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

Xavier-Elsas P, de Luca B, Queto T, Vieira BM, Masid-de-Brito D, Dahab EC, Alves Filho JC, Cunha FQ, Gaspar-Elsas MI - Mediators Inflamm. (2015)

Bottom Line: 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.By contrast, a higher IL-17A concentration (10 ng/mL) retained significant suppressive effect in both conditions, unmasking a high-end iNOS-independent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Paulo de Góes Institute for Microbiology, Federal University of Rio de Janeiro (UFRJ), 21941-590 Rio de Janeiro, RJ, Brazil.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Dependence of IL-17A on iNOS and synergism between IL-17A and exogenous NO in iNOS-deficient bone-marrow. Bone-marrow cultures were established with IL-5, alone or in association with IL-17A, from wild-type (WT) control (C57BL/6, white bars) and iNOS-deficient (iNOS-KO, black bars) mutant C57BL/6 mice (a, c) or from BALB/c wild-type mice (b) or from CD95-deficient lpr mutant BALB/c (d), as described in legend of Figure 1. Data (mean ± SEM), n = 3 (a, c, and d), n = 4 (b), are the numbers of EPO+ cells recovered at day 7. (a) Concentration-response relationship for IL-17A on WT and iNOS-deficient bone-marrow. (b) Blockade of IL-17A effect by iNOS-selective inhibitor aminoguanidine (AmGua). (c) Concentration-response relationships for IL-17A and SNP, separately and in combination, in wild-type and iNOS-deficient bone-marrow, showing synergism between IL-17A (1 ng/mL) and NO donor SNP (10−4 M) in iNOS-deficient bone-marrow. (d) Dependence of IL-17A effectiveness on CD95 (comparing with (b), white bar and first black bar). ∗P < 0.05 and ∗∗P < 0.01 for the differences between the indicated points or in (b), relative to the respective IL-5 (negative) controls.
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fig2: Dependence of IL-17A on iNOS and synergism between IL-17A and exogenous NO in iNOS-deficient bone-marrow. Bone-marrow cultures were established with IL-5, alone or in association with IL-17A, from wild-type (WT) control (C57BL/6, white bars) and iNOS-deficient (iNOS-KO, black bars) mutant C57BL/6 mice (a, c) or from BALB/c wild-type mice (b) or from CD95-deficient lpr mutant BALB/c (d), as described in legend of Figure 1. Data (mean ± SEM), n = 3 (a, c, and d), n = 4 (b), are the numbers of EPO+ cells recovered at day 7. (a) Concentration-response relationship for IL-17A on WT and iNOS-deficient bone-marrow. (b) Blockade of IL-17A effect by iNOS-selective inhibitor aminoguanidine (AmGua). (c) Concentration-response relationships for IL-17A and SNP, separately and in combination, in wild-type and iNOS-deficient bone-marrow, showing synergism between IL-17A (1 ng/mL) and NO donor SNP (10−4 M) in iNOS-deficient bone-marrow. (d) Dependence of IL-17A effectiveness on CD95 (comparing with (b), white bar and first black bar). ∗P < 0.05 and ∗∗P < 0.01 for the differences between the indicated points or in (b), relative to the respective IL-5 (negative) controls.

Mentions: We further examined whether iNOS and CD95 were required for suppression of eosinopoiesis by IL-17A (Figure 2). IL-17A, 1 ng/mL, had no effect on bone-marrow from iNOS-deficient donors, in contrast to its ability to suppress eosinopoiesis in bone-marrow from B6 wild-type control donors (Figure 2(a)). Moreover, selective iNOS inhibitor aminoguanidine, 10−4 M, prevented the effect of IL-17A, 1 ng/mL (Figure 2(b)). In the absence of IL-17A, aminoguanidine had no significant effect. The effect of iNOS inactivation (Figure 2(a)) or inhibition (Figure 2(b)) was dependent on the concentration of IL-17A in the cultures: bone-marrow from both iNOS-deficient and wild-type donors remained sensitive to IL-17A, provided that it was present at a 10-fold higher concentration (Figure 2(a)); similarly, in BALB/c bone-marrow, aminoguanidine was unable to prevent the effects of IL-17A, 10 ng/mL (not shown). Therefore, only partial blockade of IL-17A effects was achieved by interference with iNOS, suggesting the existence of two mechanisms of suppression, one operative at a lower IL-17A concentration (1 ng/mL) and mediated by iNOS and another detectable at 10-fold higher concentration (10 ng/mL), which is iNOS-independent. We further examined the possibility of synergism between exogenously introduced NO and IL-17A in the absence of iNOS: in iNOS-deficient bone-marrow, IL-17A, 1 ng/mL (a concentration insufficient to suppress eosinopoiesis; see Figure 2(a)), synergized with SNP, 10−4 M (which is also insufficient to suppress eosinopoiesis; see Figure 2(c)), resulting in significant suppression. The ability of a 10-fold higher concentration of SNP (10−3 M) to suppress eosinopoiesis, even in the absence of iNOS, is consistent with previous observations [30] that NO donors override the requirement for functional iNOS. Finally, the effects of IL-17A were abolished in bone-marrow lacking CD95 (lpr), as expected from the involvement of the iNOS-CD95L proapoptotic pathway (Figure 2(d)).


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

Xavier-Elsas P, de Luca B, Queto T, Vieira BM, Masid-de-Brito D, Dahab EC, Alves Filho JC, Cunha FQ, Gaspar-Elsas MI - Mediators Inflamm. (2015)

Dependence of IL-17A on iNOS and synergism between IL-17A and exogenous NO in iNOS-deficient bone-marrow. Bone-marrow cultures were established with IL-5, alone or in association with IL-17A, from wild-type (WT) control (C57BL/6, white bars) and iNOS-deficient (iNOS-KO, black bars) mutant C57BL/6 mice (a, c) or from BALB/c wild-type mice (b) or from CD95-deficient lpr mutant BALB/c (d), as described in legend of Figure 1. Data (mean ± SEM), n = 3 (a, c, and d), n = 4 (b), are the numbers of EPO+ cells recovered at day 7. (a) Concentration-response relationship for IL-17A on WT and iNOS-deficient bone-marrow. (b) Blockade of IL-17A effect by iNOS-selective inhibitor aminoguanidine (AmGua). (c) Concentration-response relationships for IL-17A and SNP, separately and in combination, in wild-type and iNOS-deficient bone-marrow, showing synergism between IL-17A (1 ng/mL) and NO donor SNP (10−4 M) in iNOS-deficient bone-marrow. (d) Dependence of IL-17A effectiveness on CD95 (comparing with (b), white bar and first black bar). ∗P < 0.05 and ∗∗P < 0.01 for the differences between the indicated points or in (b), relative to the respective IL-5 (negative) controls.
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Related In: Results  -  Collection

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fig2: Dependence of IL-17A on iNOS and synergism between IL-17A and exogenous NO in iNOS-deficient bone-marrow. Bone-marrow cultures were established with IL-5, alone or in association with IL-17A, from wild-type (WT) control (C57BL/6, white bars) and iNOS-deficient (iNOS-KO, black bars) mutant C57BL/6 mice (a, c) or from BALB/c wild-type mice (b) or from CD95-deficient lpr mutant BALB/c (d), as described in legend of Figure 1. Data (mean ± SEM), n = 3 (a, c, and d), n = 4 (b), are the numbers of EPO+ cells recovered at day 7. (a) Concentration-response relationship for IL-17A on WT and iNOS-deficient bone-marrow. (b) Blockade of IL-17A effect by iNOS-selective inhibitor aminoguanidine (AmGua). (c) Concentration-response relationships for IL-17A and SNP, separately and in combination, in wild-type and iNOS-deficient bone-marrow, showing synergism between IL-17A (1 ng/mL) and NO donor SNP (10−4 M) in iNOS-deficient bone-marrow. (d) Dependence of IL-17A effectiveness on CD95 (comparing with (b), white bar and first black bar). ∗P < 0.05 and ∗∗P < 0.01 for the differences between the indicated points or in (b), relative to the respective IL-5 (negative) controls.
Mentions: We further examined whether iNOS and CD95 were required for suppression of eosinopoiesis by IL-17A (Figure 2). IL-17A, 1 ng/mL, had no effect on bone-marrow from iNOS-deficient donors, in contrast to its ability to suppress eosinopoiesis in bone-marrow from B6 wild-type control donors (Figure 2(a)). Moreover, selective iNOS inhibitor aminoguanidine, 10−4 M, prevented the effect of IL-17A, 1 ng/mL (Figure 2(b)). In the absence of IL-17A, aminoguanidine had no significant effect. The effect of iNOS inactivation (Figure 2(a)) or inhibition (Figure 2(b)) was dependent on the concentration of IL-17A in the cultures: bone-marrow from both iNOS-deficient and wild-type donors remained sensitive to IL-17A, provided that it was present at a 10-fold higher concentration (Figure 2(a)); similarly, in BALB/c bone-marrow, aminoguanidine was unable to prevent the effects of IL-17A, 10 ng/mL (not shown). Therefore, only partial blockade of IL-17A effects was achieved by interference with iNOS, suggesting the existence of two mechanisms of suppression, one operative at a lower IL-17A concentration (1 ng/mL) and mediated by iNOS and another detectable at 10-fold higher concentration (10 ng/mL), which is iNOS-independent. We further examined the possibility of synergism between exogenously introduced NO and IL-17A in the absence of iNOS: in iNOS-deficient bone-marrow, IL-17A, 1 ng/mL (a concentration insufficient to suppress eosinopoiesis; see Figure 2(a)), synergized with SNP, 10−4 M (which is also insufficient to suppress eosinopoiesis; see Figure 2(c)), resulting in significant suppression. The ability of a 10-fold higher concentration of SNP (10−3 M) to suppress eosinopoiesis, even in the absence of iNOS, is consistent with previous observations [30] that NO donors override the requirement for functional iNOS. Finally, the effects of IL-17A were abolished in bone-marrow lacking CD95 (lpr), as expected from the involvement of the iNOS-CD95L proapoptotic pathway (Figure 2(d)).

Bottom Line: 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.By contrast, a higher IL-17A concentration (10 ng/mL) retained significant suppressive effect in both conditions, unmasking a high-end iNOS-independent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Paulo de Góes Institute for Microbiology, Federal University of Rio de Janeiro (UFRJ), 21941-590 Rio de Janeiro, RJ, Brazil.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus