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The Journal of Immunology, 2006, 176: 2114-2121.
Copyright © 2006 by The American Association of Immunologists

IL-3-Mediated TNF Production Is Necessary for Mast Cell Development1

Harry V. Wright*, Daniel Bailey*, Mohit Kashyap*, Christopher L. Kepley{dagger}, Marina S. Drutskaya{ddagger}, Sergei A. Nedospasov{dagger},§ and John J. Ryan2,*

* Department of Biology and {dagger} Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA 23284; {ddagger} Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia; and § Laboratory of Molecular Immunoregulation, Center for Cancer Research and Basic Research Program, Science Applications International Corp.-Frederick, National Cancer Institute, MD 21702


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells exhibit widespread distribution in the tissues, absent only in the blood. They are ideally suited to initiate protective immune responses against pathogens due to their location at the host/environment interface and their secretion of vasoactive and inflammatory mediators. These mediators include histamine, heparin, proteolytic enzymes, arachidonic acid metabolites such as PGs and leukotrienes, and an array of cytokines, including TNF (1, 2). In addition to their sentinel role, mast cells are critical effector cells responsible for the initiation and orchestration of long-lasting, cell-mediated immunity. The vasopermeability and chemotaxis attendant to mast cell activation result in the recruitment of other immune cells, including Th2 lymphocytes, neutrophils, eosinophils, basophils, and macrophages, that contribute to pathogen elimination and immunopathology (1, 2, 3, 4). This response can produce a local or systemic type I hypersensitivity reaction best characterized in atopic diseases such as asthma, allergic rhinitis, and atopic dermatitis (1, 5). Although they are best known for their role in allergic disease, mast cells have recently been implicated in the inflammatory responses associated with animal models of rheumatoid arthritis (6), multiple sclerosis (7), heart disease (8, 9), and bacterial infection (10, 11). This central role in health and disease makes our understanding of mast cell biology more crucial than ever.

Studies of rodents genetically deficient in stem cell factor (SCF)3 or IL-3 signaling have revealed a role for these signal transducers in mast cell development and function (12, 13, 14). SCF and Kit, the SCF receptor, are indispensable for normal mast cell development. IL-3, however, is not essential for mast cell development, but is required for mast cell hyperplasia in response to infection (15). In vitro culture of mouse bone marrow cells with exogenous IL-3 and SCF is an established means of deriving bone marrow mast cell (BMMC) populations (16). Both the IL-3R and Kit have been shown to activate the transcription factor Stat5 in mast cells to mediate a prosurvival signal, and our group has previously shown that mice deficient in Stat5 are likewise mast cell deficient (17). These results prompted our ongoing investigation into other regulators of mast cell growth and development.

TNF is a prototypic inflammatory mediator produced by various cell types, including mast cells, macrophages, lymphocytes, and fibroblasts (18). Elevated serum levels of TNF are associated with the pathophysiology of bacterial peritonitis, rheumatoid arthritis, inflammatory bowel disease, and ankylosing spondylitis (19, 20, 21). Initially expressed as a 26-kDa membrane-bound precursor, TNF is proteolytically cleaved to a 17-kDa mature form. Both forms have been shown to be bioactive, mediating their actions via two distinct cellular receptors, TNFRI/p55 and TNFRII/p75 (22).

In addition to its proinflammatory role, TNF assists in lymphoid organogenesis during development. TNF- or TNFRI-deficient mice exhibit defective formation of germinal centers, Peyer’s patches, and splenic microarchitecture (23, 24). TNF has also been shown to be a critical requirement in the formation of B cell follicles, follicular dendritic cell networks, and T cell-dependent Ab responses (25). Pursuant to these defects, mice that are deficient in TNF or TNFRI/II signaling have been shown to succumb to infections that wild-type mice readily resolve (25, 26, 27, 28, 29, 30). In addition to these supportive roles, TNF has been shown to promote the proliferation (31) and development (32, 33) of myeloid cells such as macrophages and dendritic cells. In contrast, TNF appears to inhibit the development of the granulocyte and erythroid lineages (34, 35, 36, 37). The role of TNF in mast cell development is unclear. This study details our examination of the effects of TNF on mast cell differentiation and survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Derivation of BMMC populations

BMMC were cultured from 6- to 12-wk-old C57BL/6 x 129 mice and C57BL/6 x 129 TNF-{alpha}–/– mice (The Jackson Laboratory) that were housed together under specific pathogen-free conditions. In some experiments a novel strain of TNF-deficient mice created via Cre-LoxP technology (38) and their littermates, also housed under specific pathogen-free conditions, were used. Data obtained using these mice did not differ from those obtained using purchased mice. The results shown include data collected from both strains of TNF-deficient mice. BMMC were prepared by culturing bone marrow cells in complete RPMI 1640 (cRPMI; Invitrogen Life Technologies; 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 1 mM HEPES; Biofluids) supplemented with IL-3 (5 ng/ml) and SCF (50 ng/ml) with and without TNF (1, 0.1, or 0.001 ng/ml; R&D Systems and PeproTech) for 3–7 wk. Mast cell phenotype was confirmed by flow cytometric analysis with Abs specific for Kit and IgE as well as by histochemical staining with Wright-Giemsa (Sigma-Aldrich).

Human bone marrow culture

Human bone marrow was purchased from Cambrex BioScience. Cells were cultured for 21 days in AIM-V medium containing IL-3 (30 ng/ml; for 7 days only), SCF (100 ng/ml; Amgen), and IL-6 (100 ng/ml) in the presence or the absence of 10 µg/ml anti-TNF (BD Pharmingen). Viable cell numbers were determined by trypan blue exclusion.

Cytokines, Abs, and flow cytometry

Murine IL-3, SCF, and TNF were purchased from PeproTech and R&D Systems. Unlabeled IgE, PE-labeled IgE, PE-labeled anti-IL-3R{alpha}, PE-labeled anti-IL-3Rbeta, and PE-labeled anti-Kit (CD117) were purchased from BD Pharmingen. Anti-mouse TNF was purchased from R&D Systems. FITC-labeled rat IgG and rat anti-mouse IgE were purchased from Southern Biotechnology Associates. Flow cytometry was performed using a FACScan equipped with CellQuest software (BD Pharmingen).

TNF measurement

Wild-type bone marrow cells cultured in IL-3 and SCF for 0–21 days were washed, starved for 4 h, and replated at a concentration of 1 x 106 cells/ml in IL-3 and/or SCF at the indicated concentrations. When signal transduction inhibitors were used, cells were preincubated in inhibitors for 30 min at 37°C. After 16 h of incubation in cytokines at 37°C, TNF concentrations in supernatants were measured using an ELISA kit (BD Biosciences). Signal transduction inhibitors and their final concentrations included: LY294002 (14 µM); PD98059 (20 µM); JNK inhibitor II (400 nM); SB203580 (6 µM); and ERK activation inhibitor peptide I (cell permeable) (25 µM). All inhibitors were purchased from Calbiochem and were solubilized in DMSO (Sigma-Aldrich).

Cell viability and apoptosis

Cells were assessed for ≥diploid (viable) or <diploid (apoptotic) DNA content by propidium iodide (PI) staining after cell fixation and permeabilization (PI-DNA staining) as previously described (34). Briefly, 200 µl of cells were removed from cultures and centrifuged in a 96-well, V-bottom plate for 5 min, then washed in PBS and fixed in 150 µl of PI fixation buffer (70% ethanol/10% FBS in 1x PBS) for 4 h to 7 days at 4°C. After fixation, cells were washed with PBS and incubated with PI-DNA staining buffer containing 100 µg/ml RNase A and 50 µg/ml PI for 2–3 h in the dark at room temperature. To assess cell numbers, samples were analyzed by flow cytometry with automated counting for a preset time (45 s/200 µl sample with 0.01-s resolution). Live cell counts included all cells outside the subdiploid DNA marker.

Caspase and 3'3'-dihexyloxacarbocyanine iodide Di(OC6)3 staining

To assess activation of caspases-3 and -9, a 200-µl aliquot of cells was removed from culture, plated in a 96-well, V-bottom plate, and centrifuged for 5 min. Forty microliters of cRPMI and 10 µl of 5x FAM-DEVD-FMK (caspase 3 indicator; Immunochemistry Technologies) or FAM-LEHD-FMK (caspase 9 indicator; Intergen) were added to cells and incubated for 1 h at 37°C. Cells were then washed twice with 1x wash buffer, resuspended in wash buffer, and analyzed by flow cytometry. Cellular Di(OC6)3 (Molecular Probes) staining was assessed by incubating cells with Di(OC6)3 at a 1-nM final concentration in cRPMI for 15 min at 37°C. Cells were washed and resuspended in PBS, then analyzed by flow cytometry. Di(OC6)3 staining and caspase activation were measured by flow cytometry.

Assessment of peritoneal mast cell numbers

Peritoneal cells were harvested by lavage of the peritoneal cavity with 4 ml of cRPMI. Vigorous massage of the peritoneum for 60 s to loosen peritoneal cells was followed by recovery of the fluid with a Pasteur pipette. Mast cell numbers were determined by flow cytometric analysis of IgE receptor/Kit coexpression and by Wright-Giemsa histochemical staining.

Tissue isolation and assessment

Mouse ear tissue samples were obtained from animals after death and fixed overnight in Carnoy’s fixative (60% (95%) ethanol, 30% chloroform, and 10% glacial acetic acid). The samples were then transferred to 75% ethanol and embedded in paraffin blocks. Five-micrometer sections were taken from these blocks, mounted to slides, and processed for staining of in situ mast cells (Wright-Giemsa; 45 s). Tissue mast cells were assessed in a blinded fashion by x1000 magnification light microscopy.

Statistics

Results are the mean ± SE for the data shown, with experiments and replications noted in the figure legends. Effects were measured by comparing with t test two data points or by ANOVA for multiple data points by SysStat9 software (SPSS). A value of p < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TNF is produced by MAC-1-positive bone marrow cells in response to IL-3 stimulation

Given the many roles of TNF in differentiation and function, we analyzed cultures of developing mast cells for the presence of TNF. Culture supernatants from bone marrow cells grown in IL-3 and SCF were measured by ELISA. We observed the production of soluble TNF in response to stimulation by IL-3 and SCF (Fig. 1A). Assays performed with either cytokine alone revealed that TNF production was induced by IL-3, but not SCF, stimulation. This response was maximal on culture days 7–10, after which it dropped sharply.


Figure 1
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  		FIGURE 1. IL-3 induces TNF production from cells of the macrophage lineage via a PI3K and MEK/ERK pathway. A, On the days indicated, bone marrow cells cultured in IL-3 and SCF were washed, starved, and replated in IL-3 and SCF (5 and 50 ng/ml, respectively), IL-3 (5 ng/ml), SCF (50 ng/ml), or medium alone. Cells were cultured for 16 h and were >95% viable as measured by PI-DNA staining analysis. Culture supernatants were measured by ELISA to detect soluble TNF-{alpha}. Data shown are the mean (minus mean of medium alone sample, generally ~50 pg) ± SE from four independent samples. *, p < 0.01 vs SCF-treated cells. B, Ten-day-old bone marrow cells were washed, starved, and exposed to inhibitors or vehicle control (DMSO) for 30 min before IL-3 (50 ng/ml) stimulation for 16 h. Culture supernatants were measured for TNF concentrations by ELISA. Cells were >90% viable at the time that supernatants were harvested, as measured by PI-DNA staining analysis. The data shown are from one representative of three experiments, each performed in triplicate and all yielding similar results. *, p < 0.01. C, Bone marrow cells cultured for 10 days in IL-3 and SCF were sorted on the basis of MAC-1 or Kit expression. Sorted cells were stimulated for 16 h with IL-3 (5 ng/ml), and culture supernatants were measured by ELISA to detect soluble TNF-{alpha}. The data shown are the mean ± SE of three samples from one of three representative experiments. *, p < 0.05 vs vehicle control.

 
To determine the IL-3R signaling pathway involved in TNF production, bone marrow cells were cultured for 10 days in IL-3 and SCF, then washed and restimulated with IL-3 in the presence of a panel of pharmacological inhibitors or vehicle control (DMSO; Fig. 1B). These experiments revealed >50% inhibition of TNF production (vs vehicle control) when cells were incubated with inhibitors of the PI3K or MAPK kinase (MEK)/ERK kinase pathways. An inhibitor of ERK, a MAPK downstream of MEK, revealed similar results, although less profound inhibition was observed in response to inhibitors of MAPK family members JNK and p38. Thus, IL-3 appeared to induce TNF production through several known signaling pathways, particularly PI3K and MEK/ERK.

As we have shown previously (39), bone marrow cells cultured in IL-3 and SCF give rise to mast cells and macrophages. These populations are easily distinguished by their expression of Kit or MAC-1, respectively. Because both mast cells and macrophages can produce TNF, we purified these populations by cell sorting to determine which lineage produced TNF in response to IL-3 stimulation. As shown in Fig. 1C, MAC-1-positive cells produced TNF in response to IL-3, whereas Kit-positive cells showed no such response. TNF was detectable in lysates from Kit-positive cells, but these levels were not altered by IL-3 stimulation (3.0 ± 2.6 and 10.1 ± 4.1 pg/ml for unstimulated vs IL-3 stimulated; p = 0.23). Thus, cells of the monocyte/macrophage lineage appear to be the source of TNF in these cultures.

TNF is an obligatory growth factor for developing mast cells in vitro

Given evidence of IL-3-elicited TNF production in bone marrow cell cultures, we determined the importance of this cytokine to cell expansion by deriving BMMC populations from TNF-deficient (knockout (KO)) bone marrow. Unlike their wild-type counterparts, TNF KO populations failed to proliferate normally when cultured in rIL-3 and SCF (Fig. 2). After 5 wk of culture, total viable cell numbers from TNF KO cultures amounted to 6% of their wild-type counterpart (4.2 x 105 vs 6.7 x 106 cells, respectively). Interestingly, although TNF production peaked during the first 7–10 days of culture (Fig. 1), significant growth retardation was not evident in TNF KO cultures until day 21.


Figure 2
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  		FIGURE 2. TNF is required for normal cell expansion in response to IL-3 and SCF. A, Bone marrow cells from wild-type and TNF KO mice were cultured in IL-3 and SCF with or without TNF (1 ng/ml). On the days indicated, a 200-µl aliquot from each of three triplicate wells was harvested for PI-DNA staining analysis. Viable cell counts were determined by excluding all cells with subdiploid DNA content and analyzing each sample for 45 s (time resolution, 0.1 s). Cell numbers were adjusted to account for dilutions due to feeding. The data shown are from one representative of five experiments, each performed with one to three replications and all yielding similar results. *, p < 0.05 when comparing TNF KO to all other populations. B, Cells were cultured as described in A, with TNF (1 ng/ml) added to TNF KO cultures on day 0 or 7 as indicated. TNF was also added to TNF KO cells on day 0, then removed on day 7 in the final sample shown. Viable cell numbers were determined on day 34 of cultured by PI-DNA staining. The data are the mean and range of two samples. C, Human bone marrow cells were cultured in IL-3 (1 wk only), SCF, and IL-6 with or without anti-TNF. On day 21, viable cell numbers were determined by trypan blue exclusion. The data shown are the mean and SD of three samples. *, p = 0.009 (as determined by Student’s t test).

 
Importantly, the capacity for normal logarithmic growth was completely restored to TNF KO populations by the addition of soluble rTNF to the culture medium. Although we used TNF at 1 ng/ml throughout our assays, concentrations of 0.01–1 ng/ml restored mast cell survival (Fig. 2A and data not shown). This expansion approximated that of wild-type cultures grown in IL-3 and SCF with or without TNF (Fig. 2, {circ} vs {square}). Also, TNF KO bone marrow cells grew normally in the presence of conditioned medium harvested from wild-type cells cultured in IL-3 and SCF (data not shown). Mirroring the production of TNF in wild-type cultures, TNF added to KO cells restored proliferation only if it was present during the first 7 days of culture (Fig. 2B). TNF addition on day 7 did not rescue proliferation, and its removal after this point still restored normal cell expansion.

Importantly, human bone marrow cells showed similar TNF dependency. The addition of a TNF-blocking Ab diminished viable cell numbers by >80% during a 21-day culture period that supported mast cell development (Fig. 2C). These findings indicate that soluble TNF is required to support in vitro mast cell development from both human and mouse bone marrow progenitors.

TNF deficiency results in apoptosis of developing mast cells

The reduction in viable cell numbers noted in TNF KO cultures could be due to increased apoptosis. To address this issue, samples of wild-type and TNF KO BMMC were harvested on day 21 of culture, when significant growth deficiencies become apparent. Apoptosis was measured by the presence of subdiploid DNA after propidium iodide staining of fixed, permeabilized cells treated with RNase A (PI-DNA staining). As shown in Fig. 3A, apoptosis was significantly increased in TNF KO cultures compared with wild-type cells (41 ± 3 vs 16 ± 1%, respectively). Importantly, the addition of TNF to the culture medium completely blocked apoptosis in TNF KO cultures, but had no significant effect on wild-type cells.


Figure 3
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  		FIGURE 3. Increased apoptosis in TNF-{alpha}-deficient bone marrow cells and implication of the mitochondrial apoptosis pathway. Bone marrow cells from wild-type and TNF KO mice were cultured in IL-3 and SCF with or without TNF (1 ng/ml). On day 21 of culture, a 200-µl aliquot from each of three triplicate wells from each condition was analyzed by PI-DNA, Di(OC6)3, and active caspase staining as described in Materials and Methods. The data shown are one representative of four experiments. *, p < 0.001 vs all other points.

 
Loss of growth factor signaling induces an apoptotic cascade that can proceed through a mitochondrial mechanism (40, 41, 42). Because TNF may serve as a growth factor, we measured changes in mitochondrial membrane potential ({Delta}{psi}m) via Di(OC6)3 staining. In 21-day-old bone marrow cultures from wild-type and TNF KO mice, Di(OC6)3 staining revealed a significant decrease in fluorescence intensity indicative of reduced {Delta}{psi}m in the absence of TNF (Fig. 3B). Fluorometric assays that measure caspase activation indicated an attendant, commensurate increase in the activity of caspase-9 and -3 in these cultures (Fig. 3, C and D). Importantly, exogenous TNF, while having no effect on wild-type BMMC, restored normal {Delta}{psi}m and caspase activity levels in TNF KO cultures. Therefore, soluble TNF is essential for preventing apoptosis of developing bone marrow cells, an activity that correlates with sustained {Delta}{psi}m.

Defective IL-3-induced survival signaling in TNF KO bone marrow cells

IL-3 promotes cell survival and protects against factor withdrawal-induced apoptosis (37). Because we found a defect in IL-3 and SCF cultures of TNF KO cells consistent with a factor withdrawal-type apoptosis, we assessed survival more precisely by examining IL-3-induced survival in short-term assays.

Wild-type and TNF KO bone marrow cells cultured for 4–21 days in IL-3 and SCF were washed and replated in IL-3 alone for 4 days, after which apoptosis was measured by PI-DNA staining (Fig. 4A). These assays revealed a progressive defect in IL-3-induced survival signaling after day 14 of culture, most notable on day 21. The addition of exogenous TNF restored survival to wild-type levels. Thus, short-term IL-3-mediated survival requires endogenous TNF production.


Figure 4
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  		FIGURE 4. TNF-{alpha}-deficient mast cells demonstrate reduced IL-3-mediated survival. A, Bone marrow cells from wild-type and TNF KO mice were cultured in IL-3 and SCF with or without TNF (1 ng/ml). On days 1–21, aliquots were washed, starved for 4 h, and replated in IL-3 (5 ng/ml) for 4 days. Cell survival was measured by PI-DNA staining analysis. The data shown are the mean ± SE of three or four experiments per point, each performed with three to six replications. *, p < 0.001 when comparing TNF KO to all other populations. B, Cells from A were harvested on day 21, washed, starved, and replated for 4 days in IL-3 at the indicated concentrations. Cell survival was measured by PI-DNA staining analysis. The data shown are from one representative of four experiments, each performed in triplicate and all yielding similar results. *, p < 0.001; **, p < 0.01; ***, p < 0.05 (when comparing TNF KO to all other populations). C, Cells from A were harvested on day 28 of culture and analyzed for expression of IL-3R {alpha}- and beta-subunits by flow cytometry. D, Cells from A were harvested on day 28 of culture, washed, starved for 6 h, and restimulated with IL-3 (50 ng/ml) for 2 min. Total cell lysates were subjected to Western blot analysis to detect tyrosine-phosphorylated and total Stat5. Numbers below the bands represent the ratio of phosphorylated to total Stat5, as determined by densitometry.

 
Using culture day 21 as an assay point, we found that TNF KO bone marrow cells were significantly less responsive to IL-3-mediated survival in concentration-response assays (Fig. 4B). Even using IL-3 at 5 ng/ml, TNF KO cells did not maintain 50% survival. The concentration of IL-3 required to sustain 70% apoptosis (30% survival) in TNF KO cells was >300-fold greater than that for wild-type cultures. The addition of soluble TNF to KO cultures greatly improved IL-3 responsiveness, and enhanced the survival of wild-type cells in limiting concentrations of IL-3 (Fig. 4B).

Importantly, the defects in IL-3-mediated survival were not caused by changes in IL-3R expression. As shown in Fig. 4C, TNF KO bone marrow cells expressed IL-3R {alpha}- and beta-chains at levels comparable to wild-type cells. Furthermore, IL-3-induced Stat5 tyrosine phosphorylation, an early event in IL-3 signaling (39, 40), was comparable in TNF KO and wild-type cultures (Fig. 4D). These data indicate that endogenous TNF may reduce the concentration of IL-3 required for survival, such that a threshold for in vitro survival cannot be reached in the absence of TNF. This defective survival signaling is not the result of reduced receptor expression or Stat5 activation.

TNF deficiency inhibits in vitro mast cell differentiation

Although IL-3-induced TNF production peaked during days 7–10 of culture (Fig. 1), by day 21 when survival deficiencies were apparent, these populations were nearly exclusively mast cells. To determine whether the defects in survival were accompanied by changes in mast cell differentiation, we measured the effects of TNF deficiency on mast cell granulation and surface Ag expression. TNF KO populations exhibited normal granulated mast cell morphology (Fig. 5A). However, analysis of these cultures for coexpression of the mast cell surface Ags Fc{epsilon}RI and Kit revealed distinct differences. TNF KO BMMC exhibited a reduced Kithigh/Fc{epsilon}RI+ population compared with wild-type cultures (Fig. 5B), and this population expressed both surface markers at a lower density. In particular, Fc{epsilon}RI expression was reduced ~50%. For example, the mean fluorescence intensity (MFI) of Fc{epsilon}RI staining shown in Fig. 5B was 116 for wild-type cells and 55 for TNF KO cells. Because so few viable cells were recovered from cultures on day 21, the functionality of these receptors was not measured. As with survival studies, the addition of exogenous TNF returned the percentage and intensity of Kit and Fc{epsilon}RI expression to wild-type levels. Therefore, the absence of TNF during mast cell development appears to have selective effects on surface marker expression without altering granulation.


Figure 5
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  		FIGURE 5. TNF KO mast cell populations have normal granulation, but reduced expression of the high affinity IgE receptor. A, Bone marrow cells from wild-type and TNF KO mice were cultured in IL-3 and SCF. On culture day 21, cells were centrifuged onto slides and stained with Wright-Giemsa for 45 s. Magnification, x1000. B, Bone marrow cells from wild-type and TNF KO mice were cultured in IL-3 and SCF with or without TNF (1 ng/ml) for 21 days before flow cytometric analysis to detect the mast cell Ags Fc{epsilon}RI and Kit. The percentage of double-positive cells is indicated. The data shown are the mean ± SE of one of four experiments, each performed in triplicate.

 
TNF deficiency selectively reduces peritoneal mast cell numbers

Given the defects in in vitro mast cell survival, it seemed possible that loss of TNF could alter in vivo mast cell numbers. Mast cells in the peritoneal cavity of TNF KO and wild-type littermates were analyzed by flow cytometry (Fig. 6A). Although total peritoneal cell numbers were comparable between the two genotypes (data not shown), Kithigh/Fc{epsilon}RI+ populations in TNF-deficient mice were reduced by >50% compared with wild-type mice (0.9 ± 0.2 and 2.0 ± 0.2%, respectively). When mast cells were quantified based on histochemical staining and morphology, the results were similar (Fig. 6). Despite these consistencies with the in vitro assays, Fc{epsilon}RI expression levels did not differ between the six wild-type and five TNF KO peritoneal mast cell populations we examined (MFI of WT, 47.1 ± 8.4; TNF KO, 65.0 ± 9.6; p = 0.19).


Figure 6
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  		FIGURE 6. TNF KO mice have reduced numbers of peritoneal mast cells. A, Peritoneal lavage was performed on wild-type and TNF KO mice. The percentage of mast cells was determined based on the expression of Fc{epsilon}RI and Kit, as detected by flow cytometry. Each point represents an individual animal. *, p < 0.001. B, Wright-Giemsa staining was used to determine the percentage of mast cells in peritoneal lavages from wild-type and TNF KO mice. The data shown are the number of mast cells per 50,000 cells from individual mice. *, p < 0.05.

 
To determine whether the reduction in mast cells was consistent in other microenvironments, we assessed mast cell numbers in skin tissues from the ears of TNF KO mice and wild-type controls. Unlike the differences in peritoneal mast cells, dermal mast cell numbers in TNF KO mice were not different from those in wild-type controls (5.6 ± 0.5 and 5.7 ± 0.1 mast cells/x1000 field, respectively). Similarly, we found no difference in mast cell numbers in the stomach or intestine (data not shown). It appears that TNF deficiency selectively reduced the peritoneal mast cell population without altering mast cell numbers in other tissues.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TNF is a pleiotropic cytokine known to play a critical role in both early and late events involved in inflammation. This includes localizing the noxious agent, amplifying the cellular and mediator responses both locally and systemically, and eliminating damaged cells (43, 44). Indeed, given the extensive roles that TNF superfamily members such as TNF have in regulating adaptive immunity, it is likely that they evolved coincident with adaptive immunity itself (43, 44). Although TNF came to prominence by virtue of its proinflammatory actions, its role in immune function is now known to extend to lymphoid organogenesis and the development and/or proliferation of many immune cell types (23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33).

Bone marrow cells cultured in IL-3 and SCF yield mixed cultures of macrophages and mast cells, lineages that often collaborate in the inflammatory response and share many properties. Because both lineages are known to produce TNF, it is interesting that IL-3 elicited TNF production from Mac-1-positive bone marrow cells, but not mast cells. It appears that TNF acts in a paracrine fashion to support mast cell development and survival. The importance of this production was clear in studies using TNF KO bone marrow, which demonstrated profound survival defects. Moreover, proof that soluble TNF could rescue the deficiency of these cultures emphasizes the plausibility of a paracrine network involving developing mast cells and macrophages. Although medium harvested from wild-type bone marrow cultures was able to rescue survival of TNF KO cells, we were unable to block this survival effect with anti-TNF Abs (data not shown). Our own opinion is that these Abs may have poor efficacy, but it is also possible that a redundant growth factor is present in these cultures and substitutes for TNF. In contrast, it was striking that Ab-mediated TNF blockade greatly reduced survival in human bone marrow cultures. These data argue that the efficacy of TNF blocking agents in treating autoimmune disease could be related to a decrease in mast cell numbers. At the minimum, the consistent role of TNF in mast cell survival across species boundaries implies that it is a conserved, and therefore important, biological control of the mast cell population.

Although we have not fully determined the means by which IL-3 induces TNF production, the PI3K and MEK/ERK pathways appear to be critical. Previous studies have shown that TNF may be regulated by the MEK pathway, but with emphasis on the downstream MAPKs p38 and JNK (45, 46). These differences may be due to the lineages studied, which include mast cells in the previous studies and macrophages in this work. Furthermore, the activation stimuli have included substance P (45), Fc{epsilon}RI (46), and IL-3.

The major effect of TNF deficiency was mast cell apoptosis, proceeding through a pathway that included mitochondrial damage and caspase activation. These events are consistent with growth factor deprivation, supporting our theory that TNF enhances IL-3-mediated survival signaling. As shown, loss of TNF expression resulted in defective IL-3-mediated survival without changes in IL-3R expression or proximal signaling events such as Stat5 activation. We recently showed that Stat5 activation was necessary for mast cell survival by maintaining the expression of Bcl-2 and Bcl-xL (17). Thus, it was a surprise that Stat5 activation appeared unaffected in mast cells with defective IL-3-mediated survival. These results indicate that Stat5-independent events downstream of the IL-3R are critical to mast cell survival.

The link between IL-3-mediated TNF production and maintenance of normal IL-3 signaling indicates the existence of a positive feedback system. The many activities ascribed to TNF include its ability to amplify cytokine signaling by enhancing the production of other cytokines in mast cells (47). This cooperativity may be based on the activation of NF-{kappa}B transcription factors by TNF and other activating stimuli. In fact, IL-3 has been shown to activate NF-{kappa}B through a Stat5-dependent mechanism (48, 49). Thus, it seems possible that loss of TNF production could reduce NF-{kappa}B activation below a threshold required for maintaining cell survival in response to IL-3, an event that would lie downstream of Stat5. These questions are the focus of our current studies.

The delayed effects of TNF can be viewed as supporting its role in commitment to the mast cell lineage. Although IL-3-mediated TNF production peaked on days 7–10 of culture, its effects on the mast cell lineage were delayed until days 18–21. Similarly, exogenous TNF only rescued proliferation of TNF KO cultures when it was added during the first 7 days of culture, after which it could be removed without impacting survival. Importantly, the peak production of TNF correlates with the development of the mast cell-committed progenitor (50). It seems possible that the committed mast cell progenitor has survival requirements distinct from the uncommitted precursor, and that the absence of TNF during commitment inhibits subsequent IL-3 signaling. This cooperativity between TNF and IL-3 is reminiscent of work published by Hu et al. (51), who showed that TNF enhances mast cell development from splenocytes. Though these studies used a distinctly different assay system, this group also found that TNF addition was necessary in the first few days of culture to enhance mast cell expansion. Although our data corroborate the importance of TNF noted by Hu et al. (51), the former study found that indomethacin prevented the mast cell-promoting effects of TNF on spleen cells. However, this cyclooxygenase inhibitor, used at 50 µM had no significant effect on mast cell survival in our bone marrow cultures (1.3 x 106 vs 0.95 x 106 viable cells when comparing IL-3 and SCF cultures with or without indomethacin; p = 0.20). Thus, the mechanism by which TNF acts may differ with the tissue microenvironment or cell lineage.

Consistent with a defect during mast cell differentiation, TNF KO mast cells expressed Kit normally, but demonstrated a 50% reduction in Fc{epsilon}RI levels. Although Kit expression is an early event in mast cell ontogeny, Fc{epsilon}RI is not detectable until commitment (50); hence, diminished Fc{epsilon}RI expression and its rescue by soluble TNF argue for defective commitment to the mast cell lineage in the absence of TNF.

Support for the importance of TNF in mast cell survival also came from in vivo studies of TNF KO mice. Peritoneal mast cell numbers in these mice were reduced 50% compared with their wild-type littermates, measurements that were consistent using both histochemical and surface Ag techniques. Interestingly, the reduction in IgE receptor expression observed in the in vitro assays was not consistent in vivo. Because IgE up-regulates the expression of its own receptor (reviewed in Ref.52), and we have recently shown that peritoneal mast cells have fully occupied IgE receptors by 8 wk of age (53), it is possible that serum IgE circumvents the reduction in Fc{epsilon}RI expression caused by loss of TNF signaling. These data indicate that the most critical role for TNF in mast cell development is its regulation of mast cell survival. Interestingly, mast cell numbers appeared to be unchanged outside of the peritoneum. Because mast cells were not increased in other tissues, it does not appear that peritoneal mast cells migrated to surrounding areas such as the stomach or intestine. However, we cannot discern from this work whether TNF deficiency affects the migration of mast cell progenitors to the peritoneum or their subsequent survival. Because the peritoneum is a rich source of macrophages, it seems possible that this tissue could have greater dependency on TNF for maintaining mast cell numbers.

The broadening use of anti-TNF therapies in inflammatory disease emphasizes the importance of understanding how this pleiotropic cytokine regulates the development and survival of inflammatory cells. As has been discussed (54, 55), TNF blockade greatly ameliorates the pathology associated with inflammatory diseases that may involve mast cells, such as rheumatoid arthritis. However, this reduction in TNF signaling is also associated with increased risk of infection (54). Because mast cells serve both to protect the host from infection and to enhance inflammation, the effects of TNF blockade may be mediated in part by its effects on mast cell survival. Understanding how TNF contributes to mast cell development and function will assist in devising new tools for clinical intervention.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by grants to the Ryan laboratory from the National Institutes of Health (1RO1AI43433 and 1R01CA91839). C.L.K. was supported by a grant from the American Lung Association and the Food Allergy and Anaphylaxis Network; M.C.B. was supported by a grant from the Russian Academy of Sciences, and with U.S. federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-12400. Back

2 Address correspondence and reprint requests to Dr. John J. Ryan, Biology Department, Virginia Commonwealth University, Box 842012, Richmond, VA 23284-2012. E-mail address: jjryan{at}saturn.vcu.edu Back

3 Abbreviations used in this paper: SCF, stem cell factor; BMMC, bone marrow-derived mast cell; cRPMI, complete RPMI 1640; Di(OC6)3, 3'3'-dihexyloxacarbocyanine iodide; KO, knockout; {Delta}{psi}m, mitochondrial membrane potential; MEK, MAPK kinase; MFI, mean fluorescence intensity; PI, propidium iodide. Back

Received for publication March 30, 2005. Accepted for publication November 22, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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