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Kelly Speiran, Daniel P Bailey,
Josephine Fernando,
Matthew Macey,
Brian Barnstein,
Motunrayo Kolawole,
Dana Curley,
Stephanie S Watowich,
Peter J Murray,
Carole Oskeritzian,
John J Ryan
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ABSTRACT: 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 Fc epsilon RI 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.
Journal of leukocyte biology 03/2009; 85(5):826-36. · 4.99 Impact Factor
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Sarah Kennedy Norton,
Brian Barnstein,
Jennifer Brenzovich, Daniel P Bailey,
Mohit Kashyap,
Kelly Speiran,
Jill Ford,
Daniel Conrad,
Stephanie Watowich,
Matthew R Moralle,
Christopher L Kepley,
Peter J Murray,
John J Ryan
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ABSTRACT: 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 FcepsilonRI 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 FcepsilonRI 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 FcepsilonRI expression and function. IL-10 may hence serve as a mediator of mast cell homeostasis, preventing excessive activation and the development of chronic inflammation.
The Journal of Immunology 04/2008; 180(5):2848-54. · 5.79 Impact Factor
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ABSTRACT: 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.
Journal of Leukocyte Biology 10/2006; 80(3):581-9. · 4.99 Impact Factor
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ABSTRACT: The objective of this study was to determine the effects of transforming growth factor (TGF)-beta1 on mast cell development.
Mast cells were cultured from mouse bone marrow in interleukin (IL)-3 + stem cell factor, in the presence or absence of TGF-beta1. We assessed mast cell development by measuring the expression of kit, T1/ST2, FcvarepsilonRI, and Fcgamma receptors. Cell morphology was determined by histochemical staining. Alterations in FcvarepsilonRI subunit expression were measured by Western blot analysis. Adoptive transfer of cultured mast cells into mast cell-deficient W/W(v) mice was used to determine if the in vivo environment could reverse the inhibitory effects of TGF-beta1.
TGF-beta1 decreased FcvarepsilonRI, c-kit, T1/ST2, and FcgammaR expression, and inhibited granule formation in developing mast cells. Accessory cells were not required for this inhibition. Smad3 deficiency did not alter the response of bone marrow cells to TGF-beta1. TGF-beta1 inhibited expression of the FcvarepsilonRI alpha subunit protein, without decreasing beta or gamma proteins. Mast cells derived in the presence of TGF-beta1 were functionally impaired, as IgE-mediated cytokine secretion was greatly reduced. The changes in granule formation and surface antigen expression were long-standing, as they were not reversed by transfer to W/W(v) mice.
TGF-beta1 may contribute to mast cell homeostasis by inhibiting maturation from bone marrow precursors. The effects of TGF-beta1 result in greatly diminished expression of cell surface markers, reduced granulation, and lack of responsiveness to IgE-mediated activation. Thus TGF-beta1 can serve as a potent and multifunctional regulator of mast cell maturation.
Experimental Hematology 12/2005; 33(11):1281-91. · 2.90 Impact Factor
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L Andrew Bouton,
Carlos D Ramirez, Daniel P Bailey,
C Fitzhugh Yeatman,
Joyce Yue,
Harry V Wright,
Jos Domen,
Roberto R Rosato,
Steven Grant,
Krista Fischer-Stenger,
John J Ryan
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ABSTRACT: The aim of this study was to determine the mechanism by which interleukin (IL)-4 + IL-10 costimulation regulates mast cell numbers to maintain immune homeostasis.
We employed mouse bone marrow-derived mast cells (BMMC) to measure the effects of IL-4 + IL-10 on survival and cell-cycle progression. p53-Deficient, bax-deficient, and bcl-2 transgenic BMMC were compared to wild-type cells to determine the role of these proteins in apoptosis. The molecular regulation of apoptosis and cell-cycle progression was investigated using flow cytometric analysis, RNase protection, and Western blotting.
IL-4 + IL-10 induced BMMC apoptosis and arrest. Apoptosis was p53-dependent. Cell death was accompanied by loss of mitochondrial membrane potential, the importance of which was demonstrated by resistance to IL-4 + IL-10-mediated cell death when Bax was deleted or Bcl-2 was overexpressed. Those cells not killed by apoptosis demonstrated a p53-independent G1 cell-cycle arrest. Apoptosis and arrest may be explained by reduced IL-3 receptor signaling.
Costimulation with IL-4 + IL-10 partly controls mast cell homeostasis through a delayed apoptosis and arrest program that is induced by a blockade of IL-3 receptor signaling. The delay in these negative effects would allow the protective effects of mast cell activation to occur for several days.
Experimental Hematology 01/2005; 32(12):1137-45. · 2.90 Impact Factor
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Sheila R Gillespie,
Randall R DeMartino,
Jingfang Zhu,
Hey Jin Chong,
Carlos Ramirez,
Christopher P Shelburne,
L Andrew Bouton, Daniel P Bailey,
Anita Gharse,
Paria Mirmonsef,
Sandra Odom,
Gregorio Gomez,
Juan Rivera,
Krista Fischer-Stenger,
John J Ryan
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ABSTRACT: FcepsilonRI 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 FcepsilonRI 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. FcepsilonRI down-regulation was functionally significant, as it diminished inflammatory cytokine production and IgE-mediated FcepsilonRI up-regulation. IL-10 and IL-4 reduced FcepsilonRI beta protein expression without altering the alpha or gamma subunits. The ability of IL-4 and IL-10 to alter FcepsilonRI expression by targeting the beta-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.
The Journal of Immunology 04/2004; 172(5):3181-8. · 5.79 Impact Factor
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ABSTRACT: The aim of this study was to assess the effects of interleukin-4 and signal transducer and activator of transcription (Stat)-6 on IL-3+SCF-induced mast cell development.
Unseparated mouse bone marrow cells were cultured in IL-3+SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-4, the use of Stat6-deficient bone marrow cells, and expression of a constitutively active Stat6 mutant were employed to assess the effects of IL-4 and Stat6 on cell viability, proliferation, and differentiation. Bax-deficient and bcl-2 transgenic bone marrow cells were used to assess the importance of the mitochondria in IL-4-mediated effects.
IL-4 elicited apoptosis and limited the cell cycle progression of developing bone marrow cells, without affecting cell differentiation. Apoptosis required that IL-4 be present during the first 8 days of the 21-day culture period. Cell death correlated with loss of mitochondrial membrane potential. Accordingly, IL-4-mediated apoptosis was inhibited by Bax deletion or bcl-2 overexpression. Lastly, Stat6 activation was both necessary and sufficient to inhibit cell survival.
IL-4 exerts potent apoptotic effects on developing mast cells and monocyte/macrophages through mitochondrial damage and Stat6 activation.
Experimental Hematology 02/2004; 32(1):52-9. · 2.90 Impact Factor
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Hey Jin Chong,
L Andrew Bouton, Daniel P Bailey,
Harry Wright,
Carlos Ramirez,
Anita Gharse,
Carole Oskeritzian,
Han Zhang Xia,
Jinfang Zhu,
William E Paul,
Chris Kepley,
Lawrence B Schwartz,
John J Ryan
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ABSTRACT: Fc receptors for IgG (FcgammaR) are widely expressed in the hematopoietic system and mediate a variety of inflammatory responses. There are two functional classes of FcgammaR, activation and inhibitory receptors. Since IgG immune complexes (IgG IC) bind each class with similar affinity, co-expression of these receptors leads to their co-ligation. Thus, expression levels of this antagonistic pair play a critical role in determining the cellular response. Murine mast cells co-express the activation receptor FcgammaRIII and the inhibitory receptor FcgammaRIIb and can be activated by IgG IC. Mast cell activation contributes to allergic and other inflammatory diseases-particularly those in which IgG IC may play important roles. Using mouse bone marrow-derived mast cells, we report that IL-4 selectively increases FcgammaRIII expression without altering FcgammaRIIb. This enhanced expression could be induced by Stat6 activation alone, and appeared to be mediated in part by increased FcgammaRIIIalpha protein synthesis without significant changes in transcription. The increase in FcgammaRIII expression was functionally significant, as it was matched by enhanced FcgammaR-mediated degranulation and cytokine production. Selective regulation of mast cell FcgammaR by interleukin-4 could alter inflammatory IgG responses and subsequently disease severity and progression.
Cellular Immunology 09/2003; 224(2):65-73. · 1.97 Impact Factor
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Christopher P Shelburne,
Margaret E McCoy,
Roland Piekorz,
Veronica Sexl,
Kwan-Ho Roh,
Sarah M Jacobs-Helber,
Sheila R Gillespie, Daniel P Bailey,
Paria Mirmonsef,
Meredith N Mann,
Mohit Kashyap,
Harry V Wright,
Hey Jin Chong,
L Andrew Bouton,
Brian Barnstein,
Carlos D Ramirez,
Kevin D Bunting,
Steven Sawyer,
Chris S Lantz,
John J Ryan
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ABSTRACT: Interleukin-3 (IL-3) and stem cell factor (SCF) are important mast cell growth and differentiation factors. Since both cytokines activate the transcription factor signal transducer and activator of transcription 5 (Stat5), a known regulator of proliferation and survival, we investigated the effects of Stat5 deficiency on mast cell development and survival. Bone marrow-derived mast cell (BMMC) populations cultured from Stat5A/B-deficient mice survived in IL-3 + SCF, but not in either cytokine alone. These cells demonstrated reduced expression of Bcl-2, Bcl-x(L), cyclin A2, and cyclin B1, with increased apoptosis and delayed cell cycle progression during IL-3 or SCF culture. Finally, the absence of Stat5 resulted in loss of in vivo mast cell development, as judged by assessments of Stat5-deficient mice and transplantation of Stat5-deficient bone marrow cells to mast cell-deficient recipient mice. These results indicate that Stat5A and Stat5B are critical regulators of in vitro and in vivo mast cell development and survival.
Blood 09/2003; 102(4):1290-7. · 9.90 Impact Factor
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Christopher P Shelburne,
Margaret E McCoy,
Roland Piekorz,
Veronica V Sexl,
Sheila R Gillespie, Daniel P Bailey,
Anita Gharse,
Paria Mirmonsef,
Meredith N Mann,
Mohit Kashyap,
Harry V Wright,
Hey Jin Chong,
L Andrew Bouton,
Carlos D Ramirez,
Chris S Lantz,
John J Ryan
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[hide abstract]
ABSTRACT: 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.
Molecular Immunology 10/2002; 38(16-18):1187-91. · 2.90 Impact Factor
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ABSTRACT: ObjectiveThe aim of this study was to assess the effects of interleukin-4 and signal transducer and activator of transcription (Stat)-6 on IL-3 + SCF-induced mast cell development.Materials and MethodsUnseparated mouse bone marrow cells were cultured in IL-3 + SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-4, the use of Stat6-deficient bone marrow cells, and expression of a constitutively active Stat6 mutant were employed to assess the effects of IL-4 and Stat6 on cell viability, proliferation, and differentiation. Bax-deficient and bcl-2 transgenic bone marrow cells were used to assess the importance of the mitochondria in IL-4-mediated effects.ResultsIL-4 elicited apoptosis and limited the cell cycle progression of developing bone marrow cells, without affecting cell differentiation. Apoptosis required that IL-4 be present during the first 8 days of the 21-day culture period. Cell death correlated with loss of mitochondrial membrane potential. Accordingly, IL-4-mediated apoptosis was inhibited by Bax deletion or bcl-2 overexpression. Lastly, Stat6 activation was both necessary and sufficient to inhibit cell survival.ConclusionIL-4 exerts potent apoptotic effects on developing mast cells and monocyte/macrophages through mitochondrial damage and Stat6 activation.
Experimental Hematology.
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Hey Jin Chong,
L. Andrew Bouton, Daniel P. Bailey,
Harry Wright,
Carlos Ramirez,
Anita Gharse,
Carole Oskeritzian,
Han-Zhang Xia,
Jinfang Zhu,
William E. Paul,
Chris Kepley,
Lawrence B. Schwartz,
John J. Ryan
[show abstract]
[hide abstract]
ABSTRACT: Fc receptors for IgG (FcγR) are widely expressed in the hematopoietic system and mediate a variety of inflammatory responses. There are two functional classes of FcγR, activation and inhibitory receptors. Since IgG immune complexes (IgG IC) bind each class with similar affinity, co-expression of these receptors leads to their co-ligation. Thus, expression levels of this antagonistic pair play a critical role in determining the cellular response. Murine mast cells co-express the activation receptor FcγRIII and the inhibitory receptor FcγRIIb and can be activated by IgG IC. Mast cell activation contributes to allergic and other inflammatory diseases—particularly those in which IgG IC may play important roles. Using mouse bone marrow-derived mast cells, we report that IL-4 selectively increases FcγRIII expression without altering FcγRIIb. This enhanced expression could be induced by Stat6 activation alone, and appeared to be mediated in part by increased FcγRIIIα protein synthesis without significant changes in transcription. The increase in FcγRIII expression was functionally significant, as it was matched by enhanced FcγR-mediated degranulation and cytokine production. Selective regulation of mast cell FcγR by interleukin-4 could alter inflammatory IgG responses and subsequently disease severity and progression.
Cellular Immunology. 224(2):65-73.
