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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

Effect of IL-17A on eosinophil production from murine bone-marrow. Bone-marrow cultures were established from BALB/c mice in the presence of IL-5 (1 ng/mL), alone or in association with the indicated concentrations of IL-17A. After the indicated periods, cells were collected, counted, and stained for eosinophil peroxidase (EPO). Total and EPO+ cell counts were performed, and data (mean ± SEM, n = 4) are the numbers of EPO+ cells present at the end of the culture. (a) Effect of varying IL-17A concentration on EPO+ cell numbers at day 7. Insert: effect of culture in the absence of IL-5; open and closed bars: cultures established, respectively, with and without IL-17A. (b) Effect of increasing duration of culture. (c) Effect of variable timing of IL-17A addition relative to the initiation of the culture. (d) Effect of blocking terminal caspases with zVAD-fmk (z-VAD) (20 μM). (e) Annexin V-FITC staining of cells in the granulocyte gate recovered from BALB/c and GATA-1 day 5 bone-marrow cultures. Data are mean ± SEM (n = 3) of the % granulocytes in the gated region (FL-1 positive, FL-2 negative). At day 2, there was no significant difference between IL-17A-exposed and control cultures from BALB/c mice (not shown). ∗P < 0.05 for the differences between the indicated points and the respective IL-5 (negative) controls.
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fig1: Effect of IL-17A on eosinophil production from murine bone-marrow. Bone-marrow cultures were established from BALB/c mice in the presence of IL-5 (1 ng/mL), alone or in association with the indicated concentrations of IL-17A. After the indicated periods, cells were collected, counted, and stained for eosinophil peroxidase (EPO). Total and EPO+ cell counts were performed, and data (mean ± SEM, n = 4) are the numbers of EPO+ cells present at the end of the culture. (a) Effect of varying IL-17A concentration on EPO+ cell numbers at day 7. Insert: effect of culture in the absence of IL-5; open and closed bars: cultures established, respectively, with and without IL-17A. (b) Effect of increasing duration of culture. (c) Effect of variable timing of IL-17A addition relative to the initiation of the culture. (d) Effect of blocking terminal caspases with zVAD-fmk (z-VAD) (20 μM). (e) Annexin V-FITC staining of cells in the granulocyte gate recovered from BALB/c and GATA-1 day 5 bone-marrow cultures. Data are mean ± SEM (n = 3) of the % granulocytes in the gated region (FL-1 positive, FL-2 negative). At day 2, there was no significant difference between IL-17A-exposed and control cultures from BALB/c mice (not shown). ∗P < 0.05 for the differences between the indicated points and the respective IL-5 (negative) controls.

Mentions: Initially, we characterized the effects of IL-17A on IL-5-induced eosinopoiesis (Figure 1), regarding its concentration-response relationship, kinetics, and window of opportunity. We further evaluated whether IL-17A had an effect in the absence of IL-5 (Figure 1(a), insert) and whether its suppressive effects, as expected in apoptotic processes, depended on caspases and were associated with increased phosphatidylserine externalization. IL-17A addition to bone-marrow cultures established from naive BALB/c donors, for 7 days, in the presence of IL-5, concentration-dependently suppressed eosinopoiesis (Figure 1(a)), with significant effects at 1 and 10 ng/mL, but not 0.1 ng/mL. Importantly, IL-17A only had a detectable effect in the presence of IL-5 (Figure 1(a), insert). While IL-5 alone supported a progressive increase in the number of eosinophils recovered up to day 7 (Figure 1(b)), IL-17A prevented this increase, with a significant effect from day 5 onwards. This type of kinetics was similar to that previously described for PGE2, the prototypical activator of the iNOS-CD95L proapoptotic mechanism [30]: like PGE2 [38], IL-17A must act at the beginning of the culture (up to 24 h) and has no significant effect if it is introduced at later times (Figure 1(c)). This effect of IL-17A (1–10 ng/mL) is abolished by zVAD-fmk, showing its dependence on caspases (Figure 1(d)). Importantly, zVAD-fmk had no effect in the absence of IL-17A. Annexin V-binding cells in the granulocyte gate were found in significantly increased numbers (Figure 1(e)), in IL-17A-exposed cultures relative to IL-5 control cultures, by day 5, when an effect of IL-17 on eosinophil numbers becomes demonstrable (Figure 1(b)), but not by day 2 (not shown). Importantly, this increase was not observed in cultures from GATA-1 donors, which do not produce eosinophils in the presence of IL-5 but do present cells in the granulocyte gate, which survive from the bone-marrow inoculum. Hence, increased Annexin V-binding in day 5 cultures is correlated with the presence of eosinophil-lineage cells and with exposure to IL-17A during the initial 24 h.


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)

Effect of IL-17A on eosinophil production from murine bone-marrow. Bone-marrow cultures were established from BALB/c mice in the presence of IL-5 (1 ng/mL), alone or in association with the indicated concentrations of IL-17A. After the indicated periods, cells were collected, counted, and stained for eosinophil peroxidase (EPO). Total and EPO+ cell counts were performed, and data (mean ± SEM, n = 4) are the numbers of EPO+ cells present at the end of the culture. (a) Effect of varying IL-17A concentration on EPO+ cell numbers at day 7. Insert: effect of culture in the absence of IL-5; open and closed bars: cultures established, respectively, with and without IL-17A. (b) Effect of increasing duration of culture. (c) Effect of variable timing of IL-17A addition relative to the initiation of the culture. (d) Effect of blocking terminal caspases with zVAD-fmk (z-VAD) (20 μM). (e) Annexin V-FITC staining of cells in the granulocyte gate recovered from BALB/c and GATA-1 day 5 bone-marrow cultures. Data are mean ± SEM (n = 3) of the % granulocytes in the gated region (FL-1 positive, FL-2 negative). At day 2, there was no significant difference between IL-17A-exposed and control cultures from BALB/c mice (not shown). ∗P < 0.05 for the differences between the indicated points and the respective IL-5 (negative) controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: Effect of IL-17A on eosinophil production from murine bone-marrow. Bone-marrow cultures were established from BALB/c mice in the presence of IL-5 (1 ng/mL), alone or in association with the indicated concentrations of IL-17A. After the indicated periods, cells were collected, counted, and stained for eosinophil peroxidase (EPO). Total and EPO+ cell counts were performed, and data (mean ± SEM, n = 4) are the numbers of EPO+ cells present at the end of the culture. (a) Effect of varying IL-17A concentration on EPO+ cell numbers at day 7. Insert: effect of culture in the absence of IL-5; open and closed bars: cultures established, respectively, with and without IL-17A. (b) Effect of increasing duration of culture. (c) Effect of variable timing of IL-17A addition relative to the initiation of the culture. (d) Effect of blocking terminal caspases with zVAD-fmk (z-VAD) (20 μM). (e) Annexin V-FITC staining of cells in the granulocyte gate recovered from BALB/c and GATA-1 day 5 bone-marrow cultures. Data are mean ± SEM (n = 3) of the % granulocytes in the gated region (FL-1 positive, FL-2 negative). At day 2, there was no significant difference between IL-17A-exposed and control cultures from BALB/c mice (not shown). ∗P < 0.05 for the differences between the indicated points and the respective IL-5 (negative) controls.
Mentions: Initially, we characterized the effects of IL-17A on IL-5-induced eosinopoiesis (Figure 1), regarding its concentration-response relationship, kinetics, and window of opportunity. We further evaluated whether IL-17A had an effect in the absence of IL-5 (Figure 1(a), insert) and whether its suppressive effects, as expected in apoptotic processes, depended on caspases and were associated with increased phosphatidylserine externalization. IL-17A addition to bone-marrow cultures established from naive BALB/c donors, for 7 days, in the presence of IL-5, concentration-dependently suppressed eosinopoiesis (Figure 1(a)), with significant effects at 1 and 10 ng/mL, but not 0.1 ng/mL. Importantly, IL-17A only had a detectable effect in the presence of IL-5 (Figure 1(a), insert). While IL-5 alone supported a progressive increase in the number of eosinophils recovered up to day 7 (Figure 1(b)), IL-17A prevented this increase, with a significant effect from day 5 onwards. This type of kinetics was similar to that previously described for PGE2, the prototypical activator of the iNOS-CD95L proapoptotic mechanism [30]: like PGE2 [38], IL-17A must act at the beginning of the culture (up to 24 h) and has no significant effect if it is introduced at later times (Figure 1(c)). This effect of IL-17A (1–10 ng/mL) is abolished by zVAD-fmk, showing its dependence on caspases (Figure 1(d)). Importantly, zVAD-fmk had no effect in the absence of IL-17A. Annexin V-binding cells in the granulocyte gate were found in significantly increased numbers (Figure 1(e)), in IL-17A-exposed cultures relative to IL-5 control cultures, by day 5, when an effect of IL-17 on eosinophil numbers becomes demonstrable (Figure 1(b)), but not by day 2 (not shown). Importantly, this increase was not observed in cultures from GATA-1 donors, which do not produce eosinophils in the presence of IL-5 but do present cells in the granulocyte gate, which survive from the bone-marrow inoculum. Hence, increased Annexin V-binding in day 5 cultures is correlated with the presence of eosinophil-lineage cells and with exposure to IL-17A during the initial 24 h.

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