Data represent mean SEM with n?=?3

Data represent mean SEM with n?=?3. M AT7519. As a positive control we used increasing concentrations of 20C50 M R-roscovitine. Apoptosis was assessed by flow cytometric analysis using annexin-V/Propidium iodide (PI) staining. The annexin-V/PI dual negative cells were considered viable, the annexin-V-positive PI-negative cells were considered apoptotic and annexin-V/PI dual positive cells were considered necrotic. AT7519, like R-roscovitine, markedly increased eosinophil apoptosis in a concentration-dependent manner (Figure 1A). However, it is apparent that AT7519 is 50 times more potent at inducing apoptosis than R-roscovitine (Figure 1A). It was also observed that at concentrations which induced similar levels of apoptosis (1 M AT7519 and 50 M R-Roscovitine) AT7519 was less likely to cause necrosis of eosinophils than R-Roscovitine (Figure 1A). Apoptosis was also assessed morphologically using light microscopy after cytocentrifugation and staining with Diff-Quick? (Figure 1B-C), confirming flow cytometric data. Open in a separate window Figure 1 The CDKi drug AT7519 induces apoptosis in primary human eosinophils in a concentration dependent manner.(A) Eosinophils were incubated for 4 h with R-roscovitine (20 MC50 M), AT7519 (1 nMC20 M) or control prior to flow cytometry analysis of AnnV/PI binding to show the percentage of viable, apoptotic and necrotic eosinophils. Data represent mean SEM with n?=?3. (B-C), Cytocentrifuge images (400xMagnification). (B), Eosinophils after 4 hours of culture; black arrows indicate healthy, viable eosinophils and back arrow head indicating an erythrocyte. (C), Eosinophils after 4 hours of AT7519 treatment (10 M); black arrows indicate apoptotic eosinophils, white arrows indicate necrotic eosinophils with extrusion of nuclei. *p 0.05, **p 0.01, ***p 0.001 versus DMSO control To address whether AT7519 induces eosinophil activation, we investigated the effect of the compound alone, and in the presence of eosinophil activating agents on two very sensitive assays of early eosinophil activation; namely i) shape change as measured by increases in forward scatter detected by flow cytometry and ii) intracellular calcium flux as measured by alterations in spectrofluorescence using Fura-2 loaded human eosinophils. AT7519 at 1 M (a concentration that markedly induces human eosinophil apoptosis) does not induce shape change or a direct increase in intracellular free calcium concentration. Furthermore, the compound does not affect the responses induced by eotaxin, platelet activating factor (PAF) or the formylated chemotactic peptice (fMLP); it neither augments nor, indeed, inhibits the responses to these agonists (data not shown). We are confident that AT7519 does not directly activate eosinophils especially since calcium flux is a key signaling pathway for subsequent eosinophil activation (e.g., LTC4 synthesis). AT7519 promotes resolution of allergic pleurisy in mice Having demonstrated that eosinophil apoptosis was markedly induced by AT7519, we investigated the ability of this agent to resolve eosinophil-dominant inflammation with AT7519 showed a marked reduction in the numbers of total leucocytes, eosinophils and mononuclear cells in the pleural cavity, consistent with enhanced resolution of established eosinophilic inflammation (Figure 2B-D). Open in a separate window Figure 2 AT7519 promotes resolution of allergic pleurisy (Figure 1A), earlier time points were chosen for pleural lavage in this set of experiments to ensure that any changes in rates of eosinophil apoptosis were observed BuChE-IN-TM-10 (See schematic representation in Figure 3A). In the AT7519 treated group there was a time-dependent decrease of eosinophil number which was mirrored by an increase in the percentage of apoptotic eosinophils as well as the percentage of macrophages containing apoptotic bodies (Figures 3B-D). At 6 h post treatment typical morphology of pleural cavity cells from vehicle treated animals demonstrating viable eosinophils and macrophages without apoptotic bodies (Figure 3E) and AT7519 treated animals demonstrating apoptotic eosinophils as well as apoptotic eosinophils inside macrophages (Figure 3F) are shown. Flow cytometric analysis of annexin-V/PI staining of pleural cells further confirmed the ability of AT7519 to induce time-dependent apoptosis.Results are expressed as the % cells per cavity, as a mean SEM of at least five mice in each group. a murine model of eosinophil-dominant inflammation and enhances resolution of established neutrophil-dependent inflammation and as well as the resolution phase of established eosinophilic inflammation as R-roscovitine. This was important to establish as the pharmacological kinase inhibition profile of these agents differs. Human eosinophils were incubated for a 4 h period with increasing concentrations from 1 nMC20 M AT7519. As a positive control we used increasing concentrations of 20C50 M R-roscovitine. Apoptosis was assessed by flow cytometric analysis using annexin-V/Propidium iodide (PI) staining. The annexin-V/PI dual negative cells were considered viable, the annexin-V-positive PI-negative cells were considered apoptotic and annexin-V/PI dual positive cells were considered necrotic. AT7519, like R-roscovitine, markedly increased eosinophil apoptosis in a concentration-dependent manner (Figure 1A). However, it is apparent that AT7519 is 50 times more potent at inducing apoptosis than R-roscovitine (Figure 1A). It was also observed that at concentrations which induced similar levels of apoptosis (1 M AT7519 and 50 M R-Roscovitine) AT7519 was less likely to cause necrosis of eosinophils than R-Roscovitine (Figure 1A). Apoptosis was also assessed morphologically using light microscopy after cytocentrifugation and staining with Diff-Quick? (Figure 1B-C), confirming flow cytometric data. Open in a separate window Figure 1 The CDKi drug AT7519 induces apoptosis in primary human eosinophils in a concentration dependent manner.(A) Eosinophils were incubated for 4 h with R-roscovitine (20 MC50 M), AT7519 (1 nMC20 M) or control prior to flow cytometry analysis of AnnV/PI binding to show the percentage of viable, apoptotic and necrotic eosinophils. Data represent mean SEM with n?=?3. (B-C), Cytocentrifuge images (400xMagnification). (B), Eosinophils after 4 hours of culture; black arrows indicate healthy, viable eosinophils and back arrow head indicating an erythrocyte. (C), Eosinophils after 4 hours of AT7519 treatment (10 M); black arrows indicate apoptotic eosinophils, white arrows indicate necrotic eosinophils with extrusion of nuclei. *p 0.05, **p 0.01, ***p 0.001 versus DMSO control To address whether AT7519 induces BuChE-IN-TM-10 eosinophil activation, we investigated the effect of the compound alone, and in the presence of eosinophil activating agents on two very sensitive assays of early eosinophil activation; namely i) shape change as measured by increases in forward scatter detected by flow cytometry and ii) intracellular calcium flux as measured by alterations in spectrofluorescence using Fura-2 loaded human eosinophils. AT7519 at 1 M (a concentration that markedly induces human eosinophil apoptosis) does not induce shape change or a direct increase in intracellular free calcium concentration. Furthermore, the compound does not affect the responses induced by eotaxin, platelet activating factor (PAF) or the formylated chemotactic peptice (fMLP); BuChE-IN-TM-10 it neither augments nor, indeed, inhibits the responses to these agonists (data not shown). We are confident that AT7519 does not directly activate eosinophils especially since calcium flux is a key signaling pathway for subsequent eosinophil activation (e.g., LTC4 synthesis). AT7519 promotes resolution of allergic pleurisy in mice Having demonstrated that eosinophil apoptosis was markedly induced by AT7519, we investigated the ability of this agent to resolve eosinophil-dominant inflammation with AT7519 showed a marked reduction in the numbers of total leucocytes, eosinophils and mononuclear cells in the pleural cavity, consistent with enhanced resolution of established eosinophilic inflammation (Figure 2B-D). Open in a separate window Figure 2 AT7519 promotes resolution of allergic pleurisy (Figure 1A), earlier time points were chosen for pleural lavage in this set of experiments to ensure that any changes in rates of eosinophil apoptosis were observed (See schematic representation in Figure 3A). In the AT7519 treated group there was a time-dependent decrease of eosinophil number which was mirrored Trp53inp1 by an increase in the percentage of apoptotic eosinophils as well as the percentage of macrophages comprising apoptotic body (Numbers 3B-D). At 6 h post treatment standard morphology of pleural cavity cells from vehicle treated animals demonstrating viable eosinophils and macrophages without apoptotic body (Number 3E) and AT7519 treated animals demonstrating apoptotic eosinophils as well as apoptotic eosinophils inside macrophages (Number 3F) are demonstrated..