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Proviral integration site for Moloney murine leukemia virus 1, but not phosphatidylinositol-3 kinase, is essential in the antiapoptotic signaling cascade.

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Presentation on theme: "Proviral integration site for Moloney murine leukemia virus 1, but not phosphatidylinositol-3 kinase, is essential in the antiapoptotic signaling cascade."— Presentation transcript:

1 Proviral integration site for Moloney murine leukemia virus 1, but not phosphatidylinositol-3 kinase, is essential in the antiapoptotic signaling cascade initiated by IL-5 in eosinophils  Nicola Andina, MD, Svetlana Didichenko, PhD, Jan Schmidt-Mende, MD, PhD, Clemens A. Dahinden, MD, Hans-Uwe Simon, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 123, Issue 3, Pages (March 2009) DOI: /j.jaci Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Pharmacologic inhibition of PI3K does not abrogate IL-5–mediated eosinophil survival. A, Immunoblotting. The 3 inhibitors LY294002, wortmannin, and IC87114 blocked IL-5–induced phosphorylation of both Akt and GSK3α/β. The immunoblots are representative of 3 independent experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. B, Viability assay. LY294002, but not wortmannin and IC87114, abrogated IL-5–mediated eosinophil survival. Blood eosinophils were analyzed after 48-hour cultures. Similar results were observed when cells were analyzed after 24-hour cultures. Values are presented as means ± SDs (n = 4). ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 IL-5 induces rapid Pim-1 expression in eosinophils in a Jak2-dependent but PI3K-independent manner. A, Immunoblotting. Pim-1 protein was expressed within 1 hour on IL-5 stimulation. B, Immunoblotting. IL-5–induced Pim-1 expression was not blocked by pharmacologic inhibition of PI3K. C, Immunoblotting. IL-5–induced Pim-1 expression was blocked by pharmacologic inhibition of Jak2 with both AG-490 (upper panel) and SD-1029 (lower panel). The duration of IL-5 stimulation is indicated in each panel. All immunoblots are representative of at least 3 independent experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 IL-5–mediated eosinophil survival is Jak2 dependent. A, Viability assay. AG-490 inhibited, in a concentration-dependent manner, the survival effect of IL-5. Blood eosinophils were analyzed after 48-hour cultures. Similar results were observed when cells were analyzed after 24-hour cultures. B, Viability assay. SD-1029 inhibited, in a concentration-dependent manner, the survival effect of IL-5. Blood eosinophils were analyzed after 24-hour cultures and therefore control survival rates are higher compared with those seen in Fig 3, A. Similar results were observed when cells were analyzed after 48-hour cultures. Values are presented as means ± SDs of 4 (Fig 3, A) and 5 (Fig 3, B) independent experiments, respectively. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Pim-1 is required for IL-5–mediated eosinophil survival. A, Viability assay. Transduction of WT Pim-1 fusion protein alone resulted in increased eosinophil survival. The fusion protein was added at the indicated times. Its uptake and degradation was assessed by means of immunoblotting. Representative experiments are shown. B, Viability assay. In contrast to WT Pim-1, DN Pim-1 completely abrogated IL-5–mediated eosinophil survival. C, Phosphatidylserine redistribution assay. Transduction of WT Pim-1 resulted in reduced eosinophil apoptosis. DN Pim-1 blocked the antiapoptotic effect of IL-5. D, DNA fragmentation assay. Transduction of WT Pim-1 alone resulted in reduced eosinophil apoptosis. DN Pim-1 blocked the antiapoptotic effect of IL-5. The assays in all panels were performed after 24-hour cultures in which PMB was present. Values in Fig 4, B, C, and D, are presented as means ± SDs (n = 6). ∗∗P < .01 and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 IL-5 maintains Mcl-1 expression levels in a Pim-1–dependent manner in eosinophils. For immunoblotting, IL-5 stimulation resulted in stable Mcl-1 expression. Transduction of WT Pim-1 alone resulted in the same Mcl-1 expression levels. DN Pim-1 abolished the stabilizing effect of IL-5 on Mcl-1 levels. The immunoblots are representative of at least 3 independent experiments. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Pim-1 is expressed by eosinophils under inflammatory conditions in vivo. A and B, Immunofluorescence. Pim-1 was readily detected in infiltrating tissue eosinophils. Tissues were obtained from patients as indicated. Eosinophils were identified by using anti-ECP antibody. Control antibodies showed no staining (data not shown). C and D, Immunofluorescence. Pim-1 was not detectable in freshly isolated blood eosinophils from healthy individuals. In contrast, blood eosinophils demonstrated evidence for Pim-1 expression after 10 hours of in vitro stimulation with IL-5. Results in all panels are representative of at least 3 independent experiments (×1,000). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 Pim-1 is expressed by blood eosinophils from a subgroup of eosinophilic patients who exhibit reduced antiapoptotic responses after IL-5 stimulation. A, Immunoblotting. Freshly isolated blood eosinophils from healthy control individuals (Normal) and from patients with hypereosinophilic syndrome (HES), atopic eczema (AE), and atopic rhinitis (AR) were analyzed for Pim-1 expression. Blood eosinophil numbers from each patient/control individual are indicated. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. B, Viability assay. Based on the Pim-1 expression levels, as shown in Fig 7, A, donors were grouped as expressing high levels of Pim-1 (Pim-1 positive; hypereosinophilic syndrome no. 3, hypereosinophilic syndrome no. 5, atopic eczema no. 3, and atopic rhinitis no. 1) or low levels of Pim-1 (Pim-1 negative; remaining donors) and plotted against the IL-5–mediated effect on eosinophil viability (viability difference between the presence and absence of IL-5 in 24-hour eosinophil cultures). ∗∗P < .01. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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