Lymphotoxin-beta receptor signaling complex: role of tumor necrosis factor receptor-associated factor 3 recruitment in cell death and activation of nuclear factor kappaB.

The binding of heterotrimeric lymphotoxin, LT alpha1 beta2, to the LTbeta receptor (LTbeta R), a member of the tumor necrosis factor receptor (TNFR) superfamily, induces nuclear factor kappaB (NF-kappaB) activation and cell death in HT29 adenocarcinoma cells. We now show that treatment with LT alpha1 beta2 or agonistic LTbeta R antibodies causes rapid recruitment of TNFR-associated factor 3 (TRAF3) to the LTbeta R cytoplasmic domain. Further, stable overexpression of a TRAF3 mutant that lacks the RING and zinc finger domains inhibits LTbeta R-mediated cell death. The inhibition is specific for LTbeta R cell death signaling, since NF-kappaB activation by LT alpha1 beta2 and Fas-mediated apoptosis are not inhibited in the same cells. The mutant and endogenous TRAF3s are both recruited at equimolar amounts to the LTbeta R, suggesting that the mutant disrupts the function of the signaling complex. These results implicate TRAF3 as a critical component of the LTbeta R death signaling complex and indicate that at least two independent signaling pathways are initiated by LTbeta R ligation.

Apoptosis mediated by the TNF-related cytokine and receptor families.

T lymphocytes use several specialized mechanisms to induce apoptotic cell death. The tumor necrosis factor (TNF)-related family of membrane-anchored and secreted ligands represent a major mechanism regulating cell death and cell survival. These ligands also coordinate differentiation of tissue to defend against intracellular pathogens and regulate development of lymphoid tissue. Cellular responses are initiated by a corresponding family of specific receptors that includes two distinct TNFR (TNFR60 and TNFR80), Fas (CD95), CD40, p75NTF, and the recently identified lymphotoxin beta-receptor (LT beta R), among others. The MHC-encoded cytokines, TNF and LT alpha, form homomeric trimers, whereas LT beta assembles into heterotrimers with LT alpha, creating multimeric ligands with distinct receptor specificities. The signal transduction cascade is initiated by transmembrane aggregation (clustering) of receptor cytoplasmic domains induced by binding to their multivalent ligands. The TRAF family of Zn RING/finger proteins bind to TNFR80; CD40 and LT beta R are involved in induction NF kappa B and cell survival. TNFR60 and Fas interact with several distinct cytosolic proteins sharing the "death domain" homology region. TNF binding to TNFR60 activates a serine protein kinase activity and phosphoproteins are recruited to the receptor forming a multicomponent signaling complex. Thus, TNFRs use diverse sets of signaling molecules to initiate and regulate cell death and survival pathways.

TNF receptor signal transduction. Ligand-dependent stimulation of a serine protein kinase activity associated with (CD120a) TNFR60.

TNF is a pluripotent cytokine that mediates activities through two distinct receptors of 55 to 60 kDa (CD120a, known as TNFR60) and 75 to 80 kDa (CD120b, known as TNFR80). These receptors share homology in the extracellular ligand binding region; however, the cytoplasmic domains are distinct and lack any inherent enzymatic activity, which suggests that ligand binding and subsequent receptor clustering leads to the association of active signaling molecules with TNFRs. To test this hypothesis, we isolated TNFRs by immunoprecipitation and examined the immune complexes for the presence of associated phosphoproteins and protein kinase activity. In the U-937 monocytic cell line, prelabeled with 32PO4, TNF induces the association of several phosphoproteins with TNFR60, but not TNFR80. The TNFR60 immune complexes also contain a TNF-dependent serine protein kinase activity, which was detected by an in vitro kinase assay, that phosphorylates proteins of 125, 97, 85, and 60 kDa, which are of apparent molecular masses that are similar to those of TNF-induced phosphoproteins that coprecipitate with TNFR60. Association of serine protein kinase activity with TNFR60 is rapid and dependent on the concentration of TNF. Proteins of molecular mass similar to the 125- and 97-kDa protein kinase substrates seem to be associated with TNFR60 immune complexes only after exposure of U-937 cells to TNF. The TNFR60-associated protein kinase activity is inhibited by staurosporine, but not by the protein kinase A and C inhibitors, HA-1004 and H7. Staurosporine greatly enhanced the sensitivity of U-937 cells to the cytotoxic effect of TNF. These results suggest a serine protein kinase(s), and, possibly, other TNF-dependent TNFR60-associated proteins may be involved in mediating signals through TNFR60 in response to ligand binding.

A lymphotoxin-beta-specific receptor.

Tumor necrosis factor (TNF) and lymphotoxin-alpha (LT-alpha) are members of a family of secreted and cell surface cytokines that participate in the regulation of immune and inflammatory responses. The cell surface form of LT-alpha is assembled during biosynthesis as a heteromeric complex with lymphotoxin-beta (LT-beta), a type II transmembrane protein that is another member of the TNF ligand family. Secreted LT-alpha is a homotrimer that binds to distinct TNF receptors of 60 and 80 kilodaltons; however, these receptors do not recognize the major cell surface LT-alpha-LT-beta complex. A receptor specific for human LT-beta was identified, which suggests that cell surface LT may have functions that are distinct from those of secreted LT-alpha.

Production of lymphotoxin (LT alpha) and a soluble dimeric form of its receptor using the baculovirus expression system.

Human LT alpha and a fusion protein (p60:Fc) comprised of the extracellular domain of the 60 kDa TNF receptor (TNFR60) fused to the Fc portion of human IgG1 were produced in insect cells infected with recombinant baculoviruses. The p60:Fc fusion produced in insect cells accumulates in culture supernatants to levels > 2 mg/l. Purified p60:Fc binds human TNF and LT alpha with high affinity (200-600 pM) and neutralizes TNF cytolytic activity at equimolar stoichiometric concentration. The data show that p60:Fc is an effective ligand-precipitating reagent which recognizes recombinant LT alpha produced in mammalian or insect cells and naturally occurring LT alpha produced in T cells. The levels of human LT alpha produced in baculovirus-infected insect cells is estimated to be approximately 20 mg/l. Insect cell-derived human LT alpha is biologically active in an L929 cytotoxicity assay and is efficiently neutralized by p60:Fc. These data demonstrate that the baculovirus system is useful for overexpressing biologically active LT alpha and p60:Fc and therefore, may be applicable to other oligomeric cytokines and soluble dimeric cytokine receptors.

Specific induction of 80-kDa tumor necrosis factor receptor shedding in T lymphocytes involves the cytoplasmic domain and phosphorylation.

The 80-kDa TNFR (TNFR80) expressed by activated human T cells is constitutively phosphorylated and undergoes limited proteolytic cleavage (shedding) at the cell surface releasing a 40-kDa soluble TNF-binding protein. Triggering of activated T cells through the TCR rapidly increased the rate of TNFR80 shedding > 20-fold more than nonstimulated cells, demonstrating that shedding is a specific, inducible process. The protein kinase inhibitor staurosporine inhibited constitutive phosphorylation and blocked inducible shedding of TNFR80, suggesting that phosphorylation may be important for cleavage of the extracellular domain. However, a deletion mutation of the entire cytoplasmic domain of human TNFR80 was shed when expressed in murine L929 cells, albeit relatively poorly compared with full length receptor. This demonstrates that the cytoplasmic domain is important but not essential for cleavage of the extracellular domain of TNFR80. Moreover, a requirement for phosphorylation of proteins other than the receptor was revealed by the finding that staurosporine completely blocked cleavage of the cytoplasmic deletion mutant. Collectively, these results demonstrate that protein phosphorylation is essential and the cytoplasmic domain is important for regulating the inducible production of soluble TNF-binding proteins by activated effector T cells.

Tumor necrosis factor (TNF) receptor expression in T lymphocytes. Differential regulation of the type I TNF receptor during activation of resting and effector T cells.

The expression of TNF-alpha receptors (TNFR) was examined on a CD4+ T cell hybridoma, transformed T cell lines, CTL clones, and activated T cells from peripheral blood to determine the basis of the immunomodulatory activity of TNF on T cell function. Analyses by ligand cross-linking and competitive binding assays with mAb to the 80-kDa receptor (TNFR-I), demonstrated that the TNFR-I was the predominant receptor expressed on activated CD4+ and CD8+ T cell subsets. However, on T cell leukemic lines, a second, non-TNFR-I binding site was identified, most likely the 55-kDa form (TNFR-II). Additional subsets of T cells were readily distinguished by their expression of TNFR-I and related members of the TNFR gene family (CD40 and CD27). Expression of the TNFR-I was dependent upon the state of T cell activation. Signaling through the TCR for Ag or IL-2R was sufficient to induce TNFR mRNA and protein expression in resting T cells. Multiple sizes of TNFR-I transcripts were detected during T cell activation; however, biosynthetic studies showed these multiple species encode a single protein of 80 kDa. These results, combined with the known ability of TNF to induce IL-2R expression, indicate that TNF and IL-2 form a reciprocating receptor amplification circuit. In contrast, differentiated effector T cells triggered through the TCR or protein kinase C initiated a rapid down-regulation (transmodulation) of the TNFR-I that preceded TNF or lymphotoxin secretion. The mechanism of transmodulation involved proteolytic processing of the mature 80-kDa receptor releasing a soluble 40-kDa fragment. This indicates that a TNF autocrine loop is not likely to form during the response of an effector T cell. Collectively, these results suggest that transcriptional and post-translational modification of the TNFR-I are important control points regulating the expression of this receptor during T cell activation.