Impaired kit- but not FcepsilonRI-initiated mast cell activation in the absence of phosphoinositide 3-kinase p85alpha gene products.

The class I(A) phosphoinositide 3-kinases (PI3Ks) consist of a 110-kDa catalytic domain and a regulatory subunit encoded by the p85alpha, p85beta, or p55gamma genes. We have determined the effects of disrupting the p85alpha gene on the responses of mast cells stimulated by the cross-linking of Kit and FcepsilonRI, receptors that reflect innate and adaptive responses, respectively. The absence of p85alpha gene products partially inhibited Kit ligand/stem cell factor-induced secretory granule exocytosis, proliferation, and phosphorylation of the serine/threonine kinase Akt. In contrast, p85alpha gene products were not required for FcepsilonRI-initiated exocytosis and phosphorylation of Akt. LY294002, which inhibits all classes of PI3Ks, strongly suppressed Kit- and FcepsilonRI-induced responses in p85alpha -/- mast cells, revealing the contribution of another PI3K family member(s). In contrast to B lymphocytes, mast cell proliferation was not dependent on Bruton's tyrosine kinase, a downstream effector of PI3K, revealing a distinct pathway of PI3K-dependent proliferation in mast cells. Our findings represent the first example of receptor-specific usage of different PI3K family members in a single cell type. In addition, because Kit- but not FcepsilonRI-initiated signaling is associated with mast cell proliferation, the results provide evidence that distinct biologic functions signaled by these two receptors may reflect differential usage of PI3Ks.

SLP-76 deficiency impairs signaling via the high-affinity IgE receptor in mast cells.

SLP-76 is an adapter protein expressed in T cells and myeloid cells that is a substrate for ZAP-70 and Syk. SLP-76-deficient mice exhibit a profound block in T-cell development. We found that although SLP-76 is expressed in mouse mast cells, SLP-76(-/-) mice have normal numbers of mast cells in their skin and bronchi. SLP-76(-/-) mice are resistant to IgE-mediated passive anaphylaxis. SLP-76(-/-) mice sensitized with IgE anti-dinitrophenyl (DNP) and then challenged with DNP-HSA developed only mild and transient tachycardia, failed to increase their plasma histamine level, and all survived the antigen challenge. Bone marrow-derived mast cells (BMMCs) from SLP76(-/-) mice failed to release beta-hexosaminidase and to secrete IL-6 after FcepsilonRI cross-linking. Tyrosine phosphorylation of phospholipase C-gamma1 (but not of Syk) and calcium mobilization in response to IgE cross-linking were reduced in SLP-76-deficient BMMCs. These results suggest that SLP-76 plays an important role in FcepsilonRI-mediated signaling in mast cells.

gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1, and suppression of early and late calcium mobilization.

We define by molecular, pharmacologic, and physiologic approaches the proximal mechanism by which the immunoglobulin superfamily member gp49B1 inhibits mast cell activation mediated by the high affinity Fc receptor for IgE (FcepsilonRI). In rat basophilic leukemia-2H3 cells expressing transfected mouse gp49B1, mutation of tyrosine to phenylalanine in either of the two immunoreceptor tyrosine-based inhibitory motifs of the gp49B1 cytoplasmic domain partially suppressed gp49B1-mediated inhibition of exocytosis, whereas mutation of both abolished inhibitory capacity. Sodium pervanadate elicited tyrosine phosphorylation of native gp49B1 and association of the tyrosine phosphatases src homology 2 domain-containing phosphatase-1 (SHP-1) and SHP-2 in mouse bone marrow-derived mast cells (mBMMCs). SHP-1 associated transiently with gp49B1 within 1 min after coligation of gp49B1 with cross-linked FcepsilonRI in mBMMCs. SHP-1-deficient mBMMCs exhibited a partial loss of gp49B1-mediated inhibition of FcepsilonRI-induced exocytosis at concentrations of IgE providing optimal exocytosis, revealing a central, but not exclusive, SHP-1 requirement in the counter-regulatory pathway. Coligation of gp49B1 with cross-linked FcepsilonRI on mBMMCs inhibited early release of calcium from intracellular stores and subsequent influx of extracellular calcium, consistent with SHP-1 participation. Because exocytosis is complete within 2 min in mBMMCs, our studies establish a role for SHP-1 in the initial counter-regulatory cellular responses whereby gp49B1 immunoreceptor tyrosine-based inhibition motifs rapidly transmit inhibition of FcepsilonRI-mediated exocytosis.

Post-transcriptional stabilization by interleukin-1beta of interleukin-6 mRNA induced by c-kit ligand and interleukin-10 in mouse bone marrow-derived mast cells.

We demostrate that a specific combination of cytokines elicits high levels of interleukin (IL)-6 gene expression in mast cells and define the cellular mechanisms of the exogenous cytokine action. The addition of c-kit ligand (KL) and IL-10 to IL-3-derived mouse bone marrow mast cells (BMMC) elicited an approximately 2-fold increase in steady-state IL-6 mRNA levels that peaked after 0.5 h and was followed by the release of approximately 0.2 ng of IL-6/10(6) cells by 5-7 h. The addition of IL-1beta to KL + IL-10 elicited a prolonged approximately 12-fold increase in the level of IL-6 mRNA by 3-5 h and an approximately 50-fold increase in the level of IL-6 protein released by 7 h. As determined by nuclear run-on analysis, KL + IL-10 stimulated IL-6 gene transcription within 0.5 h, and the addition of IL-1beta did not increase transcription. Instead, IL-1beta slowed by approximately 8-fold the decay of IL-6 mRNA as compared to its decay in BMMC stimulated with KL + IL-10 alone. The exposure of BMMC to cycloheximide 0.5 h before the addition of the three exogenous cytokines inhibited by approximately 50% the level of IL-6 mRNA generated but did not inhibit the effects of KL + IL-10, indicating that IL-1beta induces the synthesis of a protein that stabilizes IL-6 mRNA. The stabilization of IL-6 mRNA was inhibited by the addition of actinomycin D at 0.5 but not 3 h after BMMC were stimulated with IL-1beta in combination with KL + IL-10, suggesting that once transcribed, the stabilizing protein is long-lived. The addition of cycloheximide to BMMC after stimulation with KL + IL-10 with or without IL-1beta increased the levels of steady-state IL-6 mRNA compared to levels in cells without drug, indicating that in addition to stimulating IL-6 transcription, KL + IL-10 induces a protein factor that destabilizes IL-6 mRNA. Thus, there exists a novel Fcepsilon receptor type I-independent mechanism by which a mast cell can provide substantial amounts of IL-6 protein in response to the synergistic action of KL and IL-10 to induce IL-6 gene transcription, and IL-1beta to stabilize otherwise short-lived IL-6 transcripts.

A YAC contig spanning a cluster of human type III receptor protein tyrosine kinase genes (PDGFRA-KIT-KDR) in chromosome segment 4q12.

We have mapped five genes encoding protein tyrosine kinases (PTKs) to the pericentromeric region of human chromosome 4. PTK4 and TYRO4, which encode nonreceptor intracellular PTKs, are located at 4p12 and 4q13, respectively. The other three genes, PDGFRA, KIT, and KDR, encode type III transmembrane receptor PTKs for known ligands. We have developed a contig of 29 yeast artificial chromosomes (YACs) spanning approximately 2 Mb of DNA at 4q12 that includes PDGFRA, KIT, and KDR, and we have used this YAC contig to map 12 different sequence-tagged sites in this region. PDGFRA, KIT, and KDR thus constitute a cluster of genes at 4q12 encoding closely related type III receptor PTKs. Mutations of the human KIT gene result in piebaldism, an autosomal dominant disorder of melanocyte development.

Construction of a YAC contig and a STS map spanning at least seven megabasepairs in chromosome 5q34-35.

We have constructed a YAC contig containing 54 clones and a minimum of 7 Mbp of human DNA, that maps to bands q34-35 on chromosome 5. The contig was nucleated using FISH mapped cosmid clones shown to flank the t(2;5)(p23;q35) translocation breakpoint in a CD30-positive large cell lymphoma cell line. Thirty of the 54 YAC clones are non-chimeric and six span the translocation breakpoint, as determined by FISH analysis. A total of 28 YAC clone end fragments, 14 non-polymorphic YAC end STS probes and 13 polymorphic microsatellite STS markers have been used to order clones within the contig. The most distal genetic markers (D5S498 and D5S619) are separated by 15 cM based on multipoint linkage analysis. This map of overlapping clones and the set of densely spaced physical markers will promote our understanding of the 5q34-35 region and its associated genes.

Expression cloning of multiple human cDNAs that complement the phenotypic defects of ataxia-telangiectasia group D fibroblasts.

Ataxia-telangiectasia (A-T) is an inherited human disease of unknown etiology associated with neurologic degeneration, immune dysfunction, cancer risk, and genetic instability. A-T cells are sensitive to ionizing radiation and radiomimetic drugs, offering the possibility of cloning A-T genes by phenotypic complementation. We have used this sensitivity to isolate the first human cDNAs reported to complement A-T cells in culture. Complementation group D A-T fibroblasts were transfected with an episomal vector-based human cDNA library, approximately 610,000 resultant transformants were treated with the radiomimetic drug streptonigrin-resistant, and nine unrelated cDNAs were recovered from 29 surviving streptonigrin-resistant clones. Five cDNAs were mapped, but none localized to 11q23, the site of A-T complementation group A and C loci. Four of the mapped cDNAs conferred mutagen resistance to A-T D fibroblasts on secondary transfection. One cDNA was identified as a fragment of dek, a gene involved in acute myeloid leukemia. The dek cDNA fragment and pCAT4.5, a 4.5-kb cDNA that mapped to 17p11, independently complemented three different phenotypic abnormalities of A-T D fibroblasts (mutagen sensitivity, hyper-recombination, and radio-resistant DNA synthesis). The pCAT4.5 cDNA did not complement the mutagen sensitivity of an A-T group C fibroblast line, suggesting that it represents a candidate disease gene for group D A-T. Our results indicate that phenotypic complementation alone is insufficient evidence to prove that a candidate cDNA is an A-T disease gene. The complementing cDNAs may represent previously uncharacterized genes that function in the same pathway as does the A-T gene product(s) in the regulation of cellular responses to DNA damage.