Multiple myeloma disrupts the TRANCE/ osteoprotegerin cytokine axis to trigger bone destruction and promote tumor progression.

Bone destruction, caused by aberrant production and activation of osteoclasts, is a prominent feature of multiple myeloma. We demonstrate that myeloma stimulates osteoclastogenesis by triggering a coordinated increase in the tumor necrosis factor-related activation-induced cytokine (TRANCE) and decrease in its decoy receptor, osteoprotegerin (OPG). Immunohistochemistry and in situ hybridization studies of bone marrow specimens indicate that in vivo, deregulation of the TRANCE-OPG cytokine axis occurs in myeloma, but not in the limited plasma cell disorder monoclonal gammopathy of unknown significance or in nonmyeloma hematologic malignancies. In coculture, myeloma cell lines stimulate expression of TRANCE and inhibit expression of OPG by stromal cells. Osteoclastogenesis, the functional consequence of increased TRANCE expression, is counteracted by addition of a recombinant TRANCE inhibitor, RANK-Fc, to marrow/myeloma cocultures. Myeloma-stroma interaction also has been postulated to support progression of the malignant clone. In the SCID-hu murine model of human myeloma, administration of RANK-Fc both prevents myeloma-induced bone destruction and interferes with myeloma progression. Our data identify TRANCE and OPG as key cytokines whose deregulation promotes bone destruction and supports myeloma growth.

SHIP recruitment attenuates Fc gamma RIIB-induced B cell apoptosis.

Fc gammaRIIB is an inhibitory receptor that terminates activation signals initiated by antigen cross-linking of the BCR through the recruitment of SHIP. Fc gammaRIIB can also signal independently of BCR coligation to directly mediate an apoptotic response, requiring only an intact transmembrane domain. Failure to recruit SHIP, either by deletion of SHIP or mutation of Fc gammaRIIB, results in enhanced Fc gammaRIIB-triggered apoptosis. Thus, in the germinal center, where ICs are retained by FDCs, Fc gammaRIIB may be an active determinant in the negative selection of B cells whose BCRs have reduced affinity for antigen as a result of somatic hypermutation. Selection of B cells may represent the sum of opposing signals generated by the interaction of ICs with the BCR and Fc gammaRIIB through pathways modulated by SHIP.

SHIP modulates immune receptor responses by regulating membrane association of Btk.

Membrane recruitment of SHIP is responsible for the inhibitory signal generated by FcgammaRIIB coligation to the BCR. By reducing the level of PIP3, SHIP regulates the association of the tyrosine kinase Btk with the membrane through PH domain-phosphoinositol lipid interactions. Inhibition of BCR signaling by either FcgammaRIIB coligation, membrane expression of SHIP, or inhibition of P13K, conditions which result in decreased levels of PIP3, is suppressed by the expression of Btk as a membrane-associated chimera. Conversely, increasing PIP3 levels by deletion of SHIP results in increased Btk association with the membrane and hyperresponsive BCR signaling. These results suggest a central role for PIP3 in regulating the B cell stimulatory state by modulating Btk localization and thereby calcium fluxes.

Infectious complications of autologous bone marrow and peripheral stem cell transplantation for refractory leukemia and lymphoma.

We aimed to characterize the infectious complications of autologous bone marrow (AuBMT) and peripheral stem cell transplantation (PSCT) in patients with refractory leukemia and lymphoma. We performed a retrospective analysis of all patients (n = 56) with refractory leukemia or lymphoma treated with AuBMT or PSCT at Memorial Sloan-Kettering Cancer Center from January 1993 to July 1994. Records were available in 55, of whom 33 (60%) received AuBMT and 22 (40%) PSCT. Fifteen (27%) developed complicated infections, including 13 (39%) treated with AuBMT and two (9%) with PSCT. Complicated infections were caused by bacterial (11 episodes), fungal (four episodes), and viral (four episodes) pathogens. Five (9%) infections were fatal. In a multivariate model, only duration of neutropenia was significantly associated with development of complicated infection (P = 0.006). Thus, 27% of patients with refractory leukemia or lymphoma treated with AuBMT or PSCT developed complicated infections and 9% died of infection. Prolonged neutropenia was significantly associated with development of infection. Patients receiving PSCT had significantly lower rates of complicated infection, presumably due to the associated shorter duration of neutropenia. Future studies are needed to define the role of PSCT as treatment for refractory neoplastic disease.

PU.1 and an HLH family member contribute to the myeloid-specific transcription of the Fc gamma RIIIA promoter.

Expression of the low-affinity Fc receptor for IgG (murine Fc gamma RIIIA) is restricted to cells of myelomonocytic origin. We report here the promoter structure, the proximal DNA sequences responsible for transcription of Fc gamma RIIIA in macrophages and the protein factors which interact with these sequences. A 51 bp sequence, termed the myeloid restricted region (MRR), was both necessary and sufficient for conferring cell type-specific expression in macrophages. Reporter constructs containing mutations in this sequence result in the loss of MRR activity upon transfection into the macrophage cell line, RAW264.7. Two cis-acting elements have been identified and are required for full promoter function. These same elements analyzed by EMSA define two binding sites recognized by nuclear factors derived from macrophages. A 3' purine tract (-50 to -39) within the MRR binds the macrophage and B cell-specific factor, PU.1, and a second E box-like element, termed MyE, upstream of the PU.1 box (-88 to -78) binds the HLH factors TFE3 and USF. EMSA studies using RAW cell extracts suggest that both PU.1 and MyE factors may bind simultaneously to the MRR resulting in a ternary complex that is responsible, in part, for the myeloid-specific activity of the Fc gamma RIIIA promoter.

Interferon gamma-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3.

A 39-nt DNA sequence, the interferon gamma (IFN-gamma) response region (GRR), is necessary for the IFN-gamma-induced transcription of the high-affinity Fc receptor for IgG (Fc gamma RI) and sufficient for the IFN-gamma-induced transcription of transfected plasmids. By using extracts from IFN-gamma-treated cells, three protein complexes will assemble in vitro on a 9-nt core region in the 3' domain of the GRR. The sequence of this core resembles the IFN-gamma-activated sequence (GAS) described for the GBP gene. Mutations in this GAS core region prevent complex assembly and result in the loss of IFN-gamma induction of reporter constructs containing the mutation. In addition to the GAS core region, a 5' region of the GRR is necessary for optimal IFN-gamma induction and for formation of one of the DNA-protein complexes. By antibody reactivity, we show that a 91-kDa protein, first identified as a component of ISGF3, the IFN-alpha-induced transcription complex, is present in at least two of the DNA-protein complexes. IFN-alpha can induce the formation of the faster-migrating 91-kDa protein-GAS complex but not the slower-migrating complex. Furthermore, IFN-alpha does not result in appreciable transcriptional activation of Fc gamma RI or constructs containing the GRR. Thus, these data demonstrate that the IFN-gamma-activated 91-kDa protein is required for IFN-gamma induction of Fc gamma RI and suggest that an additional complex may be required for optimal expression and specificity.

Characterization of the promoter of the human gene encoding the high-affinity IgG receptor: transcriptional induction by gamma-interferon is mediated through common DNA response elements.

Expression of the high-affinity receptor for IgG (Fc gamma RI) is restricted to cells of myeloid lineage and is induced by gamma-interferon (IFN-gamma) but not by IFN-alpha/beta. The organization of the human Fc gamma RI gene has been determined and the DNA elements governing its cell type-restricted transcription and IFN-gamma induction are reported here. A 39-nucleotide sequence (IFN-gamma response region, or GRR) is defined that is both necessary and sufficient for IFN-gamma inducibility. Sequence analysis of the GRR reveals the presence of promoter elements initially defined for the major histocompatibility complex class II genes: i.e., X, H, and gamma-IRE sequences. Comparison of a number of genes whose expression is induced selectively by IFN-gamma indicates that the presence of these elements is a general feature of IFN-gamma-responsive genes. Our studies suggest that the combination of X, H, and gamma-IRE elements is a common motif in the pathway of transcriptional induction by this lymphokine.

The noradrenergic system in cultured aggregates of fetal rat brain cells: morphology of the aggregates and pharmacological indices of noradrenergic neurons.

Spherical aggregates formed rapidly in culture by re-aggregation of trypsin-dissociated brain cells from the 17-day-old fetal rat. Over about 10 days in initially random distribution of cells evolved into a 3-layered arrangement; cells with characteristics of neurons were found largely in the intermediate layer. The survival of neuronal and glial cell types was evaluated histologically and verified by electron microscopy, which revealed synaptic and myelin structures that rapidly increased in number after 18 days in culture. Levels of norepinephrine (NE) and dopamine (DA) reached peaks of 9.5 and 4.4 ng/mg protein, respectively, at culture day 21. Uptake of [3H]NE paralleled these amine levels and was blocked by desipramine or pretreatment with either reserpine or 6-OH-DA. Autoradiographs of aggregates labeled with [3H]NE showed a high density of silver grains over cells, apparently neurons, with branching processes traced for 120 micrometer. Previously accumulated [3H]NE was released under depolarizing conditions (high [K+] or vertridine) only in the presence of Ca2+. Release was induced to a lesser extent by kainic greater than glutamic acid. Thus, such aggregates appear to contain catecholaminergic neurons capable of synthesis, uptake and release of NE. The time course of development of these functions supports suggestions that aggregate preparations might be useful in studying neurochemical or morphological aspects of brain development and function in vitro.