Natural cytotoxic (NC) cell activity in basophilic cells: release of NC-specific cytotoxic factor by IgE receptor triggering

A murine interleukin 3 (IL 3)-dependent basophilic mast cell line, PT-18 (A17), and a rat basophilic leukemic cell line, RBL-2H3, were shown to be capable of selective natural cytotoxic (NC) but not natural killer (NK) cell activity. The basophilic cell types could also be augmented in their NC activity by bridging of their surface IgE receptors. IgE-mediated triggering of the basophilic cells was accomplished by coating the cells with IgE and exposing the IgE-bound cells to specific antigen or to anti-IgE monoclonal antibody. Another method of triggering was by direct binding of basophilic cells to anti-IgE receptor monoclonal antibody. Basophilic cells, triggered by these methods, not only displayed increased NC activity but also released a soluble factor capable of selectively lysing NC tumor targets, WEHI-164, but not three of the NK-sensitive targets, YAC-1, RLM1, and RBL-5. Normal C3H/HeJ mouse embryonic fibroblasts were also not lysed. Dose response and time course of the cytotoxic factor release from triggered RBL-2H3 cells were similar to those of tritiated serotonin release. As with serotonin or histamine release, the NC-specific cytotoxic factor (NCCF) was not released in the absence of extracellular calcium. Therefore, NCCF appears to be released along with other mediators during the triggering of basophilic cells by bridging of IgE receptors. The m.w. of the native form of this factor, determined by a gel filtration method, was about 43,000.     

Inhibition of the growth of IL-3-dependent mast cells from murine bone marrow by recombinant granulocyte macrophage-colony-stimulating factor

The mouse mast cell line PT-18 demonstrates [3H] thymidine uptake in the presence of either mouse IL-3 or mouse recombinant granulocyte-macrophage CSF (rGM-CSF). Experiments were thus undertaken to determine whether rGM-CSF would affect IL-3-dependent growth of mast cells from mouse bone marrow cells (BMC). BMC placed in liquid culture containing 50 U/ml of IL-3 gave rise to cultures containing up to 95% mast cells by 2 to 3 wk. The rise in percentage of mast cells was accompanied by an increase in total cell-associated histamine. In contrast, BMC grown in the presence of 50 U/ml of rGM-CSF gave rise to cultures containing primarily macrophages and granulocytes with less than 1% mast cells. The addition of increasing amounts of rGM-CSF to BMC cultures grown in the presence of IL-3 resulted in a decrease in the number of mast cells present in culture at 2 to 3 wk. Cells other than mast cells in these cultures consisted principally of granulocytes and macrophages. The rGM-CSF-related inhibition of mast cell growth was not abrogated by the addition of indomethacin to cultures. Granulocyte-macrophage cell populations added to IL-3-containing cultures did not inhibit mast cell growth. The suppressive effect of rGM-CSF on IL-3-dependent mast cell growth may indicate an important role for GM-CSF in the down-regulation of mast cell proliferation in tissues.     

IL-12/IL-18-dependent IFN-gamma release by murine dendritic cells.

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c(+) myeloid DC from 7-day cultures stimulated with TNF-alpha, IFN-alpha, IFN-gamma, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low "spontaneous" release of IL-18, and did not release IFN-gamma. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-gamma expression and release in 15-20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-gamma release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8alpha(+) and CD8alpha(-) DC (from immunocompetent and immunodeficient H-2(d) and H-2(b) mice) cultured with IL-12 and IL-18 released IFN-gamma. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-gamma release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-gamma release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-gamma generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.     

Prostaglandin E2 selectively enhances the IgE-mediated production of IL-6 and granulocyte-macrophage colony-stimulating factor by mast cells through an EP1/EP3-dependent mechanism

PGE(2) is an endogenously synthesized inflammatory mediator that is over-produced in chronic inflammatory disorders such as allergic asthma. In this study, we investigated the regulatory effects of PGE(2) on mast cell degranulation and the production of cytokines relevant to allergic disease. Murine bone marrow-derived mast cells (BMMC) were treated with PGE(2) alone or in the context of IgE-mediated activation. PGE(2) treatment alone specifically enhanced IL-6 production, and neither induced nor inhibited degranulation and the release of other mast cell cytokines, including IL-4, IL-10, IFN-gamma, and GM-CSF. IgE/Ag-mediated activation of BMMC induced the secretion of IL-4, IL-6, and GM-CSF, and concurrent PGE(2) stimulation synergistically increased mast cell degranulation and IL-6 and GM-CSF, but not IL-4, production. A similar potentiation of degranulation and IL-6 production by PGE(2), in the context of IgE-directed activation, was observed in the well-established IL-3-dependent murine mast cell line, MC/9. RT-PCR analysis of unstimulated MC/9 cells revealed the expression of EP(1), EP(3), and EP(4) PGE receptor subtypes, including a novel splice variant of the EP(1) receptor. Pharmacological studies using PGE receptor subtype-selective analogs showed that the potentiation of IgE/Ag-induced degranulation and IL-6 production by PGE(2) is mediated through EP(1) and/or EP(3) receptors. Our results suggest that PGE(2) may profoundly alter the nature of the mast cell degranulation and cytokine responses at sites of allergic inflammation through an EP(1)/EP(3)-dependent mechanism.     

Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.     

Activation- and phorbol ester-stimulated phosphorylation of a plasma membrane glycoprotein antigen expressed on mouse IL-3-dependent mast cells and serosal mast cells

As assessed by immunoprecipitation analyses, expression of the epitope recognized by the rat mAb B23.1 is approximately sevenfold greater on the surface of mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) than on serosal mast cells (SMC) obtained directly from the peritoneal cavity. Immunoprecipitation of B23.1 antibody-binding molecules from Na[125I] surface-labeled BMMC and SMC followed by sizing on SDS-polyacrylamide gels under reducing conditions demonstrated that the epitope is located on molecules of 49,000 and 47,500 Mr, respectively. An additional immunoprecipitated molecule of 42,000 Mr was detected from BMMC intrinsically radiolabeled with [35S]methionine, and pulse-chase analyses revealed that this species was a biosynthetic precursor of the 49,000 Mr cell surface form of the Ag. Treatment of the immunoprecipitated 42,000 and 49,000 Mr forms with endoglycosidase F reduced the Mr of both to 37,000, as did intrinsic radiolabeling of BMMC in the presence of tunicamycin, indicating that both the 42,000 Mr precursor form and the 49,000 Mr cell surface molecule (gp49) contained N-linked carbohydrate. Activation of [32P]orthophosphate-labeled BMMC by sensitization with mouse monoclonal IgE anti-TNP and challenge with TNP-BSA or by exposure to the calcium ionophore A23187 elicited the rapid phosphorylation of gp49 but not of its precursor forms, as did treatment of the cells with PMA. Elution of phosphorylated and immunoprecipitated gp49 from SDS-polyacrylamide gels followed by partial acid hydrolysis of the protein and phosphoamino acid analysis by high voltage thin-layer electrophoresis on cellulose plates indicated that serine, but not threonine or tyrosine, was phosphorylated upon stimulation of BMMC with IgE/Ag, calcium ionophore, or PMA. Cholera toxin did not elicit phosphorylation of gp49. These data suggest that gp49, a plasma membrane glycoprotein preferentially expressed by mouse BMMC, may be either directly or indirectly phosphorylated via protein kinase C during mast cell activation-secretion.     

IL-3-dependent mast cells attach to plate-bound vitronectin. Demonstration of augmented proliferation in response to signals transduced via cell surface vitronectin receptors.

The adhesive interactions of activated mast cells with the extracellular matrix play an important role in anchorage and cellular motility. In this report we demonstrate that IL-3-dependent bone marrow-derived mast cells adhere to plate-bound vitronectin with high affinity in a saturable and dose-dependent manner. This adhesion interaction is unique in that it does not require prior mast cell activation through Fc epsilon RI or after treatment with PMA. It is inhibited by divalent cation chelation and by competitive inhibition with a synthetic Arginine-Glycine-Aspartate-Serine tetrapeptide. Polyclonal antisera for alpha v beta 3, an integrin known to bind vitronectin, inhibits attachment to plate-bound vitronectin in a dose-dependent manner. Comparison of the adhesion interactions for vitronectin, fibronectin, and laminin indicate that adhesion to vitronectin is greater than that seen with either fibronectin or laminin, either in the presence or absence of PMA. FACS analysis using a monoclonal hamster anti-murine vitronectin receptor (alpha v) antibody followed by a fluorescein-conjugated rabbit anti-hamster IgG revealed no change in surface vitronectin receptor expression after Fc epsilon RI-mediated cell activation. Proliferation assays with correction for cell viability revealed a 25% increase in cell number above the maximal IL-3 response over a 24-h period of adhesion to a vitronectin-coated surface and a 41% increase over 96 h of adhesion to vitronectin. Binding to plate-bound vitronectin was not able to sustain cell viability in the absence of IL-3. Thus, IL-3-dependent bone marrow-derived mast cells adhere to vitronectin, an extracellular matrix protein present throughout connective tissues. This interaction generates a signal that results in the augmentation of the maximal IL-3-dependent mast cell proliferative response, thus demonstrating at least one way in which the interaction between mast cells and extracellular matrix alter the biologic responsiveness of the mast cell.     

Failure to detect IL-3-binding sites on human mast cells

IL-3, a pleiotropic lymphokine, has been termed mast cell growth factor because it promotes growth and differentiation of murine mast cells. Murine mast cells, in turn, express cell surface receptors for IL-3. Human rIL-3 has been shown to induce proliferation and differentiation of human basophils and to activate basophils via high affinity binding sites. To investigate whether human mast cells express IL-3R, binding studies with 125I-radiolabeled human rIL-3 were performed on HMC-1, a novel human mast cell line, and on pure populations (i.e., 93 to 99% purity) of human tissue mast cells obtained with mAb and C from dispersed lung (n = 2). Unexpectedly, neither enriched human lung mast cells nor HMC-1 cells bound radiolabeled human rIL-3 specifically. Moreover, human rIL-3 failed to promote uptake of [3H]thymidine, synthesis of histamine, histamine releasability, or changes in expression of mast cell differentiation Ag (YB5B8, CD54/ICAM-1, CD9/p24, CD33/gp67) on either human lung mast cells or HMC-1 cells. It is hypothesized that the fundamental difference in the biologic response to IL-3 between human and murine mast cells is due to a loss during evolution of mast cell high affinity IL-3 binding sites.     

Insulin-like growth factor-I inhibits apoptosis in IL-3-dependent hemopoietic cells.

The death of hemopoietic cells on withdrawal of CSF occurs by a mechanism known as apoptosis characterized by the early degradation of chromatin into oligonucleosome-length fragments. Insulin-like growth factor I plays a pivotal role in the regulation of somatic cell growth as a mediator of growth hormone action. Animals with low levels of circulating IGF-I are more vulnerable to infections and have diminished immune responses. To analyze the possibility of a regulatory role of IGF-I on hemopoiesis and determine its mechanism of action, we have studied the effect of this growth factor on the survival and proliferation of two IL-3-dependent hemopoietic cell lines and in IL-3-responsive primary cultures of bone marrow-derived mast cells. In IL-3-depleted cultures, IGF-I prevented DNA fragmentation and apoptotic cell death. Insulin at high concentration had a weak protective action and IGF-II was inactive in suppressing apoptosis in these IL-3-dependent hemopoietic cells. Cell proliferation was also stimulated by IGF-I in the absence of other hemopoietic growth factors although it was a weak mitogen when compared with IL-3. These results indicate that circulating or locally produced IGF-I may promote survival of both the steady state hemopoietic precursor population and cytokine-producing cells and could therefore regulate hemopoiesis acting in a concerted manner with other CSF.     

PI3-K/Akt pathway contributes to IL-6-dependent growth of 7TD1 cells

Background  

Recently, growing evidence suggests the involvement of PI 3-K/Akt in IL-6-dependent survival and proliferative responses in several types of cells. However, whether PI 3-K/Akt plays the same role in IL-6-dependent growth of 7TD1 mouse-mouse B cell hybridoma is not known.     

Characterization of IL-2-dependent cytotoxic T-cell clones. III. Inhibition of killing activity by monosaccharides

The effects of monosaccharides on the cytotoxic activity of cytotoxic T lymphocytes (CTL) and three cloned long-term cytotoxic T-lymphocyte lines (CTLL) are compared. Uncultured CTL and clones CTLL-A2 and CTLL-A11 were derived from the peritoneal cavity of C57BL/6 mice immunized against the H-2Dd determinants on the BALB/c sarcoma Meth A. Clone CTLL-R5 was derived from spleen of (BALB/c X C57BL)F1 mice immunized against a unique determinant on the BALB/c radiation-induced leukemia RL male 1. The cell-surface phenotype of the clones is Lyt-1+,2+,3+. Cytotoxic activity of CTLL-A2 and CTLL-R5 as determined by a 4-hr 51Cr-release assay was inhibited over 50% by 1 mM 2-deoxy-D-glucose. CTLL-A11 and the uncultured cytotoxic T cells were more resistant to inhibition by 2DG (40% at 20 mM). Surprisingly, it was found that the addition of D-mannose, D-galactose, D-glucose, L-fucose, alpha-methyl-D-mannose, and N-acetyl-D-glucosamine also inhibited, in a dose-related manner, the cytotoxicity of CTLL-A2 and CTLL-A11. CTLL-R5 showed a more restricted inhibition pattern: only D-mannose and D-galactose were inhibitory. The mechanism of inhibition remains to be clarified.     

Relationship of IgE receptor topography to secretion in RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking IgE-receptor complexes with anti-IgE antibody leads to degranulation. Receptor cross-linking also stimulates the redistribution of receptors on the cell surface, a process observed here by labeling the anti-IgE with 15 nm protein A-gold particles that are visible by back-scattered electron imaging in the scanning electron microscope. We report that anti-IgE binding stimulates the redistribution of IgE-receptor complexes at 37 degrees C from a dispersed topography to distributions dominated sequentially by short chains, small clusters, and large aggregates of cross-linked receptors. Cells incubated with 1 microgram/ml anti-IgE, a concentration that stimulates maximum net secretion, redistribute receptors into chains and small clusters during a 15 min incubation period. At 3 and 10 micrograms/ml anti-IgE, net secretion is reduced and the majority of receptors redistribute rapidly into clusters and large aggregates. The addition of Fab fragments with the high anti-IgE concentrations, to reduce cross-linking, delays receptor aggregation and enhances secretion. The progression of receptors from small clusters to large aggregates is prevented in cells treated with dihydrocytochalasin B to prevent F-actin assembly. These results establish that characteristic patterns of receptor topography are correlated with receptor activity. In particular, they link the formation of large receptor aggregates to reduced signalling activity. Cytoskeleton-membrane interaction is implicated in the formation or stabilization of the large receptor clusters.     

In vitro growth of bone marrow-resident T cell precursors supported by mast cell growth factor and IL-3.

The growth requirements of bone marrow-resident cells that are able to differentiate along the T cell lineage (pre-T cells) have not been well established. We recently have shown that the T cell-derived lymphokine IL-3 is able to maintain pre-T cells in vitro for at least 2 weeks. However, in our initial studies, we were not able to ascertain whether IL-3 induced pre-T cell growth during culture, or whether IL-3 simply maintained the viability of these progenitors. To address this issue, we used a multiple dose assay system to assess the level of pre-T cell activity (thymic repopulation) in a selected population of bone marrow cells (CD3-, Thy-1.2+) both before and after culture in IL-3. In addition, we tested the potential role of mast cell growth factor (MGF) in the growth and maintenance of pre-T cells in vitro. The results of these studies showed that IL-3 produced a modest, but consistent increase in the pre-T cell activity during culture. Culture of CD3-, Thy-1.2+ bone marrow cells in MGF also resulted in an increase in the total amount of detectable pre-T cell activity among the cultured cells. The most dramatic increases in pre-T cell activity, however, were induced by the culture of the selected marrow cells in both MGF and IL-3. Cultures supplemented with both cytokines produced net increases in pre-T cell activity of 40- to 75-fold after 10 days of culture. Because the increases in pre-T cell activity were not accompanied by observable increases in the size of thymic colonies produced by the pre-T cells, the increased levels of pre-T cell activity appeared to result from increases in pre-T cell numbers during culture. Thus, in addition to the other activities ascribed to MGF, this cytokine displays pre-T cell growth factor activity and can synergize with IL-3 in that capacity. The use of MGF in conjunction with IL-3 provides the best system described to date for the propagation of pre-T cells in primary bone marrow cell cultures.     

Down-regulation of wild-type p53 activity interferes with apoptosis of IL-3-dependent hematopoietic cells following IL-3 withdrawal.

Overexpression of wild-type p53 in p53-deficient leukemic cells induces apoptosis, which can be inhibited by hematopoietic survival factors. This suggests that p53 may contribute to survival factor dependence. To assess the role of wild-type p53 in mediating apoptosis following survival factor withdrawal, we interfered with endogenous p53 activity in interleukin-3 (IL-3)-dependent cells. Extended survival without IL-3 was conferred by recombinant retroviruses encoding either a full-length p53 mutant or a C-terminal p53 miniprotein, both of which can act as negative-dominant inhibitors of wild-type p53. On the other hand, excess wild-type p53 activity failed to elicit apoptosis as long as IL-3 was present. We propose that p53 is a positive, though not exclusive, mediator of survival factor dependence in hematopoietic cells.     

Human intestinal fibroblasts prevent apoptosis in human intestinal mast cells by a mechanism independent of stem cell factor, IL-3, IL-4, and nerve growth factor

In rodents, fibroblasts (FBs) mediate stem cell factor (SCF)-dependent growth of mast cells (MCs). In humans, SCF is mandatory for MC differentiation and survival. Other factors such as IL-3, IL-4, and nerve growth factor (NGF) act in synergism with SCF, thus enhancing proliferation and/or preventing apoptosis in MCs. In this study, we studied in vitro interactions between human MCs and human FBs, both isolated from the intestine and purified to homogeneity. In coculture with FBs, MCs survived for up to 3 wk, whereas purified MCs cultured alone died within a few days. TNF-alpha and IL-1beta, which both did not affect MC survival directly, enhanced FB-dependent MC growth. We provide evidence that FB-derived MC growth factors are soluble, heat-sensitive molecules which down-regulate MC apoptosis without enhancing MC proliferation. However, only low amounts of SCF were measured in FB-conditioned medium (<0.2 ng/ml). Moreover, blocking of SCF/c-kit interaction by anti-SCF or anti-c-kit Abs and neutralization of IL-3, IL-4, and NGF did not affect MC survival in the coculture system. In conclusion, our data indicate that human FBs promote survival of human MCs by mechanisms independent of SCF, IL-3, IL-4, and NGF. Such interactions between MCs and FBs may explain why MCs accumulate at sites of inflammatory bowel disease and intestinal fibrosis.     

Induction of Ym1/2 in mouse bone marrow-derived mast cells by IL-4 and identification of Ym1/2 in connective tissue type-like mast cells derived from bone marrow cells cultured with IL-4 and stem cell factor

Mast cells play an important role in allergic inflammation by releasing various bioactive mediators. The function of mast cells is enhanced by various stimuli, partly due to the induction of specific genes and their products. Although many inducible genes have been identified, a significant number of genes remain to be identified. Therefore, this study used PCR-selected cDNA subtraction to establish the profile of induced genes in the connective tissue (CT) type-like mast cells derived from bone marrow cells cultured in the presence of IL-4 and stem cell factor. Two hundred and fifty cDNA clones were obtained from the CT type-like mast cells by PCR-selected cDNA subtraction. Among them, Ym1/2, a chitinase-like protein, is one of the most abundantly induced genes. Ym1 is produced by activated macrophages in a parasitic infection, whereas its isotype, Ym2, is highly upregulated in allergic lung disease. In order to differentiate which isotype is expressed in bone marrow cells, specific primers for bone marrow-derived mast cells (BMMC), and CT type-like mast cells were used for RT-PCR. The results showed that Ym1 was constitutively expressed in bone marrow cells and gradually decreased in the presence of IL-3, whereas Ym2 was induced only in the presence of IL-4. CT type-like mast cells from bone marrow cells expressed Ym1 throughout the culture period and Ym2 was induced only by the addition of IL-4 into BMMC, indicating that IL-4 is essential for the expression of Ym1/2 genes.