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Originally published online as doi:10.1189/jlb.0405201 on July 7, 2006

Published online before print July 7, 2006
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(Journal of Leukocyte Biology. 2006;80:581-589.)
© 2006 by Society for Leukocyte Biology

Interleukin-10 induces apoptosis in developing mast cells and macrophages

Daniel P. Bailey*, Mohit Kashyap*, L. Andrew Bouton*, Peter J. Murray{dagger} and John J. Ryan*,1

* Department of Biology, Virginia Commonwealth University, Richmond; and
{dagger} Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee

1 Correspondence: Virginia Commonwealth University, Biology Department, Box 842012, Richmond, VA 23284-2012. E-mail: jjryan{at}saturn.vcu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-10 is a potent immunoregulatory cytokine capable of inhibiting the inflammatory response. As mast cells and macrophages are central effectors of inflammation, we investigated the effects of IL-10 on mast cell and macrophage development from mouse bone marrow progenitors. Bone marrow cells were cultured in IL-3 + stem cell factor (SCF), giving rise to mixed populations of mast cells and macrophages. The addition of IL-10 greatly decreased the expansion of bone marrow progenitor cells through a mechanism requiring signal tranducer and activator of transcription-3 expression. The inhibitory effects were a result of the induction of apoptosis, which occurred with caspase-3 activation and reduced mitochondrial membrane potential. Supporting a role for the mitochondrion, bone marrow cells from p53-deficient or Bcl-2 transgenic mice were partly resistant to the effects of IL-10. Further, IL-10 decreased Kit receptor expression and inhibited survival signaling by SCF or IL-3. These data indicate that IL-10 induces an intrinsic, mitochondrial apoptosis cascade in developing mast cells and macrophages through mechanisms involving blockade of growth factor receptor function. The ability of IL-10 to inhibit survival could support immune homeostasis by dampening inflammatory responses and preventing chronic inflammation.

Key Words: stat3 • hematopoietic • inflammation • caspase-3 • mitochondrial membrane potential • immune homeostasis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells are best characterized as the effectors of allergic disease; however, their role in innate and adaptive immunity is expanding. The mast cell response has recently been shown to be essential for resistance to bacterial infection and the inflammatory responses characteristic of multiple sclerosis, autoimmune arthritis, and coronary artery disease [1 2 3 4 ]. These data support the contention that mast cells participate in T helper cell type 1 (Th1) and Th2 responses. It is thus critical that we understand how mast cell development and survival are controlled. Mast cells are derived from pluripotent hematopoietic stem cells in the bone marrow and exit as committed precursors to complete their development in connective and mucosal tissues [5 ]. This process is driven by the activities of interleukin (IL)-3 and stem cell factor (SCF) in the mouse. We previously found that bone marrow cells cultured in IL-3 + SCF, without selection against adherent cells, gave rise to mixed populations of mast cells and macrophages [6 ]. This in vitro system allows for the determination of cytokine effects during cell development, when the immune response could be modified to enhance or diminish inflammation.

IL-10 is a Class 2 cytokine produced by Th2 cells, Th0 cells, CD45Rblow regulatory T cells, monocyte/macrophages, keratinocytes, some B cells, and mast cells. It is a potent, inhibitory factor, which blocks many inflammatory activities of macrophages and dendritic cells [7 , 8 ]. On mast cells, we have shown that IL-10 inhibits expression and/or function of the high-affinity receptor for immunoglobulin E (IgE), Fc receptor for IgE (Fc{epsilon}R)I, and Kit [9 , 10 ]. We have also found that combined signaling with IL-4 and IL-10 induces apoptosis of differentiated mast cells [11 , 12 ], supporting an inhibitory role for IL-10 in mast cell biology.

The binding of IL-10 to the IL-10 receptor (IL-10R) activates signal transducer and activator of transcription-3 (STAT3) via the tyrosine kinases Janus tyrosine kinase 1 and Tyk2, with activation of STAT1 and STAT5 in some systems [7 ]. Of these, STAT3 has been shown to be critical for IL-10 signaling [13 14 15 16 17 18 19 20 ]. The current study shows a relationship between IL-10-mediated STAT3 activation and the inhibition of mast cell and macrophage development from bone marrow progenitors. We found that IL-10 serves as a potent inducer of apoptosis through a STAT3-dependent pathway. These data support a role for IL-10 in inhibiting the inflammatory response by acting at the level of progenitor development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Culture system
Bone marrow cells were extracted from the femurs of C57BL/6 x 129 mice ["wild type" (WT); from Taconic Farms, Germantown, NY], STAT3 flox/–, lysMcre mice [19 , 20 ], Bax-deficient mice, p53-deficient mice (Jackson Laboratories, Bar Harbor, ME), or H2K-Bcl-2 transgenic mice [21 ]. Cells were cultured at 5 x 105 cells/ml in complete RPMI medium, consisting of RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1 mM sodium pyruvate (all from Biofluids, Rockville, MD) for 21 days. Cultures contained IL-3 (5 ng/ml) + SCF (50 ng/ml) and IL-10 at the indicated concentrations (purchased from R&D Systems, Minneapolis, MN). During the culture period, cells were fed every 4–7 days by removing and replacing half of the culture. Cultures developed into virtually pure (>95%) mast cells, based on histochemical analysis and flow cytometry staining for Fc{epsilon}RI and Kit.

Flow cytometry staining
Samples were obtained by removing a 200-µl aliquot after scraping. Samples were washed with phosphate-buffered saline (PBS) containing 3% fetal calf serum and 0.1% sodium azide (fluorescein-activated cell sorter buffer) and then incubated with 10 µl rat anti-mouse Fc receptor for IgG (Fc{gamma}R)II/Fc{gamma}RIII ascites (Clone 2.4G2) to prevent nonspecific interaction of antibodies with IgG receptors. Cells were then stained with fluorescein isothiocyanate (FITC)-labeled IgG (Southern Biotech, Inc., Birmingham, AL), phycoerythrin (PE)-labeled IgG (BD Biosciences Co., San Diego, CA), FITC-labeled anti-T1/ST2 (Morwell Diagnostics, Zurich, Switzerland), PE-labeled anti-Kit (BD Biosciences), FITC-labeled anti-IgE (Southern Biotech, Inc.), FITC-labeled anti-CD13 (BD Biosciences), or PE-labeled anti-ßIL-3/ßc (BD Biosciences). Staining with anti-IL-3R{alpha} chain was detected using PE-labeled goat anti-rat IgG (BD Biosciences).

Assessment of live cell numbers
To compare the relative number of live cells in each culture condition, samples were prepared as described for flow cytometry staining, using 200 µl cells removed from each culture. Samples were analyzed for 45 s (time resolution 0.1 s) on a BD FACScan (BD Biosciences). Live cell gating was accomplished by forward- and side-scatter (FSC and SSC, respectively) parameters, the efficacy of which was confirmed by propidium iodide (PI) exclusion. Live cell numbers in experimental conditions were compared with cells cultured in IL-3 + SCF (control conditions) to determine a percent change relative to this control group. We found this method to be more objective and consistent than similar assessments of cell numbers such as trypan blue exclusion.

PI analysis of apoptosis
Cells were assessed for ≥diploid (viable) or <diploid (apoptotic) DNA content by PI staining following cell fixation and permeabilization (PI-DNA staining) as described [11 ]. Briefly, 200 µl cells were removed from cultures and centrifuged in a 96-well v-bottom plate for 5 min and then washed in PBS and fixed in 150 µl PI fixation buffer (70% EtOH/10% FBS in 1x PBS) at least 4 h 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 45 s (0.1 s resolution). Live cell counts included all cells outside of the sub-diploid DNA marker. It is important that this technique is significantly different from the more common PI exclusion staining, which only offers live versus dead cell measurements. With fixed cells and higher PI concentrations, all DNA is stained, and the percentage of the population possessing subdiploid DNA content (i.e., apoptotic) is apparent.

Di(OC6)3 staining
Di(OC6)3 (Molecular Probes, Eugene, OR) was added to 200 µl cells at 1 nM final concentration. Samples were incubated for 30 min at 37°C in a CO2 incubator. The cells were then washed twice with PBS and analyzed by flow cytometric analysis using a FSC and SSC gate.

Histochemical analysis
Cell cultures were scraped, and 200 µl samples were removed, washed twice with PBS, and centrifuged onto microscope slides (Shandon Cytospin 2, ThermoShandon, Pittsburgh, PA), which were stained with Wright Giemsa (WG; Sigma-Aldrich, St. Louis, MO) or with acid toluidine blue (TB; 0.2% TB, 0.1 M citric acid, in 50% EtOH in dH20).

Western blot analysis
Cells were plated at 5 x 106 cells/ml and cultured in media alone or with IL-10 at 100 ng/ml for the indicated times. Whole cell lysates were prepared, and protein levels were analyzed using anti-Phospho-Stat3 (tyr 705) and anti-Stat3 (Cell Signaling Technology, Beverly, MA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
IL-10 reduces cell numbers in an IL-3- and SCF-rich environment
To determine the effects of IL-10 on mast cell development, bone marrow cells were cultured in IL-3 + SCF, with or without IL-10 for 21 days. In these cultures, IL-10 reduced relative cell numbers in a concentration-dependent manner, with a 75% decrease when IL-10 was added at 10 ng/ml (Fig. 1A ). To determine the kinetics of this effect, samples were measured at various time-points during the 21-day culture period (Fig. 1B) . The effects of IL-10 were not significant during Culture Days 0–10, but between Days 10 and 21, the live cell numbers declined rapidly, reaching a nadir of 25% of the control population. As an example of non-normalized data, timed, live cell counts from a representative experiment were 2906 versus 868 for cultures of IL-3 + SCF ± IL-10, respectively (P=.001 by Student’s t-test; n=4). The decrease in cell numbers was not caused by a reduction in proliferation, as the fraction of cells in S-phase did not decrease with the addition of IL-10, as measured by PI-DNA staining. For example, on Day 14, IL-3 + SCF cultures had 4.9 ± 0.9% S-phase, and IL-3 + SCF + IL-10 cultures had 7.3 ± 1.5% S-phase (mean±SEM, n≥8; P=0.32).


Figure 1
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  		Figure 1. Effects of IL-10 on bone marrow cell numbers in culture. (A) Concentration response of IL-10 effects on viable cell numbers. Bone marrow cells were cultured in IL-3 + SCF ± IL-10. Cultures were harvested after 21 days, and viable cell numbers were measured by PI-DNA staining and timed flow cytometric counting as described in Materials and Methods. (B) Time course of IL-10 effects. Bone marrow cells were cultured in IL-3 + SCF ± IL-10 at 10 ng/ml. Viable cell numbers were determined on the indicated days by PI-DNA staining and timed flow cytometric counting. (C) Addition/removal of IL-10 (10 ng/ml) to bone marrow cultures containing IL-3 + SCF. IL-10 was added to or removed from cultures containing IL-3 + SCF on the days indicated by the x-axis. All cultures were harvested on Day 21. Viable cell numbers were measured by PI-DNA staining and timed flow cytometric counting (*, P<.05; n≥3 by ANOVA).

 
The inhibitory effects of IL-10 required a relatively long exposure period. Not only did cell numbers remain unchanged for the first 10 days of culture, but removal of IL-10 from these cultures during the first 15 days prevented any significant decrease in cell numbers when cells were harvested on Day 21 (Fig. 1C) . Likewise, IL-10 added after the first 7 days of culture had no effect on viable cell numbers when cultures were harvested on Day 21. Thus, IL-10 greatly reduced the survival of developing mast cells, exerting its effects with delayed kinetics, which required its presence early in differentiation.

IL-10 dampens mast cell but not macrophage differentiation
We have previously found that bone marrow cells cultured in IL-3 + SCF give rise to a mixed population of macrophages and mast cells when adherent cells are retained [6 ]. To determine if IL-10 altered this pattern of development, we compared the cell populations derived from culture with and without IL-10 on Days 10 and 21. As shown in Figure 2A , the percentage of cells expressing the mast cell markers Kit and T1/ST2 was decreased significantly by IL-10 on Day 21, indicating that mast cell differentiation is hampered by these conditions. In contrast, the fraction of the population expressing the macrophage marker Mac-1 (CD11b) was not altered significantly by IL-10. These results were consistent when cell lineage was assessed by histochemical staining with WG, TB, or NSE, which also showed a 70% decrease in the percent mast cells with no change in macrophages (Fig. 2B and data not shown). The decrease in the fraction of mast cells, unmatched by an increase in macrophages, appeared to be a result of an accumulation of immature blast-like cells observed in the histochemical staining. The cumulative effects of IL-10 appear to hinder the expansion and survival of mast cells and macrophages (Fig. 1) and selectively inhibit mast cell differentiation.


Figure 2
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  		Figure 2. IL-10 inhibits mast cell but not macrophage differentiation. Bone marrow cells were cultured in IL-3 + SCF, with or without IL-10 for 21 days. (A) The percentage of cells expressing the mast cell surface antigens Kit and T1/ST2 or the macrophage antigen CD11b (Mac-1) was determined by flow cytometric analysis. Each data point represents at least four separate experiments (*, P<.05; n≥3 by ANOVA). (B) Mast cell or monocyte/macrophage morphology was determined by WG, acidic TB, or nonspecific esterase (NSE) staining. Original bar, 10 µM. Solid arrowhead indicates mast cell morphology; open arrowhead indicates monocyte/macrophage morphology.

 
STAT3 is necessary for IL-10-mediated effects on macrophage survival
Among the pathways activated by IL-10, STAT3 is essential for IL-10R signaling [19 , 20 ]. We found that IL-10 induced Stat3 tyrosine phosphorylation with rapid and transient kinetics, peaking at 30 min of stimulation (Fig. 3A ). To determine the importance of Stat3 to IL-10-mediated suppression of bone marrow cell expansion, we sought an assay using Stat3-deficient cells. STAT3-deficient mice die during embryogenesis [19 ]; hence, these mice were not a useful source of bone marrow cells in our assay. We used a lineage-restricted cre/lox system in which STAT3 is deleted in macrophages via lysozyme M-driven expression of the cre recombinase [20 ]. To avoid the complication of mixed lineage populations, which would have variable cre expression, bone marrow from these mice and their littermate controls was cultured in M-CSF ± IL-10, selectively giving rise to macrophages. Viable cells numbers were measured by PI-DNA staining and timed flow cytometric counting on Day 7 of culture. Stat3-deficient macrophages proliferated poorly compared with WT littermate control cells (258.2±20.7 vs. 3405±224.6; n=4), indicating the importance of Stat3 in M-CSF signaling. It is more important that the addition of IL-10 to these cultures had strikingly different effects. IL-10 reduced M-CSF-mediated survival by 70% in WT cultures, much like its effects in cultures containing IL-3 + SCF. However, STAT3 deletion reversed this effect completely, inducing a twofold increase in viable cell numbers (Fig. 3B) . We confirmed the loss of STAT3 expression in these cultured cells by Western blotting (Fig. 3C) . These data indicate that STAT3 expression is required for the inhibitory effects of IL-10 on developing bone marrow cells.


Figure 3
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  		Figure 3. The role of Stat3 in IL-10-mediated inhibitory effects. (A) Freshly isolated murine bone marrow cells were stimulated with IL-10 for the indicated times, after which STAT3 tyrosine phosphorylation (pY) was measured by Western blotting. (B) Bone marrow isolated from Stat3-deficient mice (Stat3 flox/–; lysMcre) or their normal littermates was cultured for 7 days in macrophage-colony stimulating factor (MCSF) ± IL-10. Live cell numbers were measured by PI-DNA staining and timed flow cytometric counting and used to determine the percent change in the presence of IL-10 (*, P<.05; n=3 by ANOVA). (C) Western blot showing deletion of STAT3 protein in STAT3 flox/–; lysMcre cells.

 
IL-10 enhances apoptosis
As IL-10 did not appear to alter S-phase progression significantly, the decrease in cell numbers that we observed was most likely caused by enhanced cell death. PI-DNA staining of fixed, permeabilized cells treated with RNase A was used to detect an increase in DNA fragmentation associated with apoptosis. As seen in Figure 4A , IL-10 had no effect on apoptosis on Days 0–10 but conveyed a significant increase to include 60% of the population between Days 10 and 21. Activation of the death-effector enzyme caspase-3 immediately precedes the onset of apoptosis. We measured caspase-3 activation midway through this period on Day 15 and found significantly higher activity in cultures containing IL-10 (Fig. 4B) . These data indicated that the reduction in live cells was caused by apoptosis.


Figure 4
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  		Figure 4. IL-10 increases bone marrow cell apoptosis and enhances caspase-3 activation. (A) Summary of increased apoptosis as judged by PI-DNA staining and an example histogram (B) of this staining from samples harvested on Day 21 of culture in IL-3 + SCF (black line) or IL-3 + SCF + IL-10 (gray line). The area designated as M1 indicates subdiploid DNA content, an indication of DNA fragmentation. (C) Summary of caspase-3 activation and an example histogram (D) of caspase-3 staining from samples harvested on Day 21 of culture in IL-3 + SCF (black line) or IL-3 + SCF + IL-10 (gray line). The area designated as M1 indicates cells with active caspase-3 (*, P<.05; n≥3 by Student’s t-test).

 
IL-10 induces changes in mitochondrial membrane stability
Changes in mitochondrial membrane potential ({Delta}{psi}M) can occur during apoptosis, often resulting from alterations in Bcl-2 gene family expression or function [22 ]. IL-10-mediated changes in {Delta}{psi}M were measured via Di(OC3)6 staining. IL-10 altered the pattern of Di(OC3)6 staining, with evidence of hypo- and hyperpolarization (Fig. 5A and B ). Thus, the presence of IL-10 resulted in altered {Delta}{psi}M.


Figure 5
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  		Figure 5. IL-10-mediated apoptosis involves a mitochondrial pathway. (A) Changes in {Delta}{psi}M were assessed on Culture Day 15 by Di(OC6)3 staining as described in Materials and Methods. A summary of these data (A) and a representative histogram (B) are shown. Cultures containing IL-3 + SCF are shown in black, and those containing IL-3 + SCF + IL-10 are in gray. The areas designated M1 and M2 represent deviation from normal mitochondrial staining and indicate hypo- and hyperpolarization, respectively. (C) Bax-deficient (Bax KO), p53-deficient (p53 KO), and H2K-Bcl-2 transgenic (Bcl-2 Tg) bone marrow cells were cultured in IL-3 + SCF ± IL-10. Viable cell numbers were assessed on Day 21 by PI-DNA staining and timed flow cytometric counting and calculated as a percentage of IL-3 + SCF cultures (*, P<.05; n≥3 by Student’s t-test).

 
We next used bone marrow cells from transgenic and gene-deficient mice to determine the importance Bcl-2 family proteins and the p53 transcription factor, which can alter their expression. Bone marrow cells from mice deficient in proapoptotic Bax or p53 expression or from mice overexpressing antiapoptotic Bcl-2 were cultured in IL-3 + SCF ± IL-10. Loss of Bax expression did not alter the IL-10-mediated decrease in cell numbers. By contrast, p53 deficiency or Bcl-2 overexpression increased survival approximately twofold; however, neither completely reversed the effects of IL-10 (Fig. 5C) . These observations indicate that the apoptotic effects of IL-10 correlate with loss of normal {Delta}{psi}M and can be partially corrected by increasing Bcl-2 expression or deleting p53.

IL-10 inhibits Kit receptor expression
Inhibition of growth factor signaling has been shown to result in p53-dependent apoptosis, which proceeds through a mitochondrial pathway, also referred to as the "intrinsic" death pathway [22 , 23 ]. IL-3 and SCF promote survival and are best known for their activities on developing mast cells. As IL-10 signaling resulted in an apoptotic response coincident with changes in {Delta}{psi}M, which was partially corrected by modifying p53 or Bcl-2, loss of IL-3 or SCF receptor expression could be the means by which IL-10 induces apoptosis in developing bone marrow cells. We monitored changes in the IL-3Rß chain and Kit, the signal transduction proteins activated by IL-3 and SCF, respectively. Based on cell surface staining, IL-10 inhibited Kit but not IL-3Rß expression (Fig. 6A ). The decrease in Kit expression, ~35% inhibition, correlated with the onset of apoptosis, beginning on Day 10 of culture. It is interesting that the addition of IL-10 on Day 14 of culture, which did not alter cell death (Fig. 1) , also diminished Kit expression by a comparable 27.8% (n=4, P<.001 vs. IL-3+SCF; data not shown). This result is consistent with our early finding that IL-10 suppresses Kit expression on mature mast cells [10 ]. These data argue that IL-10-mediated inhibition of Kit expression could be involved in apoptosis but does not strictly correlate with cell death.


Figure 6
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  		Figure 6. IL-10 alters expression and function of growth factor receptors. Bone marrow cells were cultured in IL-3 + SCF ± IL-10. (A) Expression intensity of IL-3Rßc or Kit, as determined by flow cytometry. (B) Equal numbers of viable cells cultured in IL-3 + SCF ± IL-10 for 21 days were washed and replated in IL-3 or SCF for 4 days. After PI-DNA staining, the percent viable cells was measured by flow cytometry (*, P<.05; n≥3 by ANOVA).

 
Changes in receptor signaling can occur with or without alterations in expression. It was essential that we determine if IL-10 disrupted IL-3- and SCF-mediated survival. To measure these effects, we used short-term survival assays. Bone marrow cells were cultured for 21 days in IL-3 + SCF ± IL-10, after which equal numbers of viable cells were washed and replated in decreasing concentrations of IL-3 or SCF for 4 days. The percentage of surviving and apoptotic cells was judged by the presence of subdiploid DNA, as detected by PI-DNA staining. As shown in Figure 6C , cells that had previously been cultured in IL-10 were significantly less responsive to IL-3 or SCF in these assays. Under conditions where 55–70% of the cells previously cultured in IL-3 + SCF survived in IL-3 or SCF alone, cells previously stimulated with IL-10 showed survival rates of 20%. These data indicated that IL-10 stimulation inhibited survival signaling by the Kit and IL-3R complexes, resulting in a factor-withdrawal response, which is consistent with the intrinsic, mitochondrial, apoptotic cascade we have observed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The IL-10R is expressed on a variety of hematopoietic cells including B cells, T cells, macrophages, and mast cells, where it regulates many aspects of the inflammatory response. IL-10 has been shown to be an important regulator of experimental allergic encephalomyelitis [24 25 26 ], nonobese diabetes [27 ], inflammatory arthritis [28 29 30 31 32 33 34 ], airway hyper-responsiveness [35 ], and inflammatory bowel disease [36 , 37 ]. Many studies have also found correlations between serum IL-10 and ongoing immune responses or disease progression. For example, serum IL-10 levels are elevated for at least 5 days during septicemia, and some patients demonstrate up to 2800 pg/ml [38 , 39 ]. Colorectal cancer, breast cancer, B cell lymphoma, and hepatocellular carcinoma patients show elevated serum IL-10 [40 41 42 43 ]. Similar increases are also noted in chronic hepatitis, myocardial infarction, Grave’s disease, and systemic sclerosis [44 45 46 47 ]. Systemic alterations in IL-10, coupled with its demonstrated effects on immunomodulation, argue for the clinical relevance of this cytokine. We reasoned that changes in systemic IL-10 concentrations during inflammatory responses could affect not only mature mast cells and macrophages but also their precursors.

It is important that our data could appear to contradict earlier work in this area. For example, Rennick et al. [48 , 49 ] showed IL-10 to be a cofactor for SCF-dependent mast cell progenitor growth and maturation. IL-10 was also reported to promote the growth of IL-3-dependent mast cell progenitors [50 ]. In contrast to our study, these experiments used committed mast cell precursors. It seems likely that the effects of IL-10 may be dependent on the stage of differentiation. In support of this, we found that IL-10-mediated apoptosis required that it be added to bone marrow progenitors during the first 7 days of culture, the period during which commitment to the mast cell lineage occurs [51 ].

Our focus is how Th2 cytokines such as IL-10 and IL-4 alter the inflammatory response. We have previously shown that both cytokines inhibit mast cell IgE receptor and Kit expression and can combine to induce apoptosis in mature mast cells [9 10 11 12 ]. We also found that IL-4 could induce the apoptotic death of developing mast cells [6 ]. It is important that apoptosis induced by IL-4 in developing mast cells and by IL-4 + IL-10 in mature mast cells proceeded through a mitochondrial pathway, which was blocked by Bcl-2 overexpression [6 , 12 ], consistent with a factor-withdrawal response inducing the intrinsic, apoptotic pathway. Given these results and the close association with elevated serum IL-10 in a wide range of inflammatory conditions, we assessed the role of IL-10 in progenitor development.

The most overt effect of IL-10 was a 75% reduction in viable cells, caused by an apoptotic cascade, which was consistent with the intrinsic pathway mediated by mitochondrial damage. This theory is supported by findings of caspase activation and DNA fragmentation, coincidental with changes in {Delta}{psi}M. Moreover, Bcl-2 overexpression or p53 deletion partially blocked cell death. As growth factor withdrawal induces apoptosis via a mitochondrial pathway accompanied by p53 activation [52 ], we suspected that IL-10 acts to interfere with survival signals conveyed by IL-3 and/or SCF. Consistent with this idea, exposure to IL-10 greatly reduced IL-3- or SCF-mediated survival. Although IL-10 decreased Kit receptor expression, this did not correlate entirely with apoptosis and hence could be a necessary but not sufficient event in progenitor cell death. We hypothesize that IL-10 induces the expression of inhibitory molecules, such as members of the suppressor of cytokine signaling family that dampen IL-3 and SCF signaling.

Although IL-10 can signal via several pathways, STAT3 may be the most critical of these. For example, STAT3 deletion greatly impairs IL-10 signaling in macrophages [13 , 19 , 20 ]. As we found that the percentage of macrophages did not increase with cell death, mast cells and macrophages were killed by IL-10 in our assay. Therefore, we addressed the role of Stat3 using a macrophage-restricted knockout system. In congruence with other studies about IL-10 signaling, the effects of IL-10 were completely dependent on STAT3, highlighting its importance in IL-10-mediated cell death. Although we cannot draw direct conclusions to mast cells, the shared myeloid lineage of mast cells and macrophages allows us to hypothesize that IL-10 acts via STAT3 to promote apoptosis in developing mast cells and macrophages, killing cells by blocking survival signals.

The delayed kinetics of the IL-10 response are intriguing, especially given the rapid and transient activation of Stat3 in bone marrow cells stimulated with IL-10 (Fig. 3A) . Our hypothesis is that Stat3 activation initiates a series of gene expression events, which occur in myeloid precursors prior to their lineage commitment, resulting in their apoptosis at the time of commitment and expansion. As IL-10 needs to be present throughout this period, Stat3 must sit at the apex of a pathway involving other critical signals that need to be revealed. This is our current area of investigation.

Our theory is that Th2 cytokines such as IL-10 and IL-4 use delayed kinetics in dampening the inflammatory response to balance protective immunity with chronic disease. Similar to the current study, we have found that IL-4 and IL-10 inhibit the function of mature mast cells but only after 3 days of signaling [9 , 10 , 53 ]. After 6 days of signaling, mature mast cells are killed by IL-4 + IL-10 through a mitochondrially mediated apoptosis [12 ]. It now appears that IL-4 and IL-10 also arrest precursor development through an intrinsic apoptosis pathway after 10 days. This staging of repressive events could be viewed as a hierarchical form of homeostasis, acting first on the effector cells and subsequently, the progenitors from which they develop. As stated, prolonged increases in circulating IL-10 are noted in a wide range of pathological conditions. It is interesting to note that polymorphisms in the IL-4 and IL-10 promoters as well as the IL-4R are linked to the establishment of chronic, allergic disease [54 55 56 57 58 ]. Perhaps pathological immune responses result in part from loss of homeostatic controls over mast cell function and development, which should be mediated by Th2 cytokines. Our data demonstrate that IL-10 has potent, STAT3-mediated, apoptotic effects on developing mast cells and macrophages, which may assist in preventing the establishment of chronic inflammation. Loss of these protective effects may underlie chronic inflammatory disease.


   ACKNOWLEDGEMENTS
 
This work was supported in part by generous grants to the Ryan laboratory from the American Cancer Society (IN-105), the Horsley Cancer Research Fund, the Thomas F. Jeffress and Kate Miller Jeffress Trust (J-457), and the National Institutes of Health (1RO1AI43433, 1R01CA91839).

Received April 15, 2005; revised February 2, 2006; accepted February 3, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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