Potent stimulation of eosinopoiesis in murine bone-marrow by myriadenolide is mediated by cysteinyl-leukotriene signaling.

We describe the potent effect of myriadenolide (Myr), a naturally occurring labdane diterpene, in promoting the production of eosinophils in cultured bone-marrow from several inbred mouse strains. This enhancing effect is lineage-selective and requires the eosinophil growth factors, Interleukin(IL)-5 or GM-CSF. Myr acts over a very low concentration range (10-10-10-14 M), if added at the beginning of the cell cultivation. Its enhancing effect increases between 24 h and 10 days of culture. We used both pharmacological and genetical tools to analyze its mechanism of action. Several lines of evidence show that the enhancing effect of Myr requires functional integrity of the 5-lipoxygenase (5-LO) pathway, and of CysLT1 receptors, which transduce the effects of cysteinyl-leukotrienes generated through this pathway. Myr also protects developing eosinophils from apoptosis induced by exogenous prostaglandin E2 (PGE2), but not by NO, indicating that it acts upstream of NO in the PGE2-initiated proapoptotic pathway which requires iNOS and CD95. Exposure to NO concentrations insufficient to induce apoptosis abolished the ability of eosinophils to respond to Myr, suggesting the involvement of a NO-sensitive cellular target. Myr has potential as a chemically defined research tool, which can be used to generate large numbers of eosinophils, thereby overcoming current limitations in the biochemical and molecular biological study of murine eosinophils, which has so far depended on complex, labor-intensive and long-term culture protocols for in vitro expansion. SUMMARY: Potent enhancing effects of Myr on eosinophil production in bone marrow stimulated by GM-CSF and IL-5 are mediated by the 5-LO pathway.

Blockage of Eosinopoiesis by IL-17A Is Prevented by Cytokine and Lipid Mediators of Allergic Inflammation.

Interleukin- (IL-) 17A, a pleiotropic mediator of inflammation and autoimmunity, potently stimulates bone-marrow neutrophil production. To explore IL-17A effects on eosinopoiesis, we cultured bone-marrow from wild-type mice, or mutants lacking inducible nitric oxide synthase (iNOS-/-), CD95 (lpr), IL-17RA, or IL-4, with IL-5, alone or associated with IL-17A. Synergisms between IL-17A-activated, NO-dependent, and NO-independent mechanisms and antagonisms between IL-17A and proallergic factors were further examined. While IL-17A (0.1-10 ng/mL) had no IL-5-independent effect on eosinopoiesis, it dose-dependently suppressed IL-5-induced eosinophil differentiation, by acting during the initial 24 hours. Its effectiveness was abolished by caspase inhibitor, zVAD-fmk. The effect of IL-17A (0.1-1 ng/mL) was sensitive to the iNOS-selective inhibitor aminoguanidine and undetectable in iNOS-/- bone-marrow. By contrast, a higher IL-17A concentration (10 ng/mL) retained significant suppressive effect in both conditions, unmasking a high-end iNOS-independent mechanism. Lower IL-17A concentrations synergized with NO donor nitroprusside. Eosinopoiesis suppression by IL-17A was (a) undetectable in bone-marrow lacking IL-17RA or CD95 and (b) actively prevented by LTD4, LTC4, IL-13, and eotaxin. Sensitivity to IL-17A was increased in bone-marrow lacking IL-4; adding IL-4 to the cultures restored IL-5 responses to control levels. Therefore, effects of both IL-17A and proallergic factors are transduced by the iNOS-CD95 pathway in isolated bone-marrow.

The In Vivo Granulopoietic Response to Dexamethasone Injection Is Abolished in Perforin-Deficient Mutant Mice and Corrected by Lymphocyte Transfer from Nonsensitized Wild-Type Donors.

Exogenously administered glucocorticoids enhance eosinophil and neutrophil granulocyte production from murine bone-marrow. A hematological response dependent on endogenous glucocorticoids underlies bone-marrow eosinophilia induced by trauma or allergic sensitization/challenge. We detected a defect in granulopoiesis in nonsensitized, perforin-deficient mice. In steady-state conditions, perforin- (Pfp-) deficient mice showed significantly decreased bone-marrow and blood eosinophil and neutrophil counts, and colony formation in response to GM-CSF, relative to wild-type controls of comparable age and/or weight. By contrast, peripheral blood or spleen total cell and lymphocyte numbers were not affected by perforin deficiency. Dexamethasone enhanced colony formation by GM-CSF-stimulated progenitors from wild-type controls, but not Pfp mice. Dexamethasone injection increased bone-marrow eosinophil and neutrophil counts in wild-type controls, but not Pfp mice. Because perforin is expressed in effector lymphocytes, we examined whether this defect would be corrected by transferring wild-type lymphocytes into perforin-deficient recipients. Short-term reconstitution of the response to dexamethasone was separately achieved for eosinophils and neutrophils by transfer of distinct populations of splenic lymphocytes from nonsensitized wild-type donors. Transfer of the same amount of splenic lymphocytes from perforin-deficient donors was ineffective. This demonstrates that the perforin-dependent, granulopoietic response to dexamethasone can be restored by transfer of innate lymphocyte subpopulations.

α-Galactosylceramide suppresses murine eosinophil production through interferon-γ-dependent induction of NO synthase and CD95.

BACKGROUND AND PURPOSE: α-Galactosylceramide (α-GalCer), a pleiotropic immunomodulator with therapeutic potential in neoplastic, autoimmune and allergic diseases, activates invariant natural killer T-cells throughCD1-restricted receptors for α-GalCer on antigen-presenting cells, inducing cytokine secretion. However the haemopoietic effects of α-GalCer remain little explored. EXPERIMENTAL APPROACH: α-GalCer-induced modulation of eosinophil production in IL-5-stimulated bone marrow cultures was examined in wild-type (BALB/c, C57BL/6) mice and their mutants lacking CD1, inducible NOS (iNOS), CD95 and IFN-γ, along with the effects of lymphocytes; IFN-γ; caspase and iNOS inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs) and LTD4 ; and dexamethasone. KEY RESULTS: α-GalCer (10(-6) -10(-8) M) suppressed IL-5-stimulated eosinopoiesis by inducing apoptosis. α-GalCer pretreatment in vivo (100 µg·kg(-1) , i.v.) suppressed colony formation by GM-CSF-stimulated bone marrow progenitors in semi-solid cultures. α-GalCer and dexamethasone synergistically promoted eosinophil maturation. Suppression of eosinophil production by α-GalCer was prevented by aminoguanidine and was undetectable in bone marrow lacking iNOS, CD95, CD28; or CD1d. Separation on Percoll gradients and depletion of CD3+ cells made bone marrow precursors unresponsive to α-GalCer. Responsiveness was restored with splenic lymphocytes. Experiments with (i) IFN-γ-deficient bone marrow, alone or co-cultured with spleen T-cells from wild-type, but not from CD1d-deficient, donors; (ii) IFN-γ neutralization; and (iii) recombinant IFN-γ, showed that these effects of α-GalCer were mediated by IFN-γ. Effects of α-GalCer on eosinophil production were blocked by LTD4 and NSAIDs. CONCLUSIONS AND IMPLICATIONS: α-GalCer activation of IFN-γ-secreting, CD1d-restricted lymphocytes induced iNOS-CD95-dependent apoptosis in developing eosinophils. This pathway is initiated by endogenous regulatory lymphocytes, antagonised by LTD4 , NSAIDs and aminoguanidine, and modified by dexamethasone.

Roles of 5-lipoxygenase and cysteinyl-leukotriene type 1 receptors in the hematological response to allergen challenge and its prevention by diethylcarbamazine in a murine model of asthma.

Diethylcarbamazine (DEC), which blocks leukotriene production, abolishes the challenge-induced increase in eosinopoiesis in bone-marrow from ovalbumin- (OVA-) sensitized mice, suggesting that 5-lipoxygenase (5-LO) products contribute to the hematological responses in experimental asthma models. We explored the relationship between 5-LO, central and peripheral eosinophilia, and effectiveness of DEC, using PAS or BALB/c mice and 5-LO-deficient mutants. We quantified eosinophil numbers in freshly harvested or cultured bone-marrow, peritoneal lavage fluid, and spleen, with or without administration of leukotriene generation inhibitors (DEC and MK886) and cisteinyl-leukotriene type I receptor antagonist (montelukast). The increase in eosinophil numbers in bone-marrow, observed in sensitized/challenged wild-type mice, was abolished by MK886 and DEC pretreatment. In ALOX mutants, by contrast, there was no increase in bone-marrow eosinophil counts, nor in eosinophil production in culture, in response to sensitization/challenge. In sensitized/challenged ALOX mice, challenge-induced migration of eosinophils to the peritoneal cavity was significantly reduced relative to the wild-type PAS controls. DEC was ineffective in ALOX mice, as expected from a mechanism of action dependent on 5-LO. In BALB/c mice, challenge significantly increased spleen eosinophil numbers and DEC treatment prevented this increase. Overall, 5-LO appears as indispensable to the systemic hematological response to allergen challenge, as well as to the effectiveness of DEC.

Essential roles of endogenous glucocorticoids and TNF/TNFR1 in promoting bone-marrow eosinopoiesis in ovalbumin-sensitized, airway-challenged mice.

AIMS: Stress mechanisms paradoxically contribute to allergic episodes in humans and mice. Glucocorticoids (GC) and interleukin (IL)-5 synergically upregulate murine bone-marrow eosinophil production. Here we explored the role of endogenous GC in allergen-stimulated bone-marrow eosinophil production in ovalbumin-sensitized/challenged mice. MAIN METHODS: In BALB/c or C57BL/6 mice, sensitized and intranasally challenged with ovalbumin, we monitored eosinophil numbers in freshly harvested or cultured bone-marrow, and plasma corticosterone levels. Metyrapone (MET) was used to inhibit GC synthesis, and RU486 to block GC actions. In sensitized mice challenged intraperitoneally, we examined the relationship between eosinophilia of bone-marrow and peritoneal cavity, in the absence or presence of RU486. In experiments involving in vivo neutralization of tumor necrosis factor-α (TNF) by specific antibodies, or using mice which lack functional type I TNF receptors (TNFRI), we evaluated the relationship between TNF blockade, corticosterone levels, RU486 or MET treatment and challenge-induced bone-marrow eosinophilia. KEY FINDINGS: RU486 or MET pretreatments abolished challenge-induced increases in eosinophil numbers in bone-marrow (in vivo and ex vivo), and in the peritoneal cavity. MET, but not RU486, prevented the challenge-induced increase in corticosterone levels. Challenge-induced bone-marrow eosinophilia and corticosterone surge were abolished in TNFRI-deficient mice. Anti-TNF-treatment very effectively prevented challenge-induced bone-marrow eosinophilia, in the absence of RU486 or MET, but had no independent effect in the presence of either drug. SIGNIFICANCE: Endogenous GC was essential for allergen challenge-induced increases in eosinophil numbers inside bone-marrow. This effect required TNF and TNFRI, which suggests an immunoendocrine mechanism.

Essential roles of PKA, iNOS, CD95/CD95L, and terminal caspases in suppression of eosinopoiesis by PGE2 and other cAMP-elevating agents.

Up- and downregulation of eosinopoiesis control pulmonary eosinophilia in human asthma. In mice, eosinopoiesis is suppressed in vitro by prostaglandin E2 (PGE2) and in vivo by diethylcarbamazine, through a proapoptotic mechanism sequentially requiring inducible NO synthase (iNOS) and the ligand for death receptor CD95 (CD95L). We examined the roles of iNOS, cAMP-mediated signaling, caspases, and CD95L/CD95 in suppression of eosinopoiesis by PGE2 and other agents signaling through cAMP. Bone-marrow collected from BALB/c mice, or from iNOS-, CD95-, or CD95L-deficient mutants (and wild-type controls), was cultured with interleukin-5 (IL-5), alone or associated with PGE2, cAMP-inducing/mimetic agents, caspase inhibitor zVAD-fmk, iNOS inhibitor aminoguanidine, or combinations thereof, and eosinopoiesis was evaluated at various times. PGE2, added up to 24 hours of culture, dose-dependently suppressed eosinopoiesis, by inducing apoptosis. This effect was (a) paralleled by induction of iNOS in eosinophils; (b) duplicated by sodium nitroprusside, isoproterenol, and cAMP-inducing/mimetic agents; (c) prevented by protein kinase A inhibition. NO was produced through iNOS by dibutyryl-cAMP-stimulated bone-marrow. Overall, PGE2 and isoproterenol shared a requirement for four effector elements (iNOS, CD95L, CD95, and terminal caspases), which together define a pathway targeted by several soluble up- and downmodulators of eosinopoiesis, including drugs, mediators of inflammation, and cytokines.

G-CSF suppresses allergic pulmonary inflammation, downmodulating cytokine, chemokine and eosinophil production.

AIMS: Granulocyte Colony-Stimulating Factor (G-CSF), which mobilizes hemopoietic stem cells (HSC), is believed to protect HSC graft recipients from graft-versus-host disease by enhancing Th2 cytokine secretion. Accordingly, G-CSF should aggravate Th2-dependent allergic pulmonary inflammation and the associated eosinophilia. We evaluated the effects of G-CSF in a model of allergic pulmonary inflammation. MAIN METHODS: Allergic pulmonary inflammation was induced by repeated aerosol allergen challenge in ovalbumin-sensitized C57BL/6J mice. The effects of allergen challenge and of G-CSF pretreatment were evaluated by monitoring: a) eosinophilia and cytokine/chemokine content of bronchoalveolar lavage fluid, pulmonary interstitium, and blood; b) changes in airway resistance; and c) changes in bone-marrow eosinophil production. KEY FINDINGS: Contrary to expectations, G-CSF pretreatment neither induced nor enhanced allergic pulmonary inflammation. Instead, G-CSF: a) suppressed accumulation of infiltrating eosinophils in bronchoalveolar, peribronchial and perivascular spaces of challenged lungs; and b) prevented ovalbumin challenge-induced rises in airway resistance. G-CSF had multiple regulatory effects on cytokine and chemokine production: in bronchoalveolar lavage fluid, levels of IL-1 and IL-12 (p40), eotaxin and MIP-1a were decreased; in plasma, KC, a neutrophil chemoattractant, was increased, while IL-5 was decreased and eotaxin was unaffected. In bone-marrow, G-CSF: a) prevented the increase in bone-marrow eosinophil production induced by ovalbumin challenge of sensitized mice; and b) selectively stimulated neutrophil colony formation. SIGNIFICANCE: These observations challenge the view that G-CSF deviates cytokine production towards a Th2 profile in vivo, and suggest that this neutrophil-selective hemopoietin affects eosinophilic inflammation by a combination of effects on lung cytokine production and bone-marrow hemopoiesis.

Cysteinyl-leukotriene type 1 receptors transduce a critical signal for the up-regulation of eosinophilopoiesis by interleukin-13 and eotaxin in murine bone marrow.

IL-13 and eotaxin play important, inter-related roles in asthma models. In the lungs, CysLT, produced by the 5-LO-LTC4S pathway, mediate some local responses to IL-13 and eotaxin; in bone marrow, CysLT enhance IL-5-dependent eosinophil differentiation. We examined the effects of IL-13 and eotaxin on eosinophil differentiation. Semi-solid or liquid cultures were established from murine bone marrow with GM-CSF or IL-5, respectively, and the effects of IL-13, eotaxin, or CysLT on eosinophil colony formation and on eosinophil differentiation in liquid culture were evaluated, in the absence or presence of: a) the 5-LO inhibitor zileuton, the FLAP inhibitor MK886, or the CysLT1R antagonists, montelukast and MK571; b) mutations that inactivate 5-LO, LTC4S, or CysLT1R; and c) neutralizing mAb against eotaxin and its CCR3 receptor. Both cytokines enhanced GM-CSF-dependent eosinophil colony formation and IL-5-stimulated eosinophil differentiation. Although IL-13 did not induce eotaxin production, its effects were abolished by anti-eotaxin and anti-CCR3 antibodies, suggesting up-regulation by IL-13 of responses to endogenous eotaxin. Anti-CCR3 blocked eotaxin completely. The effects of both cytokines were prevented by zileuton, MK886, montelukast, and MK571, as well as by inactivation of the genes coding for 5-LO, LTC4S, and CysLT1R. In the absence of either cytokine, these treatments or mutations had no effect. These findings provide evidence for: a) a novel role of eotaxin and IL-13 in regulating eosinophilopoiesis; and b) a role for CysLTRs in bone marrow cells in transducing cytokine regulatory signals.

Inducible nitric oxide synthase/CD95L-dependent suppression of pulmonary and bone marrow eosinophilia by diethylcarbamazine.

RATIONALE: The mechanism of action of diethylcarbamazine (DEC), an antifilarial drug effective against tropical pulmonary eosinophilia, remains controversial. DEC effects on microfilariae depend on inducible NO synthase (iNOS). In eosinophilic pulmonary inflammation, its therapeutic mechanism has not been established. We previously described the rapid up-regulation of bone marrow eosinophilopoiesis in ovalbumin (OVA)-sensitized mice by airway allergen challenge, and further evidenced the down-regulation of eosinophilopoiesis by iNOS- and CD95L-dependent mechanisms. OBJECTIVES: We investigated whether: (1) DEC can prevent the effects of airway challenge of sensitized mice on lungs and bone marrow, and (2) its effectiveness depends on iNOS/CD95L. METHODS: OVA-sensitized BALB/c mice were intranasally challenged for 3 consecutive days, with DEC administered over a 12-, 3-, or 2-day period, ending at the day of the last challenge. We evaluated: (1) airway resistance, cytokine (IFN-gamma, IL-4, IL-5, and eotaxin) production, and pulmonary eosinophil accumulation; and (2) bone marrow eosinophil numbers in vivo and eosinophil differentiation ex vivo. MEASUREMENTS AND MAIN RESULTS: DEC effectively prevented the effects of subsequent challenges on: (1) airway resistance, Th1/Th2 cytokine production, and pulmonary eosinophil accumulation; and (2) eosinophilopoiesis in vivo and ex vivo. Recovery from unprotected challenges included full responses to DEC during renewed challenges. DEC directly suppressed IL-5-dependent eosinophilopoiesis in naive bone marrow. DEC was ineffective in CD95L-deficient gld mice and in mice lacking iNOS activity because of gene targeting or pharmacological blockade. CONCLUSIONS: DEC has a strong impact on pulmonary eosinophilic inflammation in allergic mice, as well as on the underlying hemopoietic response, suppressing the eosinophil lineage by an iNOS/CD95L-dependent mechanism.