Dirk M. Anderson

A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function

Dendritic cells are rare haematopoietic cells that reside in a number of organs and tissues. By capturing, processing and presenting antigens to T cells, dendritic cells are essential for immune surveillance and the regulation of specific immunity. Several members of the tumour necrosis factor receptor (TNFR) superfamily are integral to the regulation of the immune response. These structurally related proteins modulate cellular functions ranging from proliferation and differentiation to inflammation and cell survival or death. The functional activity of dendritic cells is greatly increased by signalling through the TNFR family member CD40 (refs 7, 8). Here we report the characterization of RANK (for receptor activator of NF-κB), a new member of the TNFR family derived from dendritic cells, and the isolation of a RANK ligand (RANKL) by direct expression screening. RANKL augments the ability of dendritic cells to stimulate naive T-cell proliferation in a mixed lymphocyte reaction, and increases the survival of RANK+T cells generated with interleukin-4 and transforming growth factor (TGF)-β. Thus RANK and RANKL seem to be important regulators of interactions between T cells and dendritic cells.

Utilization of the beta and gamma chains of the IL-2 receptor by the novel cytokine IL-15.

We have recently cloned a novel cytokine, IL‐15, with shared bioactivities but no sequence homology with IL‐2. We found high affinity IL‐15 binding to many cell types, including cells of non‐lymphoid origin. Analysis of IL‐15 interaction with subunits of the IL‐2 receptor (IL‐2R) revealed that the alpha subunit was not involved in IL‐15 binding. We demonstrated directly in cells transfected with IL‐2R subunits that both the beta and gamma chains are required for IL‐15 binding and signaling. Hence, IL‐15, like IL‐2, IL‐4 and IL‐7, utilizes the common IL‐2R gamma subunit found to be defective in X‐linked severe combined immunodeficiency in humans. IL‐15 is the only cytokine other than IL‐2 that has also been shown to share the beta signaling subunit of IL‐2R. The differential ability of some cells to bind and respond to IL‐2 and IL‐15 implies the existence of an additional IL‐15‐specific component.

Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms

We have previously reported the identification of a novel mast cell growth factor (MGF) that was shown to be a ligand for c-kit and is encoded by a gene that maps near the steel locus on mouse chromosome 10. We now report the cloning of cDNAs encoding the MGF protein. The MGF protein encoded by this cDNA can be expressed in a biologically active form as either a membrane bound protein or as a soluble factor. The soluble protein promotes the proliferation of MGF-responsive cell lines and, in the presence of erythropoietin, stimulates the formation of macroscopic [corrected] erythroid and multilineage hematopoietic colonies.

The murine interleukin-4 receptor: Molecular cloning and characterization of secreted and membrane bound forms

Receptors for interleukin-4 (IL-4) are expressed at low levels on a wide variety of primary cells and cultured cell lines. Fluorescence-activated sorting of CTLL-2 cells resulted in the isolation of a subclone, CTLL 19.4, which expressed 10(6) IL-4 receptors per cell. These cells were used for the purification of IL-4 receptor protein and to prepare a hybrid-subtracted cDNA probe for isolation of cDNA clones. Three classes of IL-4 receptor cDNA were identified. The first encoded a 140 kd membrane bound IL-4 receptor containing extracellular, transmembrane, and cytoplasmic domains. The second class lacked the cytoplasmic region, and the third encoded a secreted form of the receptor. All cDNA clones expressed in COS-7 cells had IL-4 binding properties comparable to the native IL-4 receptor. The soluble form of the IL-4 receptor blocked the ability of IL-4 to induce CTLL cell proliferation and may represent a regulatory molecule specific for IL-4-dependent immune responses.

Identification and cloning of a novel IL-15 binding protein that is structurally related to the alpha chain of the IL-2 receptor.

Interleukin‐15 (IL‐15) is a novel cytokine of the four‐helix bundle family which shares many biological activities with IL‐2, probably due to its interaction with the IL‐2 receptor beta and gamma (IL‐2R beta and gamma c) chains. We report here the characterization and molecular cloning of a distinct murine IL‐15R alpha chain. IL‐15R alpha alone displays an affinity of binding for IL‐15 equivalent to that of the heterotrimeric IL‐2R for IL‐2. A biologically functional heteromeric IL‐15 receptor complex capable of mediating IL‐15 responses was generated through reconstruction experiments in a murine myeloid cell line. IL‐15R alpha is structurally similar to IL‐2R alpha; together they define a new cytokine receptor family. The distribution of IL‐15 and IL‐15R alpha mRNA suggests that IL‐15 may have biological activities distinct from IL‐2.

CD30 antigen, a marker for Hodgkin's lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF

CD30 is a surface marker for neoplastic cells of Hodgkins lymphoma and shows sequence homology to members of the tumor necrosis factor (TNF) receptor superfamily. Using a chimeric probe consisting of the extracellular domain of CD30 fused to truncated immunoglobulin heavy chains, we expression cloned the cDNA cognate from the murine T cell clone 7B9. The encoded protein is a 239 amino acid type II membrane protein whose C-terminal domain shows significant homology to TNF alpha, TNF beta, and the CD40L. Cross-hybridization to an induced peripheral blood T cell cDNA library yielded the human homolog, which is 72% identical at the amino acid level. The recombinant human ligand enhances the proliferation of CD3-activated T cells yet induces differential responses, including cell death, in several CD30+ lymphoma-derived clones. The human and murine genes map to 9q33 and the proximal region of chromosome 4, respectively.

Mast cell growth factor maps near the steel locus on mouse chromosome 10 and is deleted in a number of steel alleles

Many spontaneous, chemical-induced, and radiation-induced dominant white spotting (W) and steel (Sl) mutations have been identified in the mouse. W and Sl mutations have similar phenotypic effects including deficiencies in pigment cells, germ cells, and blood cells, Numerous studies have suggested that W acts within the affected cell while Sl instead exerts its effects in the extracellular environment. Recent findings demonstrating that W encodes the c-kit proto-oncogene, a tyrosine kinase membrane receptor, have suggested that Sl encodes a ligand for c-kit. In the accompanying article we report the identification and purification of mast cell growth factor (MGF), a c-kit ligand. Here we describe the cloning of sequences encoding MGF. Furthermore, we show that Mgf maps near Sl in the distal region of mouse chromosome 10 and is deleted in a number of Sl alleles. These findings strongly support the notion that Sl encodes the mast cell growth factor.

Embryonic RNA expression patterns of the c-kit receptor and its cognate ligand suggest multiple functional roles in mouse development.

Mutations at the dominant white spotting (W) and Steel (Sl) loci in mouse exert deleterious effects on three migratory cell lineages (primordial germ cells, melanocytes and hematopoietic stem cells) resulting in loss of pigmentation, reduced fertility and anemia. The W locus encodes the c‐kit protein tyrosine kinase (TK) receptor. More recently, the Sl locus has been shown to encode a ligand for c‐kit, which is variously known as mast cell growth factor (MGF), stem cell growth factor and c‐kit ligand. Here we report an in situ hybridization analysis comparing the expression profiles of MGF and c‐kit transcripts during mouse embryogenesis. The data are consistent with the c‐kit receptor‐ligand complex providing a homing mechanism during stem cell migration in early development and in stem cell proliferation, differentiation, or survival in late development. In the nervous system, an unexpected and complex pattern of expression is uncovered that suggests involvement of the W and Sl gene products in the organization of the neural tube and brain.

Molecular cloning and expression of the type 1 and type 2 murine receptors for tumor necrosis factor.

Clones encoding the type 1 (p80) and type 2 (p60) forms of the murine receptors for tumor necrosis factor (TNF) were isolated by cross-hybridization using probes derived from the cloned human TNF receptors. Each of the murine receptors shows strong sequence homology to the corresponding human receptor (approximately 65% amino acid identity) throughout the molecule but only modest homology, limited to ligand-binding domains, between themselves. The ligand-binding characteristics of the recombinant murine receptors mirror those of the human homologs: both receptor types bind TNF-alpha and -beta with multiple affinity classes, and the ligands cross-compete. Analysis of the murine transcripts encoding these receptors revealed the presence of RNAs for one or both forms of the receptors in all cells examined. It was also demonstrated that for both types of human TNF receptor, variably sized transcripts are observed in different cells. The murine cDNAs were further used to determine the chromosomal locations of the TNF receptor genes. They are not linked, in contrast to the ligands, and map to chromosomes 4 (type 1) and 6 (type 2).

Enhanced RANK ligand expression and responsivity of bone marrow cells in Paget’s disease of bone

Pagets disease is characterized by highly localized areas of increased osteoclast (OCL) activity. This suggests that the microenvironment in pagetic lesions is highly osteoclastogenic, or that OCL precursors in these lesions are hyperresponsive to osteoclastogenic factors (or both). To examine these possibilities, we compared RANK ligand (RANKL) mRNA expression in a marrow stromal cell line developed from a pagetic lesion (PSV10) with that in a normal stromal cell line (Saka), and expression in marrow samples from affected bones of Pagets patients with that in normal marrow. RANKL mRNA was increased in PSV10 cells and pagetic marrow compared with Saka cells and normal marrow, and was also increased in marrow from affected bones compared with uninvolved bones from Pagets patients. Furthermore, pagetic marrow cells formed OCLs at much lower RANKL concentrations than did normal marrow. Anti-IL-6 decreased the RANKL responsivity of pagetic marrow to normal levels, whereas addition of IL-6 to normal marrow enhanced RANKL responsivity. Thus, RANKL expression and responsivity is increased in pagetic lesions, in part mediated by IL-6. These data suggest that the combination of enhanced expression of RANKL in affected bones and increased RANKL sensitivity of pagetic OCL precursors may contribute to the elevated numbers of OCLs in Pagets disease.