and off rates. These efforts have yielded a more potent second-generation mAb, motavizumab, which is currently under study in phase III clinical trials. Most recently, a third generation mAb, Numax-YTE, has been generated with the intent to extend the serum half-life of the mAb in humans. If successfully developed, this drug may offer the opportunity for less frequent dosing, obviating the need for the monthly treatments that are required with palivizumab. The development of these anti-RSV approaches exemplifies the accelerated pace of drug development made possible with cutting-edge antibody engineering technologies.

Serum levels of sCD137 (4-1BB) ligand are prognostic factors for progression in acute myeloid leukemia but not in non-Hodgkin's lymphoma.

CD178 (Fas/APO-1 ligand) and CD137 ligand (CD137L) have previously been described in sera of patients with various malignancies and play an important role in the pathogenesis of various diseases. Recently, we demonstrated that low levels of soluble (s) CD137L and high levels of sCD178 correlate significantly with a long progression free survival in patients with myelodysplastic syndrome (MDS). In this study, we correlated sCD137L and sCD178 levels in sera of 42 samples of patients with acute myeloid leukemia (AML) and 46 samples of patients with non-Hodgkin's lymphoma (NHL) with stages, subtypes, and the clinical course of the diseases and determined cut-off values with maximum probability for significant differentiation between cases with higher/lower probability for progress free survival. In contrast to patients with MDS, surprisingly no correlation between sCD178 levels and different subtypes and stages or with prognosis in AML or NHL were observed. Regarding sCD137L, NHL-patients displayed lower levels compared with AML. Statistically significant higher median levels of sCD137L are present in patients with undifferentiated AML (M1/M2, 1,470 pg/mL), poor cytogenetic risk (288 pg/mL) and higher levels of BM-blasts (186 pg/mL) compared with patients with monocytoid AML (M4/M5, 89 pg/mL), intermediate cytogenetic risk (59 pg/mL) and lower levels of BM-blasts (14 pg/mL) respectively. Furthermore, in AML patients sCD137L levels correlate significantly with the probabilities to achieve complete remission (CR), stay in CR or with progress of the disease. Taken together, our data demonstrate that sCD137L can be used as a prognostic factor not only in MDS but also in AML.

4-1 BB ligand--just another costimulating molecule?

Initially, scientific interest in the 4-1BB/4-1BB Ligand system focused on the role of the 4-1BB (CD137) receptor in the costimulation of T cells. More recently, evidence is accumulating that 4-1BBL is more than "just" the ligand for a costimulatory molecule. In this review we discuss the functional properties of 4-1BB Ligand such as its preference for CD8 positive T cells and the differences to costimulation via the B7/CD28 system. Furthermore, the available data regarding its ability to transduce signals bidirectionally, i.e. also back into the ligand bearing cell, its release as a soluble form following shedding from the cell surface, and its role in the interaction of tumor cells with the immune system are reviewed.

Cell cycle phase-specific survival of CD95 ligand-challenged Jurkat cells: upregulation of heat-shock response.

An important means of regulating T-cell function occurs via physical deletion (cytolysis) of unnecessary/unwanted T cells. Among cytolytic pathways, CD95 (Fas)-based killing plays a prominent role. Although activation of T cells results in rapid upregulation of surface CD95 expression, sensitivity to CD95-based killing lags behind. To assess determinants of resistance to CD95-based killing, we used Jurkat cells as a model. Analysis of the 10% survivors of a LD(90) dose of CD95 ligand (CD95L) at 24 h demonstrated them to arise preferentially from the S + G2/M phases of the cell cycle and to remain clustered in S + G2/M without undergoing cell division. Protein immunoblot, immunocytochemistry, and RT-PCR analyses demonstrated that hsp72 was markedly upregulated in CD95L survivors within hours of CD95L challenge, indicative of a heat-shock response. Indeed, exposure of Jurkat cells to bona fide heat shock did markedly upregulate hsp72 and, upon subsequent CD95L challenge, did greatly enhance cell survival with persistent clustering to S + G2/M. These findings collectively suggest that in response to a CD95L insult, development of a heat-shock response above some critical threshold level can protect against lethality. This raises the possibility that exaggerated and/or protracted heat-shock responses under in vivo conditions may favor the survival of T cells (including autoaggressive T cells) that otherwise would be destined to die via a CD95-based pathway.

Retinoic acid and vitamin E modulate expression and release of CD178 in carcinoma cells: consequences for induction of apoptosis in CD95-sensitive cells.

CD178 (CD95-ligand) is expressed on several tumor cells and likely influences the interaction of the tumor with the host immune system. However, little is known about the mechanisms that regulate its expression on the cell surface. We have evaluated the ability of various compounds and cytokines to regulate cell surface expression and release of soluble CD178 in various carcinoma cell lines. Vitamin E succinate (VES) and retinoic acid (RA) were found to reduce CD178 surface expression, whereas interferon-gamma stimulated a slight upregulation. At 48 h, the regulation of surface CD178 by VES and RA arose from a small decrease in CD178 mRNA and to a greater extent due to an increase in the release of soluble CD178; the latter was blocked by addition of a metalloproteinase inhibitor. Accordingly, VES and RA treatment diminished the ability of tumor cells to kill CD95-sensitive cells and this effect was markedly reduced by the presence of a metalloproteinase inhibitor. Our results indicate that, in vitro, CD178 expression on the cell surface of tumor cells can be regulated by agents that alter both expression and release of the ligand. In vivo, such treatments may play an important role in the outcome of tumor sensitivity or resistance to host immune mechanisms.

Soluble CD137 (4-1BB) ligand is released following leukocyte activation and is found in sera of patients with hematological malignancies.

Expression of CD137 ligand (4-1BBL), a member of the TNF family of proteins, has been reported on several types of APCs, various carcinoma cells, and can be induced on activated T cells. In this study, we report that the soluble ligand was released constitutively at low levels from leukocytes and at higher levels following cellular activation. Release from cells was blocked by addition of a metalloproteinase inhibitor which concomitantly caused the accumulation of 4-1BBL on the cell surface. In addition, we show that a soluble form of 4-1BBL was present at high levels in the sera of some patients with various hematological diseases, but only at low levels in healthy donors. Soluble 4-1BBL was active in that it competed with recombinant 4-1BBL for binding to the 4-1BB receptor and was able to costimulate IL-2 and IFN-gamma release from peripheral T cells. These results indicate that the release of soluble 4-1BBL from the cell surface is mediated by one or more sheddases and likely regulates 4-1BB-4-1BBL interactions between cells in vivo. Cleavage of 4-1BBL to an active soluble form would alter both proximal and distal cellular responses, including cell survival and costimulatory or inflammatory responses, that are mediated through the 4-1BB pathway. This, in turn, would likely alter disease progression or outcome.

Recombinant human Fas ligand induces alveolar epithelial cell apoptosis and lung injury in rabbits.

This study investigated whether recombinant human soluble Fas ligand (rh-sFasL) induces apoptosis of primary type II pneumocytes in vitro and lung injury in vivo. Type II cells isolated from normal rabbit lung expressed Fas on their surface and became apoptotic after an 18-h incubation with rh-sFasL. Fas expression in normal rabbit lungs was localized by immunohistochemistry to alveolar and airway epithelia and alveolar macrophages. The administration of 10 microg of rh-sFasL into the right lungs of rabbits resulted 24 h later in both significantly more bronchoalveolar lavage fluid total protein and significantly more tissue changes compared with those in the left lungs, which received rh-sFasL plus Fas:Ig (a fusion protein that binds and blocks sFasL). Tissue changes included thickening of the alveolar walls, neutrophilic infiltrates, apoptotic (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive) cells in the alveolar walls, and increased expression of interleukin-8 by alveolar macrophages (as determined by immunohistochemistry). We conclude that the alveolar epithelium of normal rabbits expresses Fas and that sFasL induces lung injury and inflammation in rabbits.

Stimulation of inflammatory responses in vitro and in vivo by lipophilic HMG-CoA reductase inhibitors.

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase catalyses the rate limiting step in cholesterol biosynthesis and is markedly inhibited by the statin family of drugs. The effect of statins on lipid lowering is clearly defined, but the ability of the drugs to directly regulate inflammatory functions has not been well explored. In this report, we show that there are differences among the statins in their capacity to induce proinflammatory responses both in human monocytes in vitro, and in leukocytes in mice in vivo. Treatment of human monocytes with lipophilic statins alone stimulated the production of MCP-1, IL-8, TNF-alpha and IL-1 beta and markedly sensitized the cells to subsequent challenge with inflammatory agents. Lipophilic statins also increased the production of reactive oxygen species in monocytes. In contrast, pretreatment of cells with the hydrophilic pravastatin did not induce these heightened inflammatory responses. Furthermore, treatment of mice with lipophilic statins caused a markedly higher influx of leukocytes into the inflamed peritoneal cavity following challenge with thioglycollate. Overall, these results demonstrate that the lipophilic statins influence a regulatory pathway in monocytes that controls cytokine production and that the statins induce different pro-inflammatory responses both in vitro and in vivo.

A longitudinal study on mandibular implants supporting an overdenture: the influence of retention mechanism and anatomic-prosthetic variables on periimplant parameters.

PURPOSE: The aim of this 5-year longitudinal study was to investigate the influence of the retention mechanism for overdenture fixation on the periimplant parameters of mandibular implants. MATERIALS AND METHODS: Ninety edentulous patients, each with two interforaminal implants supporting an overdenture, participated in the study. They had received either a straight or slightly bent round clip bar, a U-shaped rigid bar with or without distal extensions, or single-ball anchors. Anatomic-morphologic and prosthetic variables were assessed related to the implant position and the type of overdenture retention. The degree of mandibular atrophy was determined using cephalometric radiographs. The angle beta between the virtual axis connecting both implants and the mandibular hinge axis was measured on mounted casts. A supporting surface was identified between bent clip bars and U-shaped extension bars. Clinical periimplant parameters were recorded, and changes of the probing attachment level after 5 years were analyzed in 70 patients with respect to the retention device and anatomic-morphologic and prosthetic variables. RESULTS: A significant increase of the Plaque Index was observed after 5 years. The mean clinical attachment loss after 5 years was about 0.2 mm. In more than 50%, beta was small, i.e., < or = 5 degrees. For these implants, loss of clinical attachment was significantly lower. The correlation between attachment loss and beta was weak, while no significant correlation was found with regard to the degree of atrophy and surface. CONCLUSION: In view of the long observation period, it was concluded that the position and retention mechanism of mandibular implants supporting an overdenture have little influence on the long-term stability of the clinical parameters.

Effectiveness of maxillary overdentures supported by implants: maintenance and prosthetic complications.

PURPOSE: The aim of this study was to evaluate the prosthetic complications with implant-supported overdentures in the maxilla. MATERIALS AND METHODS: Forty-one patients (mean age 61 years) were consecutively admitted for treatment from 1991 to 1998. A total of 173 ITI implants were placed. Four to six implants either connected with a bar (34 overdentures) or with single anchors (seven overdentures) supported the denture, and only a few parties had fewer than four implants. The overdentures had a horseshoe design and were reinforced by a cast-metal framework. The mean observation time was 3.2 years. Oral hygiene and periimplant parameters were regularly assessed, and records were kept of prosthetic maintenance service. All prosthetic complications encountered were classified related to (1) implant components and anchorage devices, (2) mechanical and structural failures of dentures, or (3) denture-related adjustments. RESULTS: Three implants did not osseointegrate, and five implants were lost after loading. Thus, the overall survival rate of the implants was 95.5%. Altogether, 85 prosthetic complications were encountered. The most frequent finding was retightening of the bar screw and adjustments of the bar retainers. Repair of dentures was not frequent and was mostly related to broken teeth. No fracture of dentures was observed. Renewal of dentures occurred twice, once after loss of all implants in one patient. Thirty-nine overdentures had been continuously worn; thus, the overall denture stability was 95%. Mucosal irritation and need for occlusal adjustment were the most frequent findings in the first year. Over time, a decrease of complications was observed. CONCLUSION: Planned maxillary overdentures supported by implants are a successful treatment modality on a short-term basis.

Inhibitors of HMG-CoA reductase sensitize human smooth muscle cells to Fas-ligand and cytokine-induced cell death.

Hydroxymethylglutaryl CoA (HMG CoA) reductase inhibitors, or statins, have been shown to reduce atherosclerotic cardiovascular morbidity and mortality. Atherosclerotic plaque lesions can be chronically inflamed and vulnerable to rupture or stable and less rupture-prone. Human smooth muscle cells (SMC) are critically important in maintaining the stability of atherosclerotic plaques. This stability may be greatly influenced by pro-inflammatory mediators such as IFN-gamma, TNF-alpha, and Il-1beta and Fas ligand (FasL) that are present in human atheroma. The purpose of the present study was to examine the effect of the statins on apoptosis of SMC. We have found that SMC are normally resistant to Fas or cytokine-induced apoptosis, but can be sensitized to these agents with pharmacological concentrations of some statins. Simvastatin and lovastatin strongly sensitized the cells to apoptotic agents while atorvastatin was less effective. In contrast to the lipophilic statins, the hydrophilic statin pravastatin did not induce this sensitization of SMC to apoptosis. Treatment of SMC with either mevalonate, the product of the HMG-CoA reductase, or geranylgeranylpyrophosphate, a down stream intermediate, prevented lipophilic statin-induced sensitization to apoptosis. These results suggest that prenylation of one or more proteins is critically involved in regulating the sensitivity of SMC to apoptotic stimuli. Our data support the emerging evidence that through this pathway the various statins may have effects which are beyond a simple lowering of the levels of circulating cholesterol.

Constitutive expression of functional 4-1BB (CD137) ligand on carcinoma cells.

Members of the TNF superfamily, including Fas, Fas ligand, and CD40, have been shown to be expressed on tumor cells. In the studies described in this work, we report that another family member, the ligand for 4-1BB (CD137), is expressed on various human carcinoma cell lines, on cells of solid tumors derived from these cell lines, and cells obtained from human tumors. Expression of 4-1BB ligand (4-1BBL) mRNA was detected by both RT-PCR and Northern blot analysis, and expression of 4-1BBL protein was detected by Western blot analysis of whole cell lysates and by FACS analysis of tumor cells and cell lines. Incubation of tumor cells with a 4-1BB-Ig fusion protein led to the production of IL-8 by the cells, demonstrating that the 4-1BBL is functionally active and signals back into the tumor cells. Furthermore, 4-1BBL expressed on the carcinoma cells functioned as a costimulatory molecule for the production of cytokines (most notably IFN-gamma) in cocultures of T cells and tumor cells. These findings suggest that 4-1BBL expressed on carcinoma cells may significantly influence the outcome of a T cell-tumor cell interaction.

Promotion of activated human B cell apoptosis and inhibition of Ig production by soluble CD95 ligand: CD95-based downregulation of Ig production need not culminate in activated B cell death.

CD95/CD95L interactions are vital to normal lymphoid homeostasis and in the protection against autoimmunity. To directly assess the effects of CD95L on activated B cell survival and Ig responses, purified human peripheral blood B cells, activated in vitro with SAC + rIL2, were incubated with a soluble CD95L fusion protein (fp) and assayed for apoptosis and IgG/IgM production. CD95L fp reproducibly increased apoptosis of these activated B cells and inhibited their Ig production. However, CD95L fp-mediated effects on activated B cell survival could be uncoupled from those on Ig production in that a soluble CD40L fp was incapable of reversing CD95L fp-mediated downregulation of Ig responses despite inhibiting CD95L fp-mediated apoptosis. Moreover, despite the specific caspase-8 inhibitor z-IETD-fmk substantially protecting transformed CL-01 B cells from CD95L fp-mediated apoptosis and permitting their ongoing proliferation, caspase-8 inhibition had no protective effects on CD95L fp-mediated inhibition of constitutive IgM production by CL-01 B cells. Collectively, these results point to a CD95-based downregulatory pathway in activated B cells that need not necessarily culminate in their death.

Differential susceptibility to CD95 (Apo-1/Fas) and MHC class II-induced apoptosis during murine dendritic cell development.

Disappearance of antigen presenting cells (APC) from the lymph node occurs following antigen specific interactions with T cells. We have investigated the regulation of CD95 (Apo-1/Fas) induced apoptosis during murine dendritic cell (DC) development. Consistent with the moderate levels of CD95 surface expression and low, or absent, MHC class II expression, immature DC in bone marrow cultures were highly sensitive to CD95 induced apoptosis, but insensitive to class II mediated apoptosis. In contrast, mature splenic, epidermal and bone marrow derived DC were fully resistant to CD95 induced cell death, but sensitive to class II induced apoptosis. Although caspase 3 and 8 activation was detected in immature DC undergoing CD95L-induced apoptosis, the pan-caspase inhibitor zVAD-fmk did not inhibit the early events of CD95-induced mitochondrial depolarisation or phosphatidyl serine exposure and only partially inhibited the killing of immature DC. In contrast, zVAD-fmk was completely effective in preventing CD95L mediated death of murine thymocytes. Collectively, these data do not support a major role of CD95: CD95L ligation in apoptosis of mature DC, but rather emphasise the existence of distinct pathways for the elimination of DC at different stages of maturation.

Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS).

The goals of this study were to determine whether the Fas-dependent apoptosis pathway is active in the lungs of patients with the acute respiratory distress syndrome (ARDS), and whether this pathway can contribute to lung epithelial injury. We found that soluble Fas ligand (sFasL) is present in bronchoalveolar lavage (BAL) fluid of patients before and after the onset of ARDS. The BAL concentration of sFasL at the onset of ARDS was significantly higher in patients who died. BAL from patients with ARDS induced apoptosis of distal lung epithelial cells, which express Fas, and this effect was inhibited by blocking the Fas/FasL system using three different strategies: anti-FasL mAb, anti-Fas mAb, and a Fas-Ig fusion protein. In contrast, BAL from patients at risk for ARDS had no effect on distal lung epithelial cell apoptosis. These data indicate that sFasL is released in the airspaces of patients with acute lung injury and suggest that activation of the Fas/FasL system contributes to the severe epithelial damage that occurs in ARDS. These data provide the first evidence that FasL can be released as a biologically active, death-inducing mediator capable of inducing apoptosis of cells of the distal pulmonary epithelium during acute lung injury.

Superantigen-driven, CD8+ T cell-mediated down-regulation: CD95 (Fas)-dependent down-regulation of human Ig responses despite CD95-independent killing of activated B cells.

Staphylococcal superantigens, including staphylococcal enterotoxin B (SEB), promote vigorous T cell-dependent Ig responses at low dose (0.01 ng/ml). In contrast, more mitogenic high dose SEB (100 ng/ml) profoundly inhibits the Ig responses. To assess the contribution of CD8+ T cells to this inhibition, high dose SEB-dependent killing of activated B cells and down-regulation of Ig responses were determined. Rapid killing (4 h) of activated B cells was effected by high dose SEB-activated CD8+ T cells (CD8*), but not by high-dose SEB-activated CD4+ T cells (CD4*), and required the presence of high dose SEB during the cytotoxicity assay. This killing was abrogated by chelation of extracellular calcium or by treatment with concanamycin A but was only modestly affected by treatment with brefeldin A, suggesting a perforin-based pathway of killing. Despite their widely disparate abilities to rapidly kill activated B cells, CD8* and CD4* demonstrated similar quantitative abilities to effect high dose SEB-dependent down-regulation of Ig responses. Antagonist anti-CD95 mAb substantially reversed high dose SEB-dependent downregulation effected by CD8* but had no appreciable effects on high dose SEB-dependent killing of activated B cells. These observations strongly suggest that the small fraction of activated B cells that secrete Ig are selectively sensitive to CD95-based killing but resistant to CD95-independent killing. This finding may help explain why clinical autoimmunity associated with increased titers of autoantibodies is a predominant feature of defects in CD95 or CD95 ligand.

A role for tumour necrosis factor-alpha, Fas and Fas-Ligand in marrow failure associated with myelodysplastic syndrome.

Apoptosis of haemopoietic cells in the marrow of patients with myelodysplastic syndrome (MDS) has been suggested as a mechanism for peripheral cytopenias. We determined the expression of Fas (CD95), Fas-Ligand (Fas-L) and TNF-alpha factors known to be involved in apoptosis, in the marrow of 44 patients with MDS and characterized their functional relevance in in vitro assays of haemopoiesis. Multidimensional flow cytometry revealed phenotypically aberrant blasts as defined by orthogonal light scatter and CD45 expression in the marrow of 24/44 patients. Among those blasts Fas expression was increased on CD34-positive cells and on cells co-expressing HLA-DR. In addition, Fas-L was expressed on some CD34+ cells of MDS patients but was never detected on CD34+ cells in normal marrow. Fas and Fas-L mRNAs as well as mRNA for TNF-alpha, known to increase Fas expression in normal marrow, were up-regulated in patients with MDS. TNF-alpha protein and sTNF-R1 levels in marrow plasma were higher in MDS patients than in controls (P<0.002 and <0.003, respectively). However, results were dependent upon disease category: TNF-alpha levels were significantly higher in patients with refractory anaemia (RA) than in patients with RA with excess blasts (RAEB) or RAEB in transformation (RAEB-T) (P=0.043). Conversely, the proportion of Fas-L-positive cells was lowest in patients with RA (P=0.037). In marrow cultures, Fas-Ig, rhuTNFR:Fc or anti-TNF-alpha antibody, by blocking Fas or TNF mediated signals, respectively, significantly increased the numbers of haemopoietic colonies compared to untreated cells (P<0.001, P<0.003, P<0.001, respectively). These results show significant dysregulation in the expression of TNF-alpha, Fas and Fas-L in the marrow from MDS patients. Altered expression of these molecules appears to be of functional relevance in the dysregulation of haemopoiesis in MDS and may be amenable to therapeutic interventions.

CD95 (Fas)-based, superantigen-dependent, CD4+ T cell-mediated down-regulation of human in vitro immunoglobulin responses.

Naturally occurring microbial superantigens (SAg) have been implicated in several human idiopathic disorders, and a compelling argument for the role of SAg in autoantibody-associated disorders, such as systemic lupus erythematosus, has been proposed. To test the effects of SAg on human in vitro Ig responses, CD4+ T cell + B cell cultures were stimulated with graded doses of staphylococcal enterotoxin B (SEB). Ig-secreting cell (IgSC) responses were very weak in CD4+ T cell + B cell cultures stimulated with SEB at the optimal mitogenic concentration (high dose SEB; 100 ng/ml) but were strong in parallel cultures stimulated with low dose SEB (0.01 ng/ml). High dose SEB actually enhanced B cell differentiation in the presence of CD4+ T cell soluble helper factors as long as the B cells were prevented from physically contacting the CD4+ T cells. However, when cell-cell contact between CD4+ T cells and B cells was permitted, high dose, but not low dose, SEB promoted increased CD4+ T cell-mediated B cell apoptosis with resulting decreases in viable CD20+ B cells and IgSC. High dose, but not low dose, SEB triggered increased levels of soluble CD95 ligand, and down-regulation of IgSC responses and incremental apoptosis of activated B cells were prevented by antagonist anti-CD95 mAb. This strongly suggests that CD4+ T cell-mediated CD95-based killing of activated B cells plays a major role in controlling SEB-driven IgSC responses. Defects in SAg-based down-regulation may contribute to autoimmune disorders such as SLE.

Analysis of the ligand binding site in Fas (CD95) by site-directed mutagenesis and comparison with TNFR and CD40.

Fas and its ligand (FasL) are members of the tumor necrosis factor receptor (TNFR) and tumor necrosis factor (TNF) superfamilies, respectively. Fas-FasL interactions trigger controlled cell death (apoptosis) in the immune system and thus play a key role in the regulation of immune responses. Structural details of the Fas-Fas ligand interaction are currently unknown. Previously, six Fas residues were identified by mutagenesis as important for ligand binding. We have now extended our mutagenesis analysis and identified additional residues which contribute to the Fas-FasL interaction. Candidate and control residues were selected based on a molecular model of the Fas extracellular region. Although residues in all three extracellular domains were identified to contribute to binding, the Fas-FasL interaction is centered on the second TNFR-like domain. Important residues were compared to critical positions in TNFR and CD40, another member of the TNFR family.