The tumor necrosis factor (TNF) superfamily of cytokines includes both soluble and membrane-bound proteins that regulate immune responses. A member of the human TNF family, BLyS (B lymphocyte stimulator), was identified that induced B cell proliferation and immunoglobulin secretion. BLyS expression on human monocytes could be up-regulated by interferon-gamma. Soluble BLyS functioned as a potent B cell growth factor in costimulation assays. Administration of soluble recombinant BLyS to mice disrupted splenic B and T cell zones and resulted in elevated serum immunoglobulin concentrations. The B cell tropism of BLyS is consistent with its receptor expression on B-lineage cells. The biological profile of BLyS suggests it is involved in monocyte-driven B cell activation.

The interleukin-1 receptor (IL-1R) signaling pathway leads to nuclear factor kappa B (NF-kappaB) activation in mammals and is similar to the Toll pathway in Drosophila: the IL-1R-associated kinase (IRAK) is homologous to Pelle. Two additional proximal mediators were identified that are required for IL-1R-induced NF-kappaB activation: IRAK-2, a Pelle family member, and MyD88, a death domain-containing adapter molecule. Both associate with the IL-1R signaling complex. Dominant negative forms of either attenuate IL-1R-mediated NF-kappaB activation. Therefore, IRAK-2 and MyD88 may provide additional therapeutic targets for inhibiting IL-1-induced inflammation.

DR3 is a death domain-containing receptor that is upregulated during T cell activation and whose overexpression induces apoptosis and NF-kappaB activation in cell lines. Here we show that an endothelial cell-derived TNF-like factor, TL1A, is a ligand for DR3 and decoy receptor TR6/DcR3 and that its expression is inducible by TNF and IL-1alpha. TL1A induces NF-kappaB activation and apoptosis in DR3-expressing cell lines, while TR6-Fc protein antagonizes these signaling events. Interestingly, in T cells, TL1A acts as a costimulator that increases IL-2 responsiveness and secretion of proinflammatory cytokines both in vitro and in vivo. Our data suggest that interaction of TL1A with DR3 promotes T cell expansion during an immune response, whereas TR6 has an opposing effect.

IntroductionThe tumor necrosis factor (TNF) family of ligands encompasses an ever-growing group of proteins, characterized by homologous cysteine-rich domains, that participates in the regulation of diverse immune and inflammatory responses. [1][2][3][4] All the members, with the exception of lymphotoxin-␣, are type II membrane proteins. Their effects are mediated either by cell contact, through the interaction of the membrane-bound form of the ligand with its corresponding receptor, or by processing and shedding of the soluble form of the ligand. 2,[5][6][7] In addition, many of the proteins, including CD27L, CD30L, OX40L, CD40L, FasL, and 4-1BBL, have moderate-sized cytoplasmic regions that are capable of delivering signals when engaged by their receptors. [7][8][9][10] The expression pattern of the family members is usually promiscuous, ranging from the broad cellular expression of TNF-␣ to a more restricted localization, such as that of CD40L expressed only on T cells. Moreover, the expression of the molecules is, in general, dependent on the activation state of the cells, being usually low or undetectable on resting cells.Recently, we described a novel member of the TNF family of ligands, B-lymphocyte stimulator (BLyS), which was identified by searching an expressed sequence tag (EST) database for homology with known TNF-like molecules. 11 The protein has been reported also as TALL-1 (TNF-and ApoL-related leukocyte-expressed ligand 1), BAFF (B-cell activator factor belonging to the TNF family), or THANK (TNF homologue that activates apoptosis, NF-B, and JNK). 12-14 The human BLyS gene encodes for a 285 amino acid (aa) protein presenting a transmembrane region between aa 47 and 73 and lacking a putative signal peptide sequence. The recombinant soluble protein (aa 134-285) binds selectively to human primary B cells and tumor cell lines of the B lineage. 11BLyS was shown to induce B-cell proliferation in standard costimulation assays with Staphylococcus aureus Cowan I (SAC I) or antihuman immunoglobulin M (IgM). BLyS administration in mice resulted in a 5-and 2-fold increase in serum IgM and IgA, respectively. 11 In addition, mice transgenic for BAFF developed autoimmune disorders such as increased germinal center formation, production of autoantibodies, and Ig deposition in kidneys. 15 Collectively, these findings suggest that BLyS has a crucial role in the humoral immune response and that regulation of BLyS expression might consequently modulate B-cell function. Our aim was, therefore, to study synthesis and release of BLyS from cells of myeloid lineage and to investigate the regulation of BLyS expression in response to cytokines. Materials and methods Medium and reagentsThe complete medium used for monocyte culture consisted of RPMI 1640 medium (Gibco BRL, Rockville, MD) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 2 M L-glutamine, and 50 g/mL gentamycin (Biofluids, Rockville, MD). The following recombinant human (rh) reagents were used: interferon-␥ (rhIFN-␥), interleukin-10 (...

High throughput cDNA sequencing has led to the identification of interferon-, a novel subclass of type I interferon that displays ϳ30% homology to other family members. Interferon-consists of 207 amino acids, including a 27-amino acid signal peptide and a series of cysteines conserved in type I interferons. The gene encoding interferon-is located on the short arm of chromosome 9 adjacent to the type I interferon gene cluster and is selectively expressed in epidermal keratinocytes. Expression of interferon-is significantly enhanced in keratinocytes upon viral infection, upon exposure to double-stranded RNA, or upon treatment with either interferon-␥ or interferon-␤. Administration of interferon-recombinant protein imparts cellular protection against viral infection in a species-specific manner. Interferon-activates the interferon-stimulated response element signaling pathway and a panel of genes similar to those regulated by other type I interferons including anti-viral mediators and transcriptional regulators. An antibody that neutralizes the type I interferon receptor completely blocks interferon-signaling, demonstrating that interferon-utilizes the same receptor as other type I interferons. Interferon-therefore defines a novel subclass of type I interferon that is expressed in keratinocytes and expands the repertoire of known proteins mediating host defense. Interferons (IFNs)1 are a family of functionally related cytokines that confer a range of cellular responses including antiviral, antiproliferative, antitumor, and immunomodulatory activities (1, 2). They are classified as type I or type II according to their structural and functional properties. Although the sole member of the type II family is IFN-␥, there are multiple members of the type I interferon class, which is divided into the IFN-␣, IFN-␤, and IFN-subclasses (1, 2). In humans, excluding psuedogenes, there are 13 non-allelic IFN-␣ genes, a single ␤ gene, and a single gene. Members of the IFN-␣ family display greater than 80% identity to each other, IFN-displays ϳ60% identity to IFN-␣, and IFN-␤ is ϳ40% identical to the other family members. The evolutionary conservation of the type I IFN genes is reflected in their common intron-less structure and their co-localization to the short arm of chromosome 9, which suggest that type I IFNs arose by gene duplication (3). The subtypes were initially categorized further by their cell of origin. IFN-␣ and IFN-genes were thought to be produced predominantly by leukocytes and IFN-␤ by fibroblasts. However, upon appropriate induction, most human cell types can generate type I IFNs (2). Exposure to a variety of agents triggers the rapid and transient production of type I IFNs, with viruses being the most efficient natural inducers (4, 5). Certain bacteria can also induce expression, as can double-stranded RNA (dsRNA) and endotoxin. In contrast, trophoblast IFNs or IFN-, which are found only in ruminant ungulate species, are not induced by viral challenge (6). These genes are expressed by the embryonic trophoecto...

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We also demonstrate that TACI interacts with nanomolar affinity with the BLyS-related tumor necrosis factor homologue APRIL for which no clear in vivo role has been described. BLyS and APRIL are capable of signaling through TACI to mediate NF-B responses in HEK293 cells. We conclude that TACI is a receptor for BLyS and APRIL and discuss the implications for B-cell biology.Members of the tumor necrosis factor superfamily of cytokines play diverse roles in the regulation of cell proliferation, differentiation, and survival. Notably, several members of this family play key roles in the regulation of the immune system (1). We and others have previously identified a novel TNF 1 -related ligand, BLyS (also known as BAFF, TALL-1, THANK, TNFSF20, and zTNF4) which is expressed on monocytes and induces B-cell proliferation and immunoglobulin secretion in vitro and in vivo (2-6). Like many members of the TNF family, BLyS has activity in vitro as a 152-amino acid soluble molecule and as a 258-amino acid transmembrane form (3). However, the biological significance of these two forms and their relative contributions in vivo remain to be resolved. More recently, transgenic mice that ectopically overexpress BLyS were shown to develop autoimmune-like phenotypes reminiscent of those observed in systemic lupus erythematosus (7-9). These findings suggest that BLyS plays an important role in the regulation of B-cell growth and humoral immunity.In order to understand the precise mechanism by which BLyS activates B-cells, the range of cell types BLyS may affect, and the potential role of BLyS as a therapeutic agent or target, we have used expression cloning to identify the receptor for BLyS. We have identified the orphan receptor TACI (10), previously characterized as being present on B-cells and a subset of T-cells, as the receptor for BLyS and show that this receptor is capable of mediating NF-B signaling in response to ligand binding. We also show that TACI interacts with another TNF family member, APRIL, which is closely related to BLyS. Parallel work by others has recently shown that TACI and a second TNFR family member, BCMA, are BLyS receptors (9, 11-14). EXPERIMENTAL PROCEDURESCell Culture and Media-HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and transfected using LipofectAMINE Plus (Life Technologies, Gaithersburg, MD) according to the manufacturer's protocol. For expression cloning screens, cells were attached to plates with poly-D-lysine.Flow Cytometry-Cells were stained with monoclonal antibodies raised against BLyS at the indicated protein concentrations, with biotinylated BLyS as described previously (2), with recombinant TACI-Fc fusion protein or with recombinant Flag-tagged proteins which were subsequently detected by the M2 anti-Flag monoclonal antibody (Sigma). Flow cytometry was performed using a FACScan instrument and associated CellQuest software (Becton Dickinson, San Jose, CA).Library Preparation, Screening, and Other DNA Manipulations-All common DNA...

The propensity score method is widely used in clinical studies to estimate the effect of a treatment with two levels on patient's outcomes. However, due to the complexity of many diseases, an effective treatment often involves multiple components. For example, in the practice of Traditional Chinese Medicine (TCM), an effective treatment may include multiple components, e.g. Chinese herbs, acupuncture, and massage therapy. In clinical trials involving TCM, patients could be randomly assigned to either the treatment or control group, but they or their doctors may make different choices about which treatment component to use. As a result, treatment components are not randomly assigned. Rosenbaum and Rubin proposed the propensity score method for binary treatments, and Imbens extended their work to multiple treatments. These authors defined the generalized propensity score as the conditional probability of receiving a particular level of the treatment given the pre-treatment variables. In the present work, we adopted this approach and developed a statistical methodology based on the generalized propensity score in order to estimate treatment effects in the case of multiple treatments. Two methods were discussed and compared: propensity score regression adjustment and propensity score weighting. We used these methods to assess the relative effectiveness of individual treatments in the multiple-treatment IMPACT clinical trial. The results reveal that both methods perform well when the sample size is moderate or large.