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PubMed1996

Preparation and characterization of soluble recombinant heterotrimeric complexes of human lymphotoxins alpha and beta.

The lymphotoxin (LT) protein complex is a heteromer of alpha (LT-alpha, also called tumor necrosis factor (TNF)-beta) and beta (LT-beta) chains anchored to the membrane surface by the transmembrane domain of the LT-beta portion. Both proteins belong to the TNF family of ligands and receptors that regulate aspects of the immune and inflammatory systems. The LT complex is found on activated lymphocytes and binds to the lymphotoxin-beta receptor, which is generally present on nonlymphoid cells. The signaling function of this receptor-ligand pair is not precisely known but is believed to be involved in the development of the peripheral lymphoid organs. To analyze the properties of this complex, a soluble, biologically active form of the surface complex was desired. The LT-beta molecule was engineered into a secreted form and co-expressed with LT-alpha using baculovirus/insect cell technology. By exploiting receptor affinity columns, the LT-alpha3, LT-alpha2/beta1, and LT-alpha1/beta2 forms were purified. All three molecules were trimers, and their biochemical properties are described. The level of LT-alpha3-like components in the LT-alpha1/beta2 preparation was found to be 0.02% by following the activity of the preparation in a WEHI 164 cytotoxicity assay. LT-alpha3 with an asparagine 50 mutation (D50N) cannot bind the TNF receptors. Heteromeric LT complexes were prepared with this mutant LT- alpha form, allowing a precise delineation of the extent of biological activity mediated by the TNF receptors. A LT-alpha3 based cytotoxic activity was used to show that the LT-alpha1/beta2 form cannot readily scramble into a mixture of forms following various treatments and storage periods. This biochemical characterization of the LT heteromeric ligands and the demonstration of their stability provides a solid foundation for both biological studies and an analysis of the specificity of the LT-bet a and TNF receptors for the various LT forms.

Amino Acid Sequence↗
PubMed1997

Cytotoxic activities of recombinant soluble murine lymphotoxin-alpha and lymphotoxin-alpha beta complexes.

Human lymphotoxin-alpha (LT alpha) is found in a secreted form and on the surface of lymphocytes as a complex with a second related protein called lymphotoxin-beta (LT beta). Both secreted human LT alpha and TNF have similar biological activities mediated via the TNF receptors, whereas the cell surface LT alpha beta complex binds to a separate receptor called the LT beta receptor (LT beta R). The murine LT alpha and LT beta (mLT alpha and mLT beta) proteins have never been characterized. When recombinant mLT alpha was produced by either of several methods, the protein had a very low specific activity relative to that of human LT alpha in the conventional WEHI 164 cytotoxicity bioassay. The weak activity observed was inhibited by a soluble murine TNF-R55 Ig fusion protein (mTNF-R55-Ig), but not by mLT beta R-Ig. Coexpression of both mLT alpha and a soluble version of mLT beta in insect cells led to an LT alpha beta form that was cytotoxic in the WEHI 164 assay via the LT beta R. To determine whether natural mLT alpha-like forms with cytotoxic activity comparable to that of secreted human LT alpha were secreted from primary spleen cells, splenic lymphocytes were activated in various ways, and their supernatants were analyzed for cytotoxic activity. Using specific Abs to distinguish between mTNF and mLT, a TNF component was readily detected; however, there was no evidence for a secreted mLT alpha cytotoxic activity using this assay. Combined, these observations suggest that secreted mLT alpha may not play a role in the mouse via interactions with TNF-R55, and the ramifications of this hypothesis are discussed.

Amino Acid Sequence↗
PubMed1997

Human TNF receptor-associated factor 5 (TRAF5): cDNA cloning, expression and assignment of the TRAF5 gene to chromosome 1q32.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are signal transducers for members of the TNF receptor superfamily. We previously identified murine TRAF5 (mTRAF5) and showed that it specifically interacts with the lymphotoxin-beta receptor (LT-beta R) and activates the transcription factor NF-kappa B. Here we have cloned the human TRAF5 homologue (hTRAF5) by cross hybridization with mTRAF5 cDNA. hTRAF5 cDNA is composed of 2894 nucleotides with a 557-amino-acid open reading frame that exhibits 77.5 and 80% identity to mTRAF5 at the nucleotide and amino acid levels, respectively. Northern blot analysis revealed that hTRAF5 mRNA is expressed in all visceral organs. Western blotting revealed that hTRAF5 protein was abundantly expressed in the human follicular dentritic cell line, FDC-1, and to a much lesser degree in several tumor cell lines. Interspecific backcross mapping revealed that Traf5 is located in the distal region of mouse chromosome 1, which shares a region of homology with human chromosome 1q. Fluorescence in situ hybridization confirmed regional localization to human chromosome 1q32.

Amino Acid Sequence↗
PubMed1995

The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family.

The cytoplasmic C-terminus of Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) is essential for B lymphocyte growth transformation and is now shown to interact with a novel human protein (LMP1-associated protein 1 [LAP1]). LAP1 is homologous to a murine protein, tumor necrosis factor receptor-associated factor 2 (TRAF2), implicated in growth signaling from the p80 TNFR. A second novel protein (EBI6), induced by EBV infection, is the human homolog of a second murine TNFR-associated protein (TRAF1). LMP1 expression causes LAP1 and EBI6 to localize to LMP1 clusters in lymphoblast plasma membranes, and LMP1 coimmunoprecipitates with these proteins. LAP1 binds to the p80 TNFR, CD40, and the lymphotoxin-beta receptor, while EBI6 associates with the p80 TNFR. The interaction of LMP1 with these TNFR family-associated proteins is further evidence for their role in signaling and links LMP1-mediated transformation to signal transduction from the TNFR family.

Alternative Splicing↗
PubMed1996

Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region.

CD40 signalings play crucial roles in B-cell function. To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned. TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins. TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-beta receptor, and LMP1 of Epstein-Barr virus. Deletion analysis showed that residues 246-269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230-245 were required. Overexpression of TRAF6 activates transcription factor NFkappaB, and its TRAF-C domain suppresses NFkappaB activation triggered by CD40 lacking residues 246-277. These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230-245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.

Amino Acid Sequence↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1997

Identification of two lymphotoxin beta isoforms expressed in human lymphoid cell lines and non-Hodgkin's lymphomas.

Two isoforms of lymphotoxin beta (LTbeta) were isolated from mRNAs of a panel of human lymphoid cell lines and tumor tissues obtained from patients with non-Hodgkin's lymphoma (NHL). The truncated LTbeta mRNA variant lacked 46 base pairs complementary to the complete sequence of exon 2, suggesting that both isoforms are produced by an alternative splicing mechanism. Skipping out of exon 2 causes a reading frame shift and a premature stop codon in the LTbeta mRNA variant. The predictive translated polypeptide would correspond to a severely shortened LTbeta protein that would lack the majority of the extracellular domain of the native molecule, thus impairing its normal complex assembly with LTalpha. These observations provide new insights into the molecular heterogeneity and biological function of LTbeta within the tumor necrosis factor and LT ligand-receptor system.

Base Sequence↗
PubMed1996

Genes in a 220-kb region spanning the TNF cluster in human MHC.

A search for new genes was performed in a 220-kb region around the tumor necrosis factor gene cluster in the human central major histocompatibility complex region using a cDNA hybridization and selection method. In addition to the seven known genes in this region, we identified a new gene that is preferentially expressed in spleen. We also identified two pseudogenes that have high degrees of homology to cytokeratin and cyclophillin, respectively. Expressed sequences for a human homologue of the mouse B144 gene were also found in the current analysis. RT-PCR analysis showed that B144 is expressed in spleen, in thymus, and prominently in the macrophage cell line, U937. We also independently identified the BAT1 gene to be the well-conserved homologue of a previously described rat liver nuclear protein.

Amino Acid Sequence↗
PubMed1994

Characterization of six new loci within the swine major histocompatibility complex class III region.

A search for new potential coding sequences was conducted within two overlapping cosmid genomic DNA clusters of about 170 and 45 kb from the swine major histocompatibility complex class III region. The sequences were detected with various probes, including pools of swine cDNA, homologous and heterologous genomic sequences, and synthetic oligonucleotides. The 170 kb cluster was centered on the tumor necrosis factor genes (TNF), and the 45 kb cluster contained the heat-shock protein 70 genes (HSP70). The TNF cluster revealed the presence of five new genes: lymphotoxin beta, BAT1, BAT2, BAT3, and a sequence related to DNA-binding factors. No sequence homologous to B144 was found in the TNF cluster, although other unidentified coding sequences may be present in this cluster. The HSP70 cluster contained a gene identified as BAT6, that is, tRNA-valyl synthetase. These results provide new evidence that the genomic maps of these various genes in the TNF and HSP70 sub-regions are similar in swine and human.

Animals↗
PubMed1997

Analysis of the first intron of TNFB gene by NcoI RFLP in Koreans.

The tumor necrosis factor B (TNFB) gene is closely liked with tumor necrosis factor A (TNFA) gene between the HLA-B and C2 genes on chromosome 6p21.3. Several genetic variabilities at the human TNFB loci have been identified, which are the NcoI restriction fragment length polymorphism (RFLP) in the first intron, amino acid substitution at codon 26 of exon 3 and EcoRI RFLP in untranslated exon 4. The NcoI RFLP of TNFB gene gives two allelic fragments of 238/259 bp and 497 bp, corresponding to TNFB*1 and TNFB*2 alleles, respectively. To investigate the frequency of NcoI RFLP in the first intron of TNFB in Koreans and to compare to that of other ethnic population, genomic DNAs were extracted from leukocytes of 305 unrelated healthy Koreans and amplified the first intron of TNFB gene by PCR. The phenotype frequencies of NcoI RFLP such as TNFB* 1/TNFB*1, TNFB*1/TNFB*2 and TNFB*2/TNFB*2 were 8.6% (n = 26), 45.2% (n = 138) and 46.2% (n = 141), respectively. The estimated allele frequencies for TNFB*1 and TNFB*2 were 0.3115 and 0.6885, respectively. The observed and expected frequencies were in good agreement with the Hardy-Weinberg's equilibrium. The heterozygosity revealed 45.2% and the allele frequencies of NcoI RFLP of TNFB in Koreans were observed comparatively similar to those of other ethnic groups.

Asian People↗
PubMed1997

Antibodies to lymphotoxin alpha (LT alpha) and LT beta recognize different glial cell types in the central nervous system.

The cytokine lymphotoxin (LT) is known to exist in two forms, secreted LT alpha and a membrane-bound LT alpha/beta complex. LT alpha shares the same receptor as tumor necrosis factor alpha and LT beta is recognized by its receptor, LT betaR. Since LT has been associated with oligodendrocyte pathology, the present study has examined the expression of these molecules by immunocytochemistry in diseased and normal CNS tissue, with a panel of monoclonal antibodies (mAb) to LT alpha, LT beta and LT betaR. Of three mAb to LT beta, two (B27 and C37) gave specific membrane staining on astrocytes, as well as lymphocytes. The third anti-LT beta mAb, B9, was selectively immunoreactive for oligodendrocytes, suggesting specific recognition sites. The reactivity was not specific for multiple sclerosis (MS) since oligodendrocytes in normal and non-MS CNS tissue also displayed positivity. MAb to LT betaR reacted with astrocytes only, giving a punctate membrane staining pattern suggestive of receptor sites. MAb to LT alpha gave strong reactivity on lymphocytes in active MS lesions and weak reactivity on microglia within lesion areas. These results show that mAb to LT alpha and LT beta recognize different cell types within the CNS. Furthermore, individual mAb against LT beta were capable of distinguishing between astrocytes and oligodendrocytes, perhaps indicative of different epitopes on LT beta. The presence of LT betaR on astrocytes suggests possible interactions between infiltrating lymphocytes and astrocytes via the LT pathway.

Antibodies, Monoclonal↗
PubMed1997

Distinct roles in lymphoid organogenesis for lymphotoxins alpha and beta revealed in lymphotoxin beta-deficient mice.

Lymphotoxin alpha (LT alpha)-deficient mice revealed critical roles for LT alpha in lymphoid organogenesis, but it is not clear whether LT alpha functions through an LT alpha homotrimer (LT alpha3) or LT alpha/beta heterotrimers. We generated LTbeta-deficient mice and found them to lack Peyer's patches, peripheral lymph nodes, splenic germinal centers, and follicular dendritic cells. Unlike LT alpha-deficient mice, LT beta-deficient mice had cervical and mesenteric lymph nodes. Furthermore, the mesenteric lymph nodes had germinal center-like regions, although these structures appeared to lack follicular dendritic cells. The absence of cervical and mesenteric lymph nodes in LT alpha-deficient mice, and yet their presence in LT beta-deficient mice and in mice deficient in tumor necrosis factor receptor types I and II, suggest that LT alpha3 may signal via an as yet unidentified receptor.

Animals↗
PubMed1995

Cloning and expression analysis of the murine lymphotoxin beta gene.

Tumor necrosis factor alpha (TNF-alpha) and soluble lymphotoxin (LT) (also called LT-alpha or TNF-beta) are cytokines with similar biological activities that are encoded by related and closely linked genes. TNF-alpha, a mediator of the inflammatory response, exists in soluble and transmembrane forms. LT-alpha can be secreted or retained at the cell surface by binding to a 33-kDa transmembrane subunit, LT-beta. The recently cloned human LT-beta gene encodes another TNF family member and is linked to the TNF/LT locus within the major histocompatibility complex locus. The cell surface LT is a heterotrimer consisting of LT-alpha and LT-beta, whose physiological function is not yet clearly defined. We now report the sequence analysis of the genomic region and cDNA of murine LT-beta gene, which is closely associated with the TNF-alpha and LT-alpha genes within the murine major histocompatibility complex locus. Unlike the TNF-alpha, LT-alpha, and human LT-beta genes, which contain four exons, the murine LT-beta contains three exons and encodes a 244-amino acid polypeptide with a 66-amino acid insert that is absent from the human homologue. In situ hybridization demonstrates constitutive expression of LT-beta in lymphoid and hematopoietic tissues. LT-beta transcription is maximal in the thymic medulla and in splenic white pulp. LT-beta mRNA is also detected in the skin and in specific regions of the brain. The LT-beta promoter region contains putative Ets-binding sites, suggesting that the expression of LT-beta may be regulated in part by Ets transcription factors whose pattern of lymphoid expression overlaps that of LT-beta.

Amino Acid Sequence↗
PubMed1997

Abnormal development of secondary lymphoid tissues in lymphotoxin beta-deficient mice.

The tumor necrosis factor (TNF) family cytokines lymphotoxin (LT) alpha and LTbeta form heterotrimers that are expressed on the surface of activated lymphocytes and natural killer cells; LTalpha homotrimers can be secreted as well. Mice with a disrupted LTalpha gene lack lymph nodes (LN), Peyer's patches (PP), and follicular dendritic cell (FDC) networks and reveal profound defects of the splenic architecture. However, it is unclear which of these abnormalities is the result of the absence in LTalpha homotrimers or LTalphabeta heterotrimers. To distinguish between these two possibilities, a mouse strain deficient in LTbeta was created employing Cre/loxP-mediated gene targeting. Mice deficient in LTbeta reveal severe defects in organogenesis of the lymphoid system similar to those of LTalpha-/- mice, except that mesenteric and cervical LN are present in most LTbeta-deficient mice. Both LTbeta- and LTalpha-deficient mice show significant lymphocytosis in the circulation and peritoneal cavity and lymphocytic infiltrations in lungs and liver. After immunization, PNA-positive B cell clusters were detected in the splenic white pulp of LTbeta-deficient mice, but FDC networks were severely underdeveloped. Collectively, these results indicate that LTalpha can signal independently from LTbeta in the formation of PNA-positive foci in the spleen, and especially in the development of mesenteric and cervical LN.

Animals↗
PubMed1994

CD40 ligation induces lymphotoxin alpha gene expression in human B cells.

CD40 plays an important role in T cell mediated B cell proliferation and isotype switching. The cytokines tumor necrosis factor (TNF)-alpha and lymphotoxin (LT)-alpha are expressed by B cells, and are known to play a role in B cell activation. We have studied TNF-alpha and LT-alpha expression in human tonsillar B cells following stimulation with anti-CD40 mAb. Anti-CD40 induced weak TNF-alpha mRNA expression but strong LT-alpha mRNA expression and had little effect on the constitutive expression of LT-beta mRNA in B cells. Induction of TNF-alpha mRNA was inhibited by actinomycin D suggesting that CD40 ligation results in transcriptional activation of the TNF-alpha and LT-alpha genes. Anti-CD40 caused minimal increase in the expression of TNF-alpha on the B cell membrane and no detectable secretion of TNF-alpha. Anti-CD40 as well as soluble CD40 ligand caused sustained induction of LT-alpha on the membrane of the B cells lasting up to 120 h but induced no detectable secretion of LT-alpha. IL-4, a cytokine known to synergize with anti-CD40 in inducing B cell proliferation and isotype switching, augmented the induction of LT-alpha mRNA and of mLT-alpha expression by anti-CD40. These results indicate that CD40 ligation vigorously induces expression of membrane LT-alpha in B cells and that membrane LT-alpha may play a role in CD40 mediated B cell activation.

Antibodies, Monoclonal↗
PubMed1997

Selective disruption of lymphotoxin ligands reveals a novel set of mucosal lymph nodes and unique effects on lymph node cellular organization.

Lymphotoxin (LT) provides a critical signal for the genesis of lymph nodes (LN) in mice. Here we show that mice treated in utero with LT beta-R-Ig, which binds to the membrane LT alpha 1 beta 2 heterotrimer, lacked most LN, yet retained a set of mucosal surface draining LN. Since mice genetically deficient in LT alpha lack all LN, including the mucosal set, we hypothesize that a novel LT alpha-dependent pathway controls their genesis. This novel set of mucosal LN cannot be discriminated on the basis of addressin expression. The discovery of LN in mice treated with LT beta-R-Ig fusion protein in utero allowed us to compare the roles of membrane LT alpha beta or soluble LT alpha/tumor necrosis factor (TNF) in the development of cellular organization in LN and spleen. Our results indicate that both membrane LT alpha beta and soluble LT alpha/TNF mediate T-B cell segregation and the organization of B cell follicles in spleen and LN. Interestingly, while antagonism of membrane LT alpha beta or soluble LT alpha/TNF prevented germinal center (GC) formation in spleen, antagonism of soluble LT alpha/TNF had no effect on LN formation. The data suggest that multiple LT/TNF ligands control B cell follicle organization in the spleen and LN of adult mice, and that the requirements for LT/TNF ligands in GC formation are distinct in the different lymphoid organs.

Animals↗
PubMed1998

Early cytokine and chemokine gene expression during Pseudomonas aeruginosa corneal infection in mice.

Using a multiprobe RNase protection assay, we examined cytokine and chemokine mRNAs that were expressed after corneal infection with Pseudomonas aeruginosa in mice. Cytokines that were upregulated included interleukin-1alpha (IL-1alpha) and -1beta, IL-1 receptor antagonist, IL-6, IL-11, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, lymphotoxin beta, transforming growth factor beta1, and tumor necrosis factor alpha. Chemokine transcripts that were upregulated included Eotaxin; gamma-interferon-inducible protein 10; monocyte chemoattractant protein 1; macrophage inflammatory proteins 1alpha, 1beta, and 2; and RANTES. Peak expression of these cytokines and chemokines was observed between 1 and 3 days after infection. These responses returned to or approached baseline preinfection levels by 7 days after ocular challenge. Identification of the various cytokines and chemokines upregulated during corneal infection provides important information relevant to unraveling the pathogenesis induced by this bacterium and provides hope that specific molecules can be targeted for therapy.

Animals↗
PubMed1995

Characterization of surface lymphotoxin forms. Use of specific monoclonal antibodies and soluble receptors.

Lymphotoxin (LT) is a cytokine related to TNF, found in human systems in both secreted and membrane bound forms. The well characterized secreted form is a trimer of a single protein, LT-alpha, whereas the surface form is composed of a complex between two related molecules, LT-alpha and LT-beta. Because there is a distinct receptor for the complex, the membrane form is believed to signal via events different from those elicited by TNF and secreted LT-alpha. By using a battery of anti-LT-alpha and LT-beta mAbs, it is clear that two LT surface forms exist on the surface of PMA-activated II-23 cells, a human T cell hybridoma. Assuming that these surface forms are trimers, a minor form appears early after induction having an apparent stoichiometry of LT-alpha 2/beta 1 and is recognized by one group of anti-LT-alpha mAbs and the p55-TNF receptor. The second and predominant form has an apparent LT-alpha 1/beta 2 composition and is recognized by a second group of pantrophic anti-LT-alpha mAbs and the LT-beta receptor. Neither of the heteromeric forms nor a putative LT-beta homotrimeric form were found to be secreted. The properties of surface LT on the II-23 cell system were similar to those of the surface LT forms on Chinese hamster ovary cells transfected with both LT-alpha and LT-beta genes and a number of lymphoid tumor lines. These experiments point toward the LT-alpha 1/beta 2 complex as the predominant membrane form of LT on the lymphocyte surface, and this complex is the primary ligand for the LT-beta receptor.

Amino Acid Sequence↗