TACI is a TRAF-interacting receptor for TALL-1, a tumor necrosis factor family member involved in B cell regulation.

We and others recently reported tumor necrosis factor (TNF) and apoptosis ligand-related leukocyte-expressed ligand 1 (TALL-1) as a novel member of the TNF ligand family that is functionally involved in B cell proliferation. Transgenic mice overexpressing TALL-1 have severe B cell hyperplasia and lupus-like autoimmune disease. Here, we describe expression cloning of a cell surface receptor for TALL-1 from a human Burkitt's lymphoma RAJI cell library. The cloned receptor is identical to the previously reported TNF receptor (TNFR) homologue transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Murine TACI was subsequently isolated from the mouse B lymphoma A20 cells. Human and murine TACI share 54% identity overall. Human TACI exhibits high binding affinities to both human and murine TALL-1. Soluble TACI extracellular domain protein specifically blocks TALL-1-mediated B cell proliferation without affecting CD40- or lipopolysaccharide-mediated B cell proliferation in vitro. In addition, when injected into mice, soluble TACI inhibits antibody production to both T cell-dependent and -independent antigens. By yeast two-hybrid screening of a B cell library with TACI intracellular domain, we identified that, like many other TNFR family members, TACI intracellular domain interacts with TNFR-associated factor (TRAF)2, 5, and 6. Correspondingly, TACI activation in a B cell line results in nuclear factor kappaB and c-Jun NH(2)-terminal kinase activation. The identification and characterization of the receptor for TALL-1 provides useful information for the development of a treatment for B cell-mediated autoimmune diseases such as systemic lupus erythematosus.

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.

The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.

ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.

Members of tumor necrosis factor receptor (TNFR) family signal largely through interactions with death domain proteins and TRAF proteins. Here we report the identification of a novel TNFR family member ATAR. Human and mouse ATAR contain 283 and 276 amino acids, respectively, making them the shortest known members of the TNFR superfamily. The receptor is expressed mainly in spleen, thymus, bone marrow, lung, and small intestine. The intracellular domains of human and mouse ATAR share only 25% identity, yet both interact with TRAF5 and TRAF2. This TRAF interaction domain resides at the C-terminal 20 amino acids. Like most other TRAF-interacting receptors, overexpression of ATAR activates the transcription factor NF-kappaB. Co-expression of ATAR with TRAF5, but not TRAF2, results in synergistic activation of NF-kappaB, suggesting potentially different roles for TRAF2 and TRAF5 in post-receptor signaling.

Osteoprotegerin: a novel secreted protein involved in the regulation of bone density.

A novel secreted glycoprotein that regulates bone resorption has been identified. The protein, termed Osteoprotegerin (OPG), is a novel member of the TNF receptor superfamily. In vivo, hepatic expression of OPG in transgenic mice results in a profound yet nonlethal osteopetrosis, coincident with a decrease in later stages of osteoclast differentiation. These same effects are observed upon administration of recombinant OPG into normal mice. In vitro, osteoclast differentiation from precursor cells is blocked in a dose-dependent manner by recombinant OPG. Furthermore, OPG blocks ovariectomy-associated bone loss in rats. These data show that OPG can act as a soluble factor in the regulation of bone mass and imply a utility for OPG in the treatment of osteoporosis associated with increased osteoclast activity.

B61 is a ligand for the ECK receptor protein-tyrosine kinase.

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Genotype and phenotype: a practical approach to the immunogenetic analysis of lymphoproliferative disorders.

Determination of cell lineage and clonality in lymphoproliferative disorders (LPD) is greatly enhanced by molecular genetic analysis in conjunction with morphologic and immunologic techniques. We now report on a technique in which we used cryostat-cut, fresh-frozen sections (CCFFS) prepared from tissues in a manner that allows DNA hybridization studies to be coordinated readily with routine morphologic and immunohistologic studies. Thirty-seven cases representing a broad spectrum of reactive and malignant LPD were examined with this method. Samples of DNA were extracted from frozen sections, subjected to Southern blot hybridization, and probed for rearrangements of the immunoglobulin (Ig) heavy-chain and the kappa and lambda light-chain genes, as well as for the T-cell receptor beta-chain gene. We also evaluated the effects of (1) diagnostic category of LPD, (2) volume of the tissue sample, and (3) fibrosis, necrosis, and ice crystal artifacts in the sample on the recovery of DNA. Ice artifact and sample size had the greatest negative impacts on the quantity and condition of DNA recovered. Of 19 samples involved by B-cell LPD, the results of immunogenetic studies were consistent with the immunophenotypes in all but one case. Of the T-cell lymphomas from which sufficient DNA was available (three out of five of the T-cell cases), all showed rearrangements of the T-cell beta-chain gene. In order to reduce sample processing time, we evaluated alternate blot hybridization methods, rapid alkaline transfers, and direct hybridization of synthetic oligonucleotides in dried agarose gels, and found that they decreased the time required for hybridization studies. In summary, the use of CCFFS as the source of DNA allows study of gene rearrangements and, at the same time, preserves frozen-tissue blocks in tumor banks for further immunologic studies. The development of time-effective methods will make the routine use of molecular-genetic analysis more practical in the diagnostic hematopathology laboratory.

Analysis of antigen receptor gene rearrangements in ethanol and formaldehyde-fixed, paraffin-embedded specimens.

Molecular hybridization analysis of DNA prepared from frozen specimens obtained from patients with lymphoproliferative disorders has aided in the determination of the lineage and clonality of the neoplastic cells in many cases. We investigated whether high molecular weight DNA suitable for nucleic acid hybridization studies could also be prepared from fixed, paraffin-embedded material. After selecting nine representative cases, we extracted DNA from frozen sections by using standard methods, and from ethanol-fixed tissue in paraffin blocks. The yields of DNA from ethanol-fixed blocks were similar to yields from frozen tissue. DNA from frozen and ethanol-fixed tissues was subjected to Southern blot hybridization and probed for rearrangements of immunoglobulin heavy, and kappa, and lambda light chain genes, as well as for the T cell receptor beta-chain gene. In each of the cases, comparable results were obtained, regardless of the source of DNA. DNA extracted from ethanol-fixed blocks stored for 2 years gave identical results. We also prepared DNA from formaldehyde-fixed, paraffin-embedded tissue obtained from six of the patients. Five of the specimens yielded spoolable DNA (average recovered, 313 micrograms), but the DNA was degraded more than that obtained from the frozen or ethanol-fixed specimens. Formaldehyde-treated DNA gave variable results in Southern blot hybridization studies, and less than half of the results were interpretable. We conclude that ethanol-fixed, paraffin-embedded tissues provide an excellent source of DNA for nucleic-acid hybridization studies, and that they are easily handled and stored.

Oligonucleotide-directed mutagenesis using plasmid DNA templates and two primers.

Oligonucleotide-directed mutagenesis using plasmid vectors has been simplified by introducing two changes to the previous method. First, template preparation has been simplified by using the covalently closed circular plasmid DNA directly for mutagenesis, eliminating the need for a wholly or partially single-stranded circular DNA template. Second, two primers are used, eliminating the need for producing the covalently closed molecule during in vitro replication. The advantages of the approach are discussed.