Recombinant human (rh) stem cell factor and rhIL-4 stimulate differentiation and proliferation of CD3+ cells from umbilical cord blood and CD3+ cells enhance FcepsilonR1 expression on fetal liver-derived mast cells in the presence of rhIL-4

We previously reported that rhIL-4 induced apoptosis and rhIL-6 mediated protection of human mast cells derived from cord blood mononuclear cells. Based on the result, we attempted to obtain the phenotypes and differentiation of CD3+ cells from cord blood by investigating their cell surface markers in the presence of rhSCF plus rhIL-4. The effect of co-cultured CD3+ cells on fetal liver mast cells (FLMCs) was also determined. Phenotypes from cord blood-derived cells were analyzed by flow cytometry and cell numbers were determined. Fetal liver mast cells were cultured with cord blood-derived cells (mainly CD3+) in the presence of rhSCF and/or rhIL-4 and were analyzed to determine cell number and expression of Kit+ and FcepsilonR1. The percentage of CD3+ cells from cord blood-derived cells on day 0 was about 41 +/- 13.5%, following monocytes and granulocytes. CD3+ cells increased in number (1.5-fold) and purity (90%), whereas other cell types did not survive. More than 60% of CD3+ cells from cord blood at day 0 were CD4(-)CD8-. These double-negative cells dramatically decreased by 1 week of culture, while CD4+CD8+ cells increased in number and purity through 3 weeks of culture, and then decreased as greater numbers of single-positive T cells emerged. We also found that FcepsilonR expression on FLMC increased in the presence of rhIL-4, but was not affected by the T cells that developed from cord blood mononuclear cells. The results indicate that IL-4, a Th2 type cytokine, together with rhSCF, can induce T cell proliferations, differentiation, and maturation from cord blood progenitor cells.     

Neutralizing endogenous IL-6 renders mast cells of the MCT type from lung, but not the MCTC type from skin and lung, susceptible to human recombinant IL-4-induced apoptosis

Human cord blood-derived mast cells undergo apoptosis upon exposure to recombinant human (rh)IL-4 and become resistant to rhIL-4-induced apoptosis when cultured in the presence of rhIL-6. The current study extends these effects of rhIL-4 to different populations of human mast cells, namely fetal liver-derived mast cells, lung-derived mast cells, and skin-derived mast cells. Endogenous production of IL-6 appears to protect fetal liver-derived mast cells and those of the MC(T) phenotype from rhIL-4-mediated apoptosis, because neutralization of IL-6 renders these mast cells sensitive. In contrast, mast cells of the MC(TC) phenotype from skin and lung were resistant to IL-4-mediated apoptosis, even after neutralization of endogenous IL-6. MC(TC) cells were CD124(low), whereas those of the MC(T) cells were CD124(high). These observations extend the phenotypic differences between MC(T) and MC(TC) types of human mast cells to include different functional responses to IL-4.     

IL-3-mediated TNF production is necessary for mast cell development

Mouse mast cell development and survival are largely controlled by the cytokines IL-3 and stem cell factor (SCF). We have found that IL-3 stimulation of bone marrow cells induces the production of TNF via a PI3K- and MAPK kinase/ERK-dependent pathway. Specifically, Mac-1-positive cells were responsible for TNF production, which peaked on days 7-10 of culture and decreased rapidly thereafter. The importance of IL-3-induced TNF secretion was demonstrated by the failure of TNF-deficient bone marrow cells to survive for >3 wk when cultured in IL-3 and SCF, a defect that was reversed by the addition of soluble TNF. The development of human mast cells from bone marrow progenitors was similarly hampered by the addition of TNF-blocking Abs. Cell death was due to apoptosis, which occurred with changes in mitochondrial membrane potential and caspase activation. Apoptosis appeared to be due to loss of IL-3 signaling, because TNF-deficient cells were less responsive than their wild-type counterparts to IL-3-mediated survival. In vitro cultured mast cells from TNF-deficient mice also demonstrated reduced expression of the high affinity IgE receptor, which was restored to normal levels by the addition of soluble TNF. Finally, TNF-deficient mice demonstrated a 50% reduction in peritoneal mast cell numbers, indicating that TNF is an important mast cell survival factor both in vitro and in vivo.     

Degranulation and cytokine expression in human cord blood-derived mast cells cultured in serum-free medium with recombinant human stem cell factor

Human cord blood-derived mast cells (HCMC) grown in medium with serum and recombinant human stem cell factor (rhSCF) with or without interleukin (IL)-6 are less mature than human skin mast cells (HSMC). We found that c-kit-positive HCMC cultured for 8-10 weeks with rhSCF in serum-free medium became sensitive to basic secretatogues and expressed the serine protease, chymase, which is preferentially expressed in HSMC. The HCMC release beta-hexosaminidase (beta-HEX) within 1 min of stimulation with compound 48/80 or substance P, and release was suppressed by pertussis toxin. Approximately 34% of the HCMC in the serum-free culture stained positively with chymase antibody. Chymase and c-kit levels, and responsiveness to basic secretagogues, increased substantially after an additional 2 weeks in a serum-free environment with rhIL-6 and rhSCF. Moreover, Fc(epsilon)RI-dependent activation of the HCMC resulted in induction of cytokines and cyclooxygenase-2. These results show that HCMC can differentiate into a phenotype morphologically and functionally similar to HSMC if exposed to SCF in serum-free medium.     

Pseudomonas aeruginosa-induced human mast cell apoptosis is associated with up-regulation of endogenous Bcl-xS and down-regulation of Bcl-xL

Mast cells play a critical role in the host defense against bacterial infection. Recently, apoptosis has been demonstrated to be essential in the regulation of host response to Pseudomonas aeruginosa. In this study we show that human mast cell line HMC-1 and human cord blood-derived mast cells undergo apoptosis as determined by the ssDNA formation after infection with P. aeruginosa. P. aeruginosa induced activation of caspase-3 in mast cells as evidenced by the cleavage of D4-GDI, an endogenous caspase-3 substrate and the generation of an active form of caspase-3. Interestingly, P. aeruginosa treatment induced up-regulation of Bcl-x(S) and down-regulation of Bcl-x(L). Bcl-x(S), and Bcl-x(L) are alternative variants produced from the same Bcl-x pre-mRNA. The former is proapoptotic and the latter is antiapoptotic likely through regulating mitochondrial membrane integrity. Treatment of mast cells with P. aeruginosa induced release of cytochrome c from mitochondria and loss of mitochondrial membrane potentials. Moreover, P. aeruginosa treatment reduced levels of Fas-associated death domain protein-like IL-1beta-converting enzyme-inhibitory proteins (FLIPs) that are endogenous apoptosis inhibitors through counteraction with caspase-8. Thus, human mast cells undergo apoptosis after encountering P. aeruginosa through a mechanism that likely involves both the Bcl family protein mitochondrial-dependent and the FLIP-associated caspase-8 pathways.     

IL-9 enhances the growth of human mast cell progenitors under stimulation with stem cell factor

We examined the effects of IL-9 on human mast cell development from CD34(+) cord blood (CB) and peripheral blood cells in serum-deprived cultures. IL-9 apparently enhanced cell production under stimulation with stem cell factor (SCF) from CD34(+) CB cells. A great majority of the cultured cells grown with SCF + IL-9 became positive for tryptase at 4 wk. In methylcellulose cultures of CD34(+) CB cells, IL-9 increased both the number and size of mast cell colonies grown with SCF. Furthermore, SCF + IL-9 caused an exclusive expansion of mast cell colony-forming cells in a 2-wk liquid culture of CD34(+) CB cells, at a level markedly greater than for SCF alone. Clonal cell cultures and RT-PCR analysis showed that the targets of SCF + IL-9 were the CD34(+)CD38(+) CB cells rather than the CD34(+)CD38(-) CB cells. IL-9 neither augmented the SCF-dependent generation of progeny nor supported the survival of 6-wk-cultured mast cells. Moreover, there was no difference in the appearance of tryptase(+) cells and histamine content in the cultured cells between SCF and SCF + IL-9. The addition of IL-9 increased numbers of mast cell colonies grown with SCF from CD34(+) peripheral blood cells in children with or without asthma. It is of interest that mast cell progenitors of asthmatic patients responded to SCF + IL-9 to a greater extent than those of normal controls. Taken together, IL-9 appears to act as a potent enhancer for the SCF-dependent growth of mast cell progenitors in humans, particularly asthmatic patients.     

Demonstration of the origin of human mast cells from CD34+ bone marrow progenitor cells.

It has been established that murine mast cells are derived from a pluripotent bone marrow stem cell. In humans, the corresponding pluripotent cell is included in the CD34+ bone marrow population. To determine whether human mast cells arise from CD34+ human progenitor cells, enriched CD34+ cells were cultured over agarose surfaces (interphase cultures) or cocultured with mouse 3T3 fibroblasts in the presence of recombinant human (rh) IL-3. The presence of both mast cells and basophils was determined using a variety of histochemical and immunohistologic techniques, including immunogold labeling for IgE receptors and mast cell tryptase. Mast cells and basophils continued to appear in cultures when T cell, B cell, macrophage, and eosinophil committed progenitor cells were removed, but were not seen in cultures from which CD34+ cells were removed. CD34+ cells layered over agarose in the presence of rhIL-3 were shown to give rise to cultures that contained mast cells (1 to 5%) and basophils (25 to 40%). Cultures supplemented with rhIL-4 showed no additional increase in mast cells or basophils. CD34+ cells cocultured with 3T3 fibroblasts in the presence of rhIL-3 gave rise to mast cells within the fibroblast monolayer, which by 6 wk comprised up to 46% of the monolayer. CD34-cells on 3T3 fibroblasts gave rise to few mast cells (2% of the monolayer). Mast cell granules from interphase cultures contained homogeneous electron-dense material. In contrast, mast cells within 3T3 monolayers at 6 wk contained a variety of granule morphologies, including scroll, mixed, reticular, dense core, or homogeneous patterns. We conclude that both human mast cells and basophils arise from CD34+ human progenitor cells.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human Mast cell progenitors can be infected by macrophagetropic human immunodeficiency virus type 1 and retain virus with maturation in vitro

Mast cells are critical components of innate and adaptive immunity that differentiate in tissues in situ from circulating committed progenitor cells. We now demonstrate that human cord blood-derived mast cell progenitors are susceptible to infection with macrophagetropic (M-tropic) and dualtropic human immunodeficiency virus type 1 (HIV-1) isolates but not with T-cell-tropic (T-tropic) strains. Mast cell progenitors (c-kit(+) CD13(+) cells with chloroacetate esterase activity) were purified from 4-week-old cultures of cord blood mononuclear cells maintained in stem cell factor, interleukin-6 (IL-6), and IL-10 using a CD14 depletion column. These progenitors expressed CCR3, CCR5, and CXCR4, as well as low levels of CD4. When infected in vitro with viruses pseudotyped with different HIV and simian immunodeficiency virus envelope glycoproteins, only M-tropic and dualtropic, but not T-tropic, viruses were able to enter mast cell progenitors. Both the CCR5-specific monoclonal antibody 2D7 and TAK-779, a nonpeptide inhibitor of CCR5-mediated viral entry, blocked HIV-1 strain ADA infection by >80%. Cultures infected with replication-competent virus produced progressively increasing amounts of virus for 21 days as indicated by p24 antigen detection. Mast cell progenitors that were exposed to an M-tropic, green fluorescent protein-expressing HIV-1 strain exhibited fluorescence indicative of viral entry and replication on a single-cell level and retained virus production during differentiation. The trafficking of mast cell progenitors to multiple tissues, combined with the long life span of mature mast cells, suggests that they could provide a widespread and persistent HIV reservoir in AIDS.     

IL-4 induces the proteolytic processing of mast cell STAT6

IL-4 is a potent, pleiotropic cytokine that, in general, directs cellular activation, differentiation, and rescue from apoptosis. However, in mast cells, IL-4 induces the down-regulation of activation receptors and promotes cell death. Mast cells have been shown to transduce IL-4 signals through a unique C-terminally truncated isoform of STAT6. In this study, we examine the mechanism through which STAT6 is processed to generate this isoform. We demonstrate that STAT6 processing in mast cells is initiated by IL-4-induced phosphorylation and nuclear translocation of full-length STAT6 and subsequent cleavage by a nuclear serine-family protease. The location of the protease in the nucleus ensures that the truncated STAT6 has preferential access to bind DNA. IL-4-responsive target genes in mast cells are identified by chromatin immunoprecipitation of STAT6, including the IL-4 gene itself. These results suggest a molecular explanation for the suppressive effects of IL-4 on STAT6-regulated genes in mast cells.     

Intracellular IL-15 controls mast cell survival

The regulation of mast cell activities and survival is a central issue in inflammatory immune responses. Here, we have investigated the role of mouse interleukin-15, a pro-inflammatory and pleiotropic cytokine, in the control of mast cell survival and homeostasis. We report that aged IL-15−/− mice show a reduced number of peritoneal mast cells compared to WT mice. Furthermore, IL-15 deficiency in bone marrow derived mouse mast cells (BMMCs) results in increased susceptibility to apoptosis mediated by growth factor deprivation and A-SMase-treatment. IL-15−/− BMMCs show a constitutive stronger mRNA and protein expression as well as enzymatic activity of the members of the mitochondrial apoptotic pathways including acidic lysosomal aspartate protease cathepsin D (CTSD), endogenous acid sphingomyelinase (A-SMase), caspase-3 and -7 compared to wild type (WT) BMMCs. Furthermore, IL-15−/− BMMCs constitutively generate more A-SMase-derived ceramide than WT controls and display a decreased expression of pro-survival sphingosin-1-phosphate (SPP) both in cytosol and membrane cell fractions. Furthermore, pre-treatment of mast cells with imipramine or pepstatin A, inhibitors of the intracellular acid sphingomyelinase and cathepsin D pathways respectively, increases survival in IL-15−/− BMMCs. These findings suggest that intracellular IL-15 is a key regulator of pathways controlling primary mouse mast cell homeostasis.     

IL-15 expression on RA synovial fibroblasts promotes B cell survival

Introduction

The purpose of this study was to examine the role of RA Synovial Fibroblast (RASFib) IL-15 expression on B cell survival.

Methods

Magnetically sorted peripheral blood memory B cells from 15 healthy subjects were cocultured with RASFib.

Results

RASFib constitutively expressed membrane IL-15. Survival of isolated B cells cultured for 6 days, below 5%, was extended in coculture with RASFib to 52+/−8% (p<0.001). IL-15 neutralizing agents but not isotype controls, reduced this rate to 31+/−6% (p<0.05). Interestingly, rhIL-15 had no effect on isolated B cells but significantly increased their survival in coculture with RASFib. In parallel, B cell IL-15R chains were upregulated in cocultures. BAFF and VCAM-1, that are expressed on RASFib, were tested as potential candidates involved in upregulating B cell IL-15R. Culture of B cells in the presence of rhBAFF or rhVCAM-1 resulted in significantly increased survival, together with upregulation of all three IL-15R chains; in parallel, rhIL-15 potentiated the anti-apoptotic effect of BAFF and VCAM-1. Both BAFF and VCAM-1 neutralizing agents downmodulated the effect of RASFib on B cell survival and IL-15R expression. In parallel, rhIL-15 had a lower effect on the survival of B cells cocultured with RASFib in the presence of BAFF or VCAM-1 neutralizing agents. Peripheral blood B cells from 15 early RA patients demonstrated an upregulated IL-15R and increased survival in cocultures.

Conclusion

IL-15 expression on RASFib significantly contributes to the anti-apoptotic effect of RASFib on B cells. IL-15 action is facilitated by BAFF and VCAM-1 expressed on RASFib, through an upregulation of IL-15R chains.     

IgE enhances Fc epsilon receptor I expression and IgE-dependent release of histamine and lipid mediators from human umbilical cord blood-derived mast cells: synergistic effect of IL-4 and IgE on human mast cell Fc epsilon receptor I expression and mediator release

We investigated the effects of IgE versus IL-4 on Fc epsilon RI surface expression in differentiated human mast cells derived in vitro from umbilical cord blood mononuclear cells. We found that IgE (at 5 micrograms/ml) much more strikingly enhanced surface expression of Fc epsilon RI than did IL-4 (at 0.1-100 ng/ml); similar results were also obtained with differentiated mouse mast cells. However, IL-4 acted synergistically with IgE to enhance Fc epsilon RI expression in these umbilical cord blood-derived human mast cells, as well as in mouse peritoneal mast cells derived from IL-4-/- or IL-4+/+ mice. We also found that: 1) IgE-dependent enhancement of Fc epsilon RI expression was associated with a significantly enhanced ability of these human mast cells to secrete histamine, PGD2, and leukotriene C4 upon subsequent passive sensitization with IgE and challenge with anti-IgE; 2) preincubation with IL-4 enhanced IgE-dependent mediator secretion in these cells even in the absence of significant effects on Fc epsilon RI surface expression; 3) when used together with IgE, IL-4 enhanced IgE-dependent mediator secretion in human mast cells to levels greater than those observed in cells that had been preincubated with IgE alone; and 4) batches of human mast cells generated in vitro from umbilical cord blood cells derived from different donors exhibited differences in the magnitude and pattern of histamine and lipid mediator release in response to anti-IgE challenge, both under baseline conditions and after preincubation with IgE and/or IL-4.     

A critical role for mast cells and mast cell-derived IL-6 in TLR2-mediated inhibition of tumor growth

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.     

IL-13 regulates vascular cell adhesion molecule-1 expression in human osteoblasts

Activated T cells (Act T) produce multiple cytokines that affect osteoblast function as well as osteoclastogenesis. One of these cytokines, IL-13, is a multifunctional cytokine elaborated by Act T that regulates vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. VCAM-1 has also been implicated in osteoclast formation by myeloma cells. We therefore studied whether IL-13 regulates VCAM-1 in human osteoblastic cells since these cells express RANKL, the major osteoclastogenic factor and osteoclast precursors are found adjacent to osteoblasts. Human T cells were activated in the absence or presence of Cyclosporin A (CsA), an inhibitor of the production of most activated T cell cytokines. Conditioned media were assayed for IL-13 by ELISA. Act T produced IL-13 and, unlike other T cell cytokines, this was elevated 3-fold by CsA. Exposure of human osteoblasts (hOB) to doses of recombinant human IL-13 (rhIL-13, 0-10 ng/ml) resulted in an increase of VCAM-1 mRNA (up to 5-fold) within 4 h with a maximum stimulation at 1 ng/ml. CsA had no effect on basal hOB VCAM-1 mRNA expression. Examination of VCAM-1 on the cell surface of hOB, by immunocytochemistry, revealed increasing levels of surface expression of the protein within 16 h after stimulation with doses of rhIL-13 (0.1-10 ng/ml) which were reflective of the mRNAs. IL-6 production was also stimulated in a dose dependent manner with a maximum of 2.5-fold with 1 ng/ml rhIL-13 within 16 h. Since both VCAM-1 and IL-6 showed similar responses to IL-13, IL-6 was examined for its ability to induce VCAM-1. Immunocytochemistry demonstrated no effect of IL-6 on VCAM-1 expression. These data demonstrate that during pathological processes associated with T cell activation, such as rheumatoid arthritis or possibly post-menopausal osteoporosis, T cells may play a pivotal role in osteoclast precursor adhesion to osteoblasts as a first step prior to RANKL signaling.     

Pseudomonas aeruginosa exotoxin A induces human mast cell apoptosis by a caspase-8 and -3-dependent mechanism

Mast cells play an important role in both allergy and innate immunity. Recently, we demonstrated an active interaction between human mast cells and Pseudomonas aeruginosa leading to the production of multiple cytokines. Here, we show that both primary cultured human cord blood-derived mast cells and the human mast cell line HMC-1 undergo apoptosis as determined by single-stranded DNA (ssDNA) formation after stimulation with P. aeruginosa exotoxin A (ETA), a major toxin produced by this bacterium. ETA-induced ssDNA formation was completely inhibited by Z-VAD (where Z is benzyloxycarbonyl), which blocks multiple caspases, suggesting a role for caspases in this process. Active caspase-3 formation in mast cells after an ETA challenge was detected by both Western blotting and flow cytometry analysis. ETA-induced caspase-3 activity in human mast cells was demonstrated by the detection of a characteristic 23 kDa product of D4-GDI (where GDI is guanine nucleotide dissociation inhibitor), an endogenous caspase-3 substrate. Interestingly, a specific caspase-8 inhibitor, Z-IETD-fmk (where fmk is fluoromethyl ketone), blocked ETA-induced cleavage of D4-GDI, but a caspase-9 inhibitor (Z-LEHD-fmk) did not. Treatment of mast cells with caspase-3 inhibitor Z-DEVD-fmk or caspase-8 inhibitor Z-IETD-fmk reduced the generation of ssDNA induced by ETA, suggesting a role for caspase-8 and -3 in ETA-induced mast cell apoptosis. Furthermore, treatment of mast cells with ETA induced decreases of the short form and a long form (p43) of Fas-associated death domain protein (FADD)-like interleukin-1beta-converting enzyme (FLICE) (caspase-8)-inhibitory proteins (FLIPs), which are endogenous caspase-8 inhibitors. Taken together, these results suggest that ETA-induced mast cell apoptosis involves down-regulation of antiapoptotic proteins, FLIPs, and activation of caspase-8 and -3 pathways.     

Murine and human IL-7 activate STAT5 and induce proliferation of normal human pro-B cells

The role of IL-7 in lymphoid development and T cell homeostasis has been extensively documented. However, the role of IL-7 in human B cell development remains unclear. We used a xenogeneic human cord blood stem cell/murine stromal cell culture to study the development of CD19+ B-lineage cells expressing the IL-7R. CD34+ cord blood stem cells were cultured on the MS-5 murine stromal cell line supplemented with human G-CSF and stem cell factor. Following an initial expansion of myeloid/monocytoid cells within the initial 2 wk, CD19+/pre-BCR- pro-B cells emerged, of which 25-50% expressed the IL-7R. FACS-purified CD19+/IL-7R+ cells were larger and, when replated on MS-5, underwent a dose-dependent proliferative response to exogenous human IL-7 (0.01-10.0 ng/ml). Furthermore, STAT5 phosphorylation was induced by the same concentrations of human IL-7. CD19+/IL-7R- cells were smaller and did not proliferate on MS-5 after stimulation with IL-7. In a search for cytokines that promote human B cell development in the cord blood stem cell/MS-5 culture, we made the unexpected finding that murine IL-7 plays a role. Murine IL-7 was detected in MS-5 supernatants by ELISA, recombinant murine IL-7 induced STAT5 phosphorylation in CD19+/IL-7R+ pro-B cells and human B-lineage acute lymphoblastic leukemias, and neutralizing anti-murine IL-7 inhibited development of CD19+ cells in the cord blood stem cell/MS-5 culture. Our results support a model wherein IL-7 transduces a replicative signal to normal human B-lineage cells that is complemented by additional stromal cell-derived signals essential for normal human B cell development.     

Partial purification of a mast cell growth-enhancing activity and its separation from IL-3 and IL-4

A growth factor acting synergistically with IL-3 on thiol-sensitive "mucosal type" bone marrow-derived mast cell lines, and therefore termed mast cell growth enhancing activity, is present in PWM stimulated spleen cell conditioned medium. Mast cell growth enhancing activity can be partially purified and completely separated from IL-3, IL-4, and IL-5, and for the most part from IL-6 and GM-CSF using strong cation exchange and Procion red affinity chromatography. Mast cell growth enhancing activity binds to Con A-Sepharose and can be digested with trypsin and chymotrypsin. It shows a Mr ranging from 37 to 43 kDa under nonreducing SDS-PAGE and a main isoelectric point ranging from 6.2 to 7.3.     

IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release

IL-6 enhances the differentiation of pluripotent hematopoietic stem cells but predominantly affects the differentiation of hematopoietic cells in the granulocyte-macrophage lineage. We have previously shown that multinucleated cells (MNC) with many features of the osteoclast phenotype form in long term human marrow cultures. Addition of rhIL-6 (10 to 100 pg/ml) to these cultures significantly increased MNC formation, with greater than 80% of the MNC expressing an Ag that cross-reacts with the mAb 23c6. This antibody preferentially binds to osteoclasts. rhIL-6 did not enhance MNC formation in marrow cultures treated with 1,25 dihydroxyvitamin D3, a potent stimulator of MNC formation, but significantly increased the percentage of MNC that cross-reacted with the 23c6 mAb. Addition of antihuman IL-1 to cultures treated with rhIL-6 totally inhibited the increase in MNC formation stimulated by rhIL-6. In contrast, anti-IL-1 did not affect MNC formation stimulated by 1,25 dihydroxyvitamin D3. Further, conditioned media from marrow cultures exposed to rhIL-6 contained elevated levels of IL-1 beta (500 pg/ml compared to 23 pg/ml in control cultures 15 h after IL-6 addition). These results suggest that the capacity of rhIL-6 to stimulate formation of MNC which cross-react with 23c6 is mediated by induction of release of IL-1 beta.