The Quality of Well-Being scale in asymptomatic HIV-infected patients. HNRC Group. HIV Neural Behavioral Research Center

Mice bearing mutations at either of two loci, dominant White spotting(W) or Steel(Sl), exhibit development defects in hematopoietic, melanocytic and germ cells. Genetics studies have shown that the SI locus encodes the Steel factor (SF), which is the ligand for the tyrosine kinase receptor c-kit, the product of the W locus. SF is synthesized in membrane-bound form and can be processed to produce a soluble form. Cell-cell interaction is important in the production of normal blood cells in vivo and in vitro and in the cellular expansion of leukemic cells. We discuss here how SF decreases the requirements in cell interaction for blast colony formation in acute myeloblastic leukemia (AML) and the presence of membrane-bound SF possibly contributes to the density-dependent growth of the AML blasts. We explain that SF is mainly a survival factor for hematopoietic cells, of little proliferative effect, which maintains CD34+ hematopoietic cells in an undifferentiated state. These properties would potentially allow the maintenance of hematopoietic cells in culture for the purpose of marrow purging or gene therapy. The activation of the c-kit signal transduction pathway may play a significant role in the development of many types of non-hematological malignancies by disrupting normal cell-cell interactions and allowing the growth of cancer cell populations. In summary, the properties of the SF indicate it has a role for survival signals during the process of normal differentiation, AML proliferation and in the maintenance of many c-kit+ tumors.     

Stem cell factor enhances the adhesion of AML cells to fibronectin and augments fibronectin-mediated anti-apoptotic and proliferative signals

Acute myeloid leukaemia (AML) cells express the SCF receptor c-kit (CD117) on their cell surface and demonstrate enhanced adhesion to fibronectin (FN) following exposure to stem cell factor (SCF). Increased adhesion occurs within 5 min, is dose dependent, and persists beyond 2 h. Baseline and enhanced adhesion occur through the surface FN receptor very late antigen-5 (VLA-5, CD49e/CD29) which is expressed by AML cells. Unstimulated AML cells exposed to FN undergo less apoptosis than controls (inhibition 22.5 +/- 7.0%, P = 0.02, n = 8). Exposure to SCF alone without FN also inhibits AML cell apoptosis (by 19.0 +/- 7.7% compared to controls, P = 0.06, n = 8). Simultaneous exposure to SCF and FN increases the inhibition of AML cell apoptosis to 37.8 +/- 7.9% (P = 0.005 compared to control, P = 0.04 compared to FN alone, P = 0.06 compared to SCF alone) demonstrating that SCF not only enhances the propensity of AML cells to adhere to FN, but also results in an additive survival benefit following FN contact. Some but not all the reduction in apoptosis is mediated through VLA-5. The combination of SCF and FN also affects proliferation, resulting in a synergistic enhancement of AML cell proliferation in half the cases studied. When normal CD34+ human haemopoietic progenitors were studied, FN had little effect on their apoptosis and failed to enhance the anti-apoptotic effect of SCF. It did, however, synergise with SCF in promoting CD34+ cell proliferation. Exposure of AML cells to SCF and FN, both of which can be found in high concentration in the bone marrow stroma, inhibits apoptosis. Cytokines and extracellular matrix proteins augment each others' effects since SCF enhances adhesion to fibronectin, which in turn augments the survival signal delivered by the cytokine alone. Cytokine and adhesion receptors can combine to affect cell characteristics including proliferation and survival.     

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.     

Expression of stem cell factor (SCF) and SCF receptor (c-kit) in synovial membrane in arthritis: correlation with synovial mast cell hyperplasia and inflammation

OBJECTIVE: Stem cell factor (SCF), the ligand for the SCF receptor (c-kit) expressed on precursors and mature mast cells (MC), is a major agonist for human MC (e.g., SCF induces MC development, chemotaxis, activation, proliferation of MC precursors, mediates MC adhesion, and changes MC releasability). We investigated expression of SCF and c-kit in synovial membrane with particular reference to the mechanism of local MC hyperplasia and inflammation in arthritis. METHODS: We conducted single and double labeling immunohistochemistry (ABC, APAAP, indirect immunofluorescence techniques) with antibodies to SCF, c-kit, MC tryptase, Ki-67 antigen (marker for proliferating cells), and CD68 (monocyte/macrophage marker). Synovial specimens analyzed were from 31 patients: traumatic arthritis (TrA, n=9), osteoarthritis (OA, n=12), and rheumatoid arthritis (RA, n=10). Control experiments were performed on human lung, skin, and buccal mucosa tissues, on the HMC-1 mast cell line, and isolated lung MC. Morphometry was performed by computerized image analysis. RESULTS: Synovial c-kit expression was found to be restricted to MC, whereas SCF is detected in synovial lining cells, stromal fibroblasts, monocyte/macrophages, endothelial cells, and in vascular basement membranes. SCF staining was localized to MC as well, but it was not possible to specify whether this represents SCF produced by or bound (via c-kit) to MC. In inflamed synovial membranes/areas, SCF was found to be redistributed into the extracellular matrix. Redistribution of SCF was accompanied by degranulation and/or accumulation of c-kit+ MC, the hyperplasia of which correlated positively with histologic inflammation/inflammatory cell densities, but did not appear to involve MC proliferation in situ. These findings appeared to be common for all the conditions (TrA, OA, RA) studied. CONCLUSION: In addition to the demonstration/characterization of SCF and c-kit protein expression in human synovium, results of this study suggest the hypothesis that, in arthritis, local mobilization of SCF may play a role in the development of synovial MC hyperplasia without inducing in situ proliferation of MC, and that the synovial SCF/MC c-kit system may contribute to the local nonspecific inflammatory response/arthritic flares in TrA, OA, and RA.     

Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants

Hematopoiesis is viewed as a differentiating system emanating from a pluripotent hematopoietic stem cell capable of both self-renewal and differentiation. By identifying and characterizing a novel and highly specific in vitro mitogenic response to the N-acetyl glucosamyl/sialic acid specific, stem cell-binding lectin wheat germ agglutinin (WGA), we demonstrate the existance of a rare (0.1%), plastic adherent precursor in rat bone marrow capable of proliferation (two to seven divisions) in response to WGA. Stimulated cells possess a lineage (lin)low/- immunophenotype and immature blastoid morphology (WGA blasts). A subsequent proliferative response to stem cell factor (SCF), the ligand for the proto-oncogene receptor tyrosine kinase c-kit, is characterized by an initial maturation in immunophenotype and subsequent self-renewal of cells (SCF blasts) without differentiation for at least 50 generations. Although granulocyte colony-stimulating factor (G-CSF), interleukin (IL) -6, IL-7, and IL-11 synergize with SCF to increase blast colony formation, cytokines such as granulocyte-macrophage CSF or IL-3 are without significant effect. At all time points in culture, however, cells rapidly differentiate to mature neutrophils with dexamethasone or to mainly monocytes/macrophages in the presence of 1alpha,25-dihydroxyvitamin D3, characterized by cell morphology and cytochemistry. Removal of SCF during blast maturation, self-renewal, or induction of differentiation phases results in apoptotic cell death. Data indicate a pivotal role for SCF/c-kit interaction during antigenic maturation, self-renewal, and apoptotic protection of these lineage-restricted progenitors during non-CSF-mediated induction of differentiation. This approach provides a source of many normal, proliferating myelomonocytic precursor cells, and introduces possible clinical applications of ex vivo expanded myeloid stem cells.     

Regulation of cell-matrix adhesion by receptor tyrosine kinases

Cell-cell and cell-matrix adhesive interactions mediated by integrins play crucial roles in leukocyte migration to inflamed tissues, and also in cell migration during embryogenesis. Much remains to be learned about the molecular mechanisms of regulation of adhesion mediated by integrins. Recently we found that steel factor and c-kit induce adhesion to fibronectin by VLA-5 in mast cells. Activation of adhesiveness is transient, and occurs at concentrations of steel factor 100-fold lower than required for growth stimulation. This suggests that regulation of adhesion is an important biological function of steel factor and c-kit. Other receptor tyrosine kinases such as the PDGF receptor can substitute for c-kit. Signaling through receptor tyrosine kinases may offer a general mechanism for the regulation of integrin avidity.     

Role of dimerization of the membrane-associated growth factor kit ligand in juxtacrine signaling: the Sl17H mutation affects dimerization and stability-phenotypes in hematopoiesis

The Kit ligand (KL)/Kit receptor pair functions in hematopoiesis, gametogenesis, and melanogenesis. KL is encoded at the murine steel (Sl) locus and encodes a membrane growth factor which may be proteolytically processed to produce soluble KL. The membrane-associated form of KL is critical in mediating Kit function in vivo. Evidence for a role of cytoplasmic domain sequences of KL comes from the Sl17H mutation, a splice site mutation that replaces the cytoplasmic domain with extraneous amino acids. Using deletion mutants and the Sl17H allele, we have investigated the role of the cytoplasmic domain sequences of KL in biosynthetic processing and cell surface presentation. The normal KL protein products are processed for cell surface expression, where they form dimers. Both Sl17H and the cytoplasmic deletion mutants of KL were processed to the cell surface; however, the rate of transport and protein stability were affected by the mutations. Deletion of cytoplasmic domain sequences of KL did not affect dimerization of KL. In contrast, dimerization of the Sl17H protein was reduced substantially. In addition, we have characterized the hematopoietic cell compartment in Sl17H mutant mice. The Sl17H mutation has only minor effects on hematopoiesis. Tissue and peritoneal mast cell numbers were reduced in mutant mice as well as in myeloid progenitors. Interestingly, long-term bone marrow cultures from Sl17H mice did not sustain the long-term production of hematopoietic cells. In addition, homing of normal hematopoietic progenitors to the spleen of irradiated Sl17H/Sl17H recipient mice was diminished in transplantation experiments, providing evidence for a role of Kit in homing or lodging. These results demonstrate that the membrane forms of KL exist as homodimers on the cell surface and that dimerization may play an important role in KL/Kit-mediated juxtacrine signaling.     

Mouse bone marrow stromal cell line MC3T3-G2/PA6 with hematopoietic-supporting activity expresses high levels of stem cell antigen Sca-1

The murine clonal preadipose cell line, MC3T3-G2/PA6 (PA6), has the ability to support in vitro proliferation of hematopoietic stem cells defined as colony-forming units in spleen (CFU-S). In order to ascertain the relationship between the hematopoietic-supporting activity of PA6 cells and their expression, we cultured a number of these cells for over 45 weeks and investigated the level at which they expressed several cell surface markers and membrane-bound growth factors. Besides expressing stem cell factor (SCF) and macrophage colony-stimulating factor (M-CSF), PA6 cells were found by flow cytometry analysis to express high levels of stem cell antigen-1 (Sca-1). The expression level of Sca-1 in PA6 cells correlated with the ability of the latter to support hematopoiesis, whereas no such correlation was observed in the case of SCF and M-CSF expression. A cDNA clone encoding the protein recognized by anti-Sca-1 antibody was isolated from PA6 cells by expression cloning, so that its nucleotide sequence encoded the protein identical to mouse alloantigen Ly-6A.2. Genetically engineered COS-7 cells, transformed by the expression vector carrying the Ly-6A.2 gene, suppressed proliferation of murine lineage marker-negative (Lin) bone marrow cells by themselves and synergistically augmented proliferation of these cells in the presence of SCF. These results suggest that Ly-6A.2 regulates the proliferation of hematopoietic progenitor cells, and is one of the molecules organizing the hematopoietic microenvironment provided by stromal cells.     

Associations between TGF-beta1 receptors in human bone marrow stromal cells

Bone marrow stromal cells are required for sustained haemopoiesis. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine present in the bone marrow microenvironment which regulates the expression of several cytokines, cytokine receptors and cell adhesion elements. The TGF-beta receptors type I and II, and endoglin, mediate TGF-beta1 binding to the membrane of human bone marrow stromal cells. [125I]TGF-beta1-affinity labelling experiments showed that three different anti-endoglin monoclonal antibodies co-immunoprecipitated a 68 kD TGF-beta1-labelled polypeptide together with TGF-beta1/endoglin complexes. Here, we have shown that the 68 kD receptor corresponds to the type I receptor, indicating that endoglin and the type I receptor associate on the membrane of these cells upon ligand binding. The expression of endoglin by stromal cells was found to be up-regulated by TGF-beta1, but not by IL-1beta. The association of endoglin with signalling components of the TGF-beta receptor system on the membrane of bone marrow stromal cells might modulate TGF-beta1 access to the signalling pathways, and therefore it could regulate TGF-beta1-mediated stromal cellular responses.     

Expression and regulation of c-kit receptor and response to stem cell factor in childhood malignant T-lymphoblastic cells

The cytokine stem cell factor (SCF) synergizes with IL-7 to enhance the proliferation of thymocytes. We therefore investigated the role of the SCF receptor, the protooncogene c-kit, in the pathogenesis of pediatric T-lineage malignancies. Expression and regulation of c-kit in cells from children with non-Hodgkin's lymphoma (T-NHL) or acute lymphoblastic leukemia (T-ALL) and the proliferative effect of SCF on these cells were examined in seven cell lines and 21 biopsy tumor cell preparations. Inducibility of c-kit receptors by SCF, IL-1beta, IL-2, IL-7, TGF-beta, TNF-alpha, PMA or calcium ionophore A23187 was studied by flow cytometry (FCM). C-kit receptors were detected in three out of seven T-lymphoblastic cell lines and in nine out of 21 biopsy tumor cell preparations. Upregulation of c-kit could be induced by cultivation, and to a higher extent by cultivation and addition of IL-1beta, TNF-alpha, TGF-beta or A23187. Downregulation of c-kit occurred in the presence of SCF or PMA. SCF caused a downregulation of c-kit receptors in eight of nine, and a proliferative response in three of 11 c-kit-positive T-lymphoblastic cell preparations. We conclude that c-kit is able to transduce a growth stimulatory signal in some T-lymphoblastic cells and that its expression may not be detectable in a resting metabolic or proliferative state.     

Immunophenotypic characterization of stromal cells in aspirated human bone marrow samples

The presence of stromal cells was investigated in aspirated bone marrow prepared by the same method as that used for the initiation of human long-term bone marrow culture (hLTBMC). In previous studies, we performed immunocytochemical staining of cytocentrifuge cell preparations using a panel of antibodies with which we characterized stromal cell populations in hLTBMC. This approach allowed morphological as well as immunophenotypic assessment of cells of interest. Morphologically distinctive cell populations expressing vascular cell adhesion molecule-1 and low-affinity nerve growth factor receptor (NGFR) were observed to be present, but no cells expressing alpha-smooth muscle actin were found. Few macrophages were present, consistent with the origin of hLTBMC stroma-adherent macrophages from monocytes and their precursor cells rather than from mature macrophages among the culture-initiating cells. In the absence of double immunostaining, it was not possible to deduce whether CD34+ cells, which were present in varying numbers in the cytocentrifuge preparations, included stromal as well as primitive hematopoietic cells. In addition to single cells, multicellular tissue fragments containing a variety of stromal cell types were detected in many samples. Their presence raises the possibility that at least some components of hLTBMC stroma may arise by explant growth from complex tissue fragments containing vascular and fibroblastic elements. Overall, our results indicate that demonstration of a variety of stroma-associated antigens, in particular NGFR, provides a useful new tool for identifying stromal elements in aspirated bone marrow.     

Generation of a novel stem cell factor-dependent mast cell progenitor

Tissue mast cell development requires stem cell factor (SCF), whereas helminth-induced intestinal mucosal mast cell hyperplasia also requires T cell-derived factors such as IL-3. We generated progenitor mast cells (PrMC) from mouse bone marrow cells (BMC) in vitro with a triad of SCF, IL-6, and IL-10 that exhibit IL-3-mediated mitogenic and maturation responses. SCF/IL-6/IL-10 transiently elicited a cell subpopulation with the phenotype (c-kit(high)Thy-1(low)) of fetal blood promastocytes at 3 wk of culture that progressed within 1 wk to FcepsilonRI-bearing PrMC, designated PrMCTriad. PrMCTriad lacked mouse mast cell carboxypeptidase A (mMC-CPA) protein, required SCF for IL-3-driven thymidine incorporation, and responded to SCF plus IL-3 with strong mMc-CPA immunoreactivity, clarifying distinct sequential roles for SCF and IL-3 in mast cell development. PrMCTriad, arising from BMC through promastocytes, are metamastocytes that acquire microenvironmentally determined phenotypic features.     

Stromal cell-derived factor-1 alpha and stem cell factor/kit ligand share signaling pathways in hemopoietic progenitors: a potential mechanism for cooperative induction of chemotaxis

Stromal cell-derived factor (SDF-1alpha), the ligand for CXCR4, is a chemokine that acts as a potent chemoattractant for hemopoietic progenitor cells. Stem cell factor/kit ligand (SCF/KL), an early acting cytokine, has recently been reported to enhance the chemotaxis induced by SDF-1alpha. However, very little is known about downstream signaling events following these receptor-ligand interactions. To investigate these events, we utilized a model progenitor cell line, CTS, which expresses both the CXCR4 and c-kit receptors. We observed strong Ca2+ mobilization and enhancement of chemotaxis following treatment with SDF-1alpha or SCF/KL. A combination of these factors enhanced this chemotaxis in CTS cells as well as in CD34+ bone marrow cells. Prior treatment of CTS cells with pertussis toxin inhibited the SDF-1alpha-induced chemotaxis, suggesting that SDF-1alpha signaling involves a pertussis-sensitive Gi-coupled protein. SDF-1alpha treatment resulted in a rapid phosphorylation of the focal adhesion molecules RAFTK (related adhesion focal tyrosine kinase), paxillin, and p130cas, which then declined within minutes. SCF/KL alone or in combination with SDF-1alpha induced a rapid and sustained effect on phosphorylation of these substrates. SDF-1alpha treatment resulted in a rapid and robust activation of p44/42 mitogen-activated protein kinase compared with the relatively weak and delayed effect of SCF/KL treatment. Interestingly, a delayed but sustained activation of mitogen-activated protein kinase activation was observed when the factors were used in combination. Such cooperativity in downstream signaling pathways may explain the enhanced chemotaxis of progenitors observed with SDF-1alpha in combination with SCF/KL.     

A pharmacodynamic study of morphine and its glucuronide metabolites after single morphine dosing in cancer patients with pain

Our previous results showed that hematopoietic stem cells from 16-week-old Sl/Sl(d) mice are not as competitive as congenic +/+ control stem cells. Possible explanations for these findings are that the Steel stem cells are either inherently defective or lose competitive ability by residence in an environment lacking membrane-bound Steel factor. In the present report, any long-term effects of the Steel microenvironment were eradicated by transferring neonatal Sl(d)/Sl(d) marrow and spleen cells into an irradiated but otherwise normal adult hematopoietic microenvironment. Host cells were completely replaced by donor cells within 6 weeks. Eight months after transplantation, the Sl(d)/Sl(d) and similarly treated +/+ littermate control cells from the primary recipient marrow were competed against genetically marked normal cells in an irradiated secondary host. The Steel cells were as competitive as the control cells demonstrating that Steel stem cells are not inherently defective. Results suggest that the stem cells, when retained in the mutant environs into adulthood, are either reduced in number or phenotypically altered by lack of the membrane-bound Steel factor.     

Expression of the c-kit gene is critical for migration of neonatal rat gonocytes in vitro

Rat gonocytes migrate to the basement membrane during the first postnatal week, a change in position crucial for their survival. These cells express the c-kit gene from the day of birth through Day 5 in vivo and develop the ability to migrate in Sertoli cell-gonocyte cocultures. In this study, we asked whether c-kit expression and synthesis of Kit protein are required for pseudopod production by gonocytes in vitro. To determine whether gonocyte migration in vitro is invariably accompanied by c-kit expression, we quantified percentages of gonocytes expressing c-kit with increasing time in vitro and correlated these data with pseudopod development by individual cells. We also determined the effect of exposure to Kit antibodies on gonocyte migration in vitro, and, conversely, asked whether addition of exogenous stem cell factor (SCF), the Kit ligand, stimulates pseudopod development. We found that 1) increasing numbers of gonocytes express c-kit with increasing time in vitro; 2) once these cells begin migrating in vitro, the appearance of a pseudopod on a gonocyte is absolutely correlated with kit expression by that cell; 3) incubating cocultures with Kit antibodies significantly reduces the number of cells with pseudopods, without any detectable decrease in numbers of gonocytes; and 4) addition of exogenous SCF to cocultures prepared on Day 5 results in a transient but significant increase in the percentage of gonocytes with pseudopods even though we found that Sertoli cells in the cultures produce endogenous SCF. Thus, our findings provide evidence to support a role for c-kit expression by neonatal gonocytes and, presumably, SCF expression by neonatal Sertoli cells in stimulating migration of these germ cells in vitro.