Daniel P. Bailey

IL-10 Suppresses Mast Cell IgE Receptor Expression and Signaling In Vitro and In Vivo

Mast cells are known for their roles in allergy, asthma, systemic anaphylaxis, and inflammatory disease. IL-10 can regulate inflammatory responses and may serve as a natural regulator of mast cell function. We examined the effects of IL-10 on in vitro-cultured mouse and human mast cells, and evaluated the effects of IL-10 on FcεRI in vivo using mouse models. IgE receptor signaling events were also assessed in the presence or absence of IL-10. IL-10 inhibited mouse mast cell FcεRI expression in vitro through a Stat3-dependent process. This down-regulation was consistent in mice tested in vivo, and also on cultured human mast cells. IL-10 diminished expression of the signaling molecules Syk, Fyn, Akt, and Stat5, which could explain its ability to inhibit IgE-mediated activation. Studies of passive systemic anaphylaxis in IL-10-transgenic mice showed that IL-10 overexpression reduced the IgE-mediated anaphylactic response. These data suggest an important regulatory role for IL-10 in dampening mast cell FcεRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.

IL-10 Inhibits FcεRI Expression in Mouse Mast Cells

FcεRI expression and function is a central aspect of allergic disease. Using bone marrow-derived mouse mast cell populations, we have previously shown that the Th2 cytokine IL-4 inhibits FcεRI expression and function. In the current study we show that the Th2 cytokine IL-10 has similar regulatory properties, and that it augments the inhibitory effects of IL-4. FcεRI down-regulation was functionally significant, as it diminished inflammatory cytokine production and IgE-mediated FcεRI up-regulation. IL-10 and IL-4 reduced FcεRI β protein expression without altering the α or γ subunits. The ability of IL-4 and IL-10 to alter FcεRI expression by targeting the β-chain, a critical receptor subunit known to modulate receptor expression and signaling, suggests the presence of a Th2 cytokine-mediated homeostatic network that could serve to both initiate and limit mast cell effector function.

Catecholamines in murine bone marrow derived mast cells

Cultured murine bone marrow derived mast cells (BMMC) were found to store high levels of dopamine (3753+/-844 pg/10(7) cells) and occasionally produce norepinephrine and epinephrine. The catecholamine synthesis inhibitor, alpha-methyl-para-tyrosine, decreased intracellular catecholamine concentrations, and activation with ionomycin stimulated dopamine release. Neither dopaminergic receptor antagonists nor exogenous dopamine < or =10 microM affected IL-3-induced cell proliferation. High exogenous dopamine (20-100 microM) decreased proliferation and increased apoptosis, and the anti-oxidant ascorbic acid prevented these effects. Increased expression of the anti-apoptotic factor Bcl-2 or loss of pro-apoptotic Bax expression attenuated dopamine-induced apoptosis, suggesting the apoptosis proceeds through a mitochondrial pathway.

Interleukin‐10 induces apoptosis in developing mast cells and macrophages

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 macrophge 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.

Endogenous suppression of mast cell development and survival by IL-4 and IL-10.

Mast cell development is an important component of atopic and chronic inflammatory diseases such as asthma, multiple sclerosis, rheumatoid arthritis, and atherosclerosis. In this study, we found that IL‐4 and IL‐10 were produced constitutively in cultures of developing mast cells, correlating with mast cell purity. Deletion of either gene increased mast cell numbers and FcεRI expression during culture in IL‐3 + stem cell factor (SCF). By adding exogenous IL‐4 and IL‐10 to bone marrow (BM) cultures containing IL‐3 + SCF, we found that IL‐4 + IL‐10 suppressed mast cell development through mechanisms not used by either cytokine alone. IL‐4 + IL‐10 elicited a rapid cell death coincidental with reduced Kit receptor expression and signaling and enhanced mitochondrial damage and caspase activation. IL‐4 or IL‐10 costimulation, unlike either cytokine alone, altered mast cell ontogeny to yield predominantly macrophages in cultures that typically produce mast cells. This effect was observed consistently with unseparated BM cells, purified mouse BM stem cells, and erythrocyte‐depleted human umbilical cord blood cells. These experiments demonstrated a major role for Stat6 and Stat3, but not the Stat3‐induced transcriptional repressor Ets variant gene 3. Genetic background was also a critical factor, as BALB/c‐derived BM cells were completely resistant to IL‐10‐mediated killing and expressed lower levels of IL‐10R. Collectively, these results support the theory that IL‐4 and IL‐10 function as endogenous regulators of mast cell progenitor development, consistent with a role in immune homeostasis. Loss of this homeostasis, perhaps via genetic polymorphism, could contribute to the etiology of mast cell‐associated disease.

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.

Stat5: an essential regulator of mast cell biology.

Interleukin-3 (IL-3) and stem cell factor (SCF) are important mast cell growth and differentiation factors. Since both cytokines activate the transcription factor Stat5, a known regulator of proliferation and survival, we investigated the effects of Stat5 deficiency on mast cell development and survival. This article will review data presented at The Fourth International Workshop on Signal Transduction in the Activation and Development of Mast Cells and Basophils. The full set of data is now in preparation for publication. We find that the absence of Stat5 A and B results in a total loss of in vivo mast cell development. Bone marrow-derived mast cell (BMMC) populations can be cultured and maintained from Stat5-deficient mice in IL-3+SCF, but not in either cytokine alone. The absence of Stat5 resulted in aberrant control of Bcl-2, Bcl-x(L) and cyclin A2, with increased apoptosis and delayed cell cycle progression after IL-3 or SCF stimulation. These results indicate that Stat5 A and B are critical regulators of in vitro and in vivo mast cell biology.