Addition Of Stem Cell Factor Research Articles

Characterization of adhesive interactions between mast cells and laminin isoforms: evidence of a principal role for alpha 6 integrin.

Mast cells are known to adhere to laminin, although there is limited information on the characteristics of this event. To further examine this adhesive interaction, we thus determined the adherence of murine mast cell lines and primary bone marrow cultured mast cells (BMCMC) to murine laminin (mLN), human placental laminin-1 (hLN), merosin (laminin-2) and various laminin fragments, concentrating on activating stimuli, the involvement of integrins, and the effect on mast cell activation. Murine mast cells were found to adhere to both mLN and hLN and to merosin, not only following exposure to phorbol 12-myristate 13-acetate (PMA), but also after Fc epsilon RI aggregation or addition of stem cell factor (SCF). Adhesion to laminin was partially inhibited by soluble E8 and PA22-2, both fragments of laminin that promote mast-cell adhesion when bound on surfaces. Mast-cell lines and BMCMC consistently expressed high levels of alpha 6 integrin. Antibody to alpha 6 blocked spontaneous and inhibited activated mast-cell adhesion to hLN, and inhibited mast-cell adhesion to mLN and its fragment E8. Mast-cell adhesion to both laminin isoforms increased Fc epsilon RI-mediated mast-cell secretion. These observations demonstrate that mast-cell attachment to laminin is promoted by physiological stimuli, is mediated principally by alpha 6 integrin, and results in enhanced cell activation.

Abnormal Expression of Mouse Mast Cell Protease 5 Gene in Cultured Mast Cells Derived From Mutant mi/mi Mice

AbstractMast cells contain a lot of mast cell-specific proteases. We have reported that the expression of mouse mast cell protease 6 (MMCP-6) is remarkably reduced in both cultured mast cells (CMCs) and skin mast cells of mi/mi mutant mice. In the present study, we found that the expression of MMCP-5 was reduced in CMCs but not in skin mast cells of mi/mi mice, and we compared the regulation mechanisms of MMCP-5 with those of MMCP-6. The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF ). The consensus sequence recognized and bound by bHLH-Zip transcription factors is CANNTG. The overexpression of the normal (+) MITF but not of mi-MITF normalized the poor expression of the MMCP-5 gene in mi/mi CMCs, indicating the involvement of +-MITF in transactivation of the MMCP-5 gene. Although +-MITF directly bound CANNTG motifs in the promoter region of the MMCP-6 gene and transactivated it, the binding of +-MITF to the CAGTTG motif in the promoter region of the MMCP-5 gene was not detectable. The +-MITF appeared to regulate the transactivation of the MMCP-5 gene indirectly. Moreover, addition of stem cell factor to the medium normalized the expression of the MMCP-5 but not of the MMCP-6 gene in mi/mi CMCs. Despite the significant reduction of both MMCP-5 and MMCP-6 expressions in mi/mi CMCs, their regulation mechanisms appeared to be different.

Functional and morphological characterization of immunomagnetically selected CD34+ hematopoietic progenitor cells.

We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony-forming cells and their precursors, detected as long-term culture-initiating cells (LTC-IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8-fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC-IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34-enriched fractions. In five cases CD34+ selected cells grown in a stroma-free long-term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM-CSF every 48 h, showed a 15 (+/- 15) and 31 (+/- 21) mean colony forming unit-granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well-differentiated myeloid cells (MPO+), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37 degrees C, the majority of the cells showed no iron particles, but about 30% of the cells were iron-labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short-term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.

Major Histocompatibility Complex Class II-Mediated Inhibition of Hematopoiesis in Long-Term Marrow Cultures Involves Apoptosis and Is Prevented by c-kit Ligand

Expression of major histocompatibility complex (MHC) class II molecules is developmentally regulated and lineage dependent. Their role in hematopoiesis is not well defined. Previous studies in a canine model showed that dogs given 920 cGy of total body irradiation, transplanted with autologous marrow, and treated with anti-MHC class II monoclonal antibody (MoAb) immediately posttransplant experienced only a transient granulocyte recovery that was followed by graft failure. In the present study, the effect of anti-MHC class II MoAbs on canine in vitro hematopoiesis was investigated. Anti-MHC class II MoAb H81.9 or B1F6 (both recognizing nonpolymorphic determinants) had no inhibitory effect when added directly to colony-forming unit-granulocyte-macrophage (CFU-GM) grown in agar. However, the addition of intact MoAb or as F(ab')2 fragments to long-term marrow cultures (LTMCs) resulted in a dose-dependent inhibition of the generation of CFU-GM among nonadherent cells. Inhibition was most profound with MoAb added at the time of initiation of culture. However, even if MoAb was added 3 weeks after recharging LTMCs, CFU-GM generation rapidly decreased. In addition, the number of adherent cells in LTMCs decreased; predominantly fibroblast-like cells with prominent cytoplasmic vesiculation remained. Acridine orange/ethidium bromide staining and TdT-mediated deoxyuridine triphosphate-digoxigenin nick end labeling (TUNEL) tests showed an increase in the proportion of apoptotic cells in both the nonadherent and adherent compartments. Binding of anti-MHC class II MoAb to unfractionated marrow cells resulted in an increase in free (Ca2+)i; no changes in tyrosine phosphorylation pattern were observed. The addition of stem cell factor (SCF), but not granulocyte colony-stimulating factor or granulocyte-macrophage colony-stimulating factor, to LTMCs prevented apoptosis, and the generation of CFU-GM was indistinguishable from controls. Similarly, a supportive adherent layer was maintained. Thus, anti-MHC class II MoAbs interfere with hematopoiesis both in vitro and in vivo. The mechanism involves programmed cell death in subpopulations of adherent and nonadherent cells. Inhibition of hematopoiesis is abrogated by exogenous SCF.

Transforming growth factor-beta prevents stem cell factor-mediated rescue of mast cells from apoptosis after IL-3 deprivation.

IL-3-dependent mast cells undergo apoptosis upon removal of IL-3, an event that is prevented by the addition of stem cell factor (SCF) acting through its receptor c-kit, suggesting that SCF provides a mechanism to allow mast cells to survive and to differentiate in tissues in the relative absence of IL-3. This observation is consistent with the thesis that the microenvironment, in part, controls mast cell number and viability by modulating SCF production and release. The purpose of the present study was to determine whether a second factor, TGF-beta 1, was capable of modifying the SCF-mediated survival pathway. TGF-beta 1 (1 and 10 ng/ml), known to be an important regulator of cell growth and function, did inhibit the SCF-mediated rescue from apoptosis in IL-3-deprived mast cells. TGF-beta 1 exerted its inhibitory effect on SCF-mediated rescue from apoptosis, even when added 4 h after the addition of SCF. In contrast, TGF-beta 1 had no substantial effect on the viability of mast cells that were grown in the presence of IL-3. TGF-beta 1 also had no noticeable effect on viability and proliferation of a growth factor-independent mast cell line. The inhibitory effect of TGF-beta 1 was neutralized by specific anti-TGF-beta mAb. TGF-beta 1 did not affect the expression of c-kit, as determined by using flow cytometric analysis of mast cells labeled with FITC-conjugated anti-c-kit. These results demonstrate how SCF and TGF-beta may act in concert to regulate mast cell numbers under physiologic or pathologic conditions.

Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis).

Proliferating primordial germ cells (PGCs) isolated from mouse embryos soon after their arrival in the genital ridges would only survive in vitro at temperature of less than 30 degrees C (De Felici, M. and McLaren, A. (1983). Exp. Cell. Res. 144, 417-427; Wabik-Sliz, B. and McLaren, A. (1984). Exp. Cell. Res. 154, 530-536) or when co-cultured on cell feeder layers (Donovan, P. J., Stott, D., Godin, I., Heasman, J. and Wylie, C. C. (1986). Cell 44, 831-838; De Felici, M. and Dolci, S. (1991). Dev. Biol. 147, 281-284). In the present paper we report that mouse PGC death in vitro occurs with all the hallmarks of programmed cell death or apoptosis. We found that after 4-5 hours in culture many PGCs isolated from 12.5 dpc fetal gonads assumed a nuclear morphology and produced membrane bound fragments (apoptotic bodies) typical of apoptotic cells. In addition, PGCs in culture accumulated high level of tissue transglutaminase (tTGase; an enzyme that is induced and activated during apoptosis) and showed extensive degradation of DNA to oligonucleosomal fragments, which is characteristic of apoptosis. The physiological relevance of this mechanism of PGC death is supported by the finding that some PGCs undergoing apoptosis, as revealed by the high level of tTGase expression, were detected in the embryo. Most importantly, we show that the addition of stem cell factor (SCF) or leukemia inhibitory factor (LIF) to the culture medium, two cytokines known to favour PGC survival and/or proliferation in vitro, markedly reduced the occurrence of apoptosis in PGCs during the first hours in culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Expansion of Primitive Human Hematopoietic Progenitors in a Perfusion Bioreactor System with IL-3, IL-6, and Stem Cell Factor

Present methods for long-term hematopoietic culture (LTHC) employ a static culture environment which is not well-characterized. Primitive long-term culture-initiating cell (LTC-IC) numbers have been shown to decline in conventional static human LTHC, even with exogenous cytokine combinations. We have expanded human hematopoietic cells from umbilical cord blood on a preformed marrow stroma with synergistic cytokine combinations in a novel perfusion bioreactor system, which continuously maintained culture conditions within desired ranges. Interleukin-3 (IL-3) and interleukin-6 (IL-6) in perfusion culture resulted in rapid 7-day expansion of granulocyte-macrophage colony forming units (CFU-GM, 11-fold), erythroid burst-forming units (BFU-E, 2.5-fold), and granulocyte-erythroid-macrophage colony forming units (CFU-Mix, 2.4-fold), compared to 6-fold, 1.4-fold, and no expansion, respectively, in static cultures. Addition of stem cell factor (SCF) to IL-3/IL-6 in static culture increased the extent of CFU-GM expansion (to 9-fold), but did not result in BFU-E or CFU-Mix expansion. In perfusion cultures with IL-3/IL-6/SCF, much greater expansions of CFU-GM (18-fold) and CFU-Mix (5.3-fold) were obtained. More importantly, expansion of LTC-IC (nearly 3-fold in two of three experiments) was only obtained with IL-3/IL-6/SCF and perfusion. The ability to expand hematopoietic cells while maintaining or expanding primitive progenitors has potential clinical applications in bone marrow transplantation and gene therapy.

Human mast cells but not human basophils coculture with mouse 3T3 fibroblasts

Human mast cells and basophils have been shown to arise from CD34+ pluripotent progenitor cells in the presence of rhIL-3 (1). Two different culture systems using CD34+ cells, the agarose interphase culture and 3T3 fibroblast/CD34 + coculture, give rise to mast cells and basophils but with markedly different results. (1,2). By 3 weeks in interphase agarose cultures, CD34+ cells give rise to approximately 25-45% basophils and 1-5% tryptase positive mast cells. These mast cells have homogeneous granule patterns that resemble immature mast cells. With the addition of stem cell factor (rhSCF) (3) to rhIL-3, total cell number increases and mast cell maturation is promoted without altering the percentages of mast cells and basophils (4). These mast cells have tryptase-positive granules, with characteristic patterns seen in mature mast cells. In contrast, CD34+ cells cocultured with mouse 3T3 fibroblasts give rise by 6 weeks to approximately 50% tryptase positive mast cells which adher to the monolayer and possess granule scroll patterns. No basophils are ever identified by six weeks in cocultures.