The Involvement of JAK2 and Pim-1 in IL-5-Mediated Eosinophil Survival

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1 The Involvement of JAK2 and Pim-1 in IL-5-Mediated Eosinophil Survival Natasha Ivey Hampton University Paul Bertics Ph.D. and Cynthia Koziol Departments of Biomolecular Chemistry and Chemical and Biological Engineering School of Medicine and Public Health College of Engineering Abstract Eosinophils are considered an effector cell in the fibrosis and airway thickening found to be a part of asthma pathogenesis. Eosinophilic inflammation of the airway is a key feature of both allergic and non-allergic asthma. In the eosinophil, signaling pathways including the JAK/STAT are stimulated by IL-5. In other cell systems both JAK2 and Pim-1 have been suggested to be involved in cell survival, but the exact mechanisms by which these molecules may mediate IL-5- dependent eosinophil survival have yet to be demonstrated. Therefore, we hypothesize that IL-5-dependent eosinophil survival is mediated through JAK2 and Pim-1-dependent mechanisms. Our studies demonstrate that the JAK inhibitors AG490 and JAK Inhibitor I decrease STAT5 phosphorylation following IL-5 stimulation at concentrations of 30 mm and 300 nm, respectively. Furthermore, AG490 attenuates IL-5- dependent eosinophil survival at a concentration of 30 mm. JAK Inhibitor I shows little to no effect on IL-5-dependent eosinophil survival. These preliminary studies suggest a role for JAK2 in IL-5-dependent eosinophil survival and the role of Pim-1 in this process has yet to be elucidated. Introduction Millions of people suffer from the sneezing and wheezing of allergies and asthma, diseases that have over the last 30 years become increasingly prevalent in some parts of the world. Increased hygiene and a lack of exposure to various microorganisms may be affecting the immune systems of many populations particularly in highly developed countries like the US. This is the essence of the "hygiene hypothesis". The hygiene hypothesis suggests that the more hygienic one becomes, the more susceptible one is to various allergic inflammation diseases (1). This thought argues that rising incidence of asthma and several other diseases may be, at least in part, the result of evolutionary changes that have made us susceptible. The eosinophil is a granulocytic leukocyte that is produced in the bone marrow and traffics to tissues, predominantly those with a mucosal interface with the environment (2). Eosinophils are increased in number in allergic disease both systemically and locally (3-5). Recent studies suggest that the inflammatory response in asthma is associated with increased concentrations of cytokines found in fluid obtained from the lungs of asthmatics after exposure to allergen (6). Primary eosinophil-active cytokines are Interleukin-5 (IL-5), Interleukin-3 (IL-3), and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). Allergic inflammation in asthma is associated with eosinophilia and an increased production of IL-5 (7). Interleukin-5 promotes eosinophil differentiation, enhances the level of blood eosinophils by facilitating the movement of eosinophils from the bone marrow into the blood, plays a central role in effector functions and enhances eosinophil survival through the suppression of apoptotic cell death (8). Studies have shown that IL-5 stimulates the activation of signaling pathways in the eosinophil. One such pathway is the JAK/STAT pathway, which has been reported to be associated with many different processes including cell survival, differentiation, and proliferation (9) (Figure 1). Pim-1 is a serine/threonine kinase that has also been shown to play a role in cell survival, differentiation, and proliferation (10). Figure 1. JAK/STAT Signaling Pathway - IL-5 stimulation activates the JAK/STAT pathway. This leads to the phosphorylation and dimerization of STAT. STAT then enters the nucleus where transcription begins and Pim-1 is expressed. Pim-1 is suggested to play a role in cell survival. 21

2 Several signal transduction pathways may be associated with the regulation of Pim-1 expression. Recent studies provide evidence that Pim-1 protein expression is mediated by IL-5 (11) and that Pim-1 gene expression is STAT5-dependent in other cell systems (12,13). IL-5 stimulates Pim-1 expression in blood eosinophils whereas Pim-1 protein expression is constitutive in blood and airway eosinophils following allergen challenge. The constitutive expression of Pim-1 found in eosinophils following allergen challenge is greater in the airway eosinophils when compared to their blood counterparts (11). This phenomena coincides with increased viability of blood and airway eosinophils that is seen after allergen challenge (14). Accordingly, we hypothesized that IL-5- dependent eosinophil survival is mediated through JAK2 and Pim-1-dependent mechanisms. Materials and Methods Reagents Percoll was purchased from Pharmacia (Piscataway, NJ) and anti-cd16-conjugated paramagnetic microbeads were acquired from Miltenyi Biotech (Auburn, CA). IL-5 was obtained from R&D Systems (Minneapolis, MN). We acquired anti-phosphotyrosine 694 STAT 5 (9351S) from Cell Signaling Technology. From Santa Cruz Biotechnology (Santa Cruz, CA) we obtained anti-stat5b, which detects b isoform (sc-835) and HRP-conjugated goat anti-rabbit IgG. Chemiluminescence substrate reagents SuperSignal West Pico, Dura, Femto and Restore Western blot stripping buffer were obtained from Pierce (Rockford, IL). The JAK2 selective inhibitors AG490 and JAK inhibitor I were purchased from Calbiochem (San Diego, CA). - glycerophosphate and mammalian protease inhibitor cocktail were obtained from Sigma-Aldrich (St. Louis, MO). Annexin V-FITC and Propidium Iodide were acquired from BD PharMingen (San Jose, CA). Human Subjects Subjects providing blood for eosinophil purification ranged from 18 to 55 years in age. Healthy, nonallergic, nonasthmatic subjects whom had no history of allergies or other medical conditions, and were taking no medications, were skin test negative. Allergic subjects were skin test positive (>3mm by skin prick method) for at least one of the common allergens tested (cat dander, ragweed, dust mite); they had history of seasonal or perennial allergic rhinitis but had no clinical history to asthma. The only medications used by subjects were antihistamines and/or Flonase, as needed. Allergic asthma patients were skin test positive. Isolation of Eosinophils Eosinophils were purified from heparinized peripheral blood of volunteer human donors, with eosinophils composing 2-10% of the peripheral blood leukocytes. Through Percoll monolayer (1.09 g/ml) and lysis of erythrocytes by hypotonic shock a granulocyte mixture was obtained from the leukocyte buffy coat after centrifugation. By incubation of the granulocyte mixture with anti-cd16-conjugated paramagnetic microbeads the suspension was depleted of neutrophils. The resulting suspension was > 99% eosinophils (10). Flow Cytometry For analysis of cell viability, blood eosinophils (5 x 10 5 /well) were suspended in media (RPMI + 25 mm HEPES + 0.1% HSA) in a 48 well plate and incubated for 30 minutes at 37 C. The samples were incubated with varying concentrations of JAK inhibitor I (0-1 µm), AG490 (0-300 µm), or DMSO control and stimulated with 100 pm IL-5 or carrier (PBS + 0.1% HAS) for 40 hours. Cells were lifted and the wells were washed with cold 1X sterile PBS. Cells were then centrifuged for 5 minutes at 520 x g at 4 C. Supernatant was removed and pellet was resuspended in 1X Annexin Binding Buffer and placed in flow tubes for analysis. Viability was assessed via staining with Annexin V-FITC and Propidium Iodide uptake. For analysis, 10,000 events were collected using a Becton Dickinson Biosciences (San Jose, CA) FACScan II flow cytometer, and data analyses were performed using CellQuest software (BD Biosciences). The percentage of eosinophil survival at the time point was determined by comparing the number of viable eosinophils remaining to the original number of viable eosinophils at time zero. Immunoblotting Blood eosinophils (1-2 X 10 6 /well) were suspended in media (RPMI + 25 mm HEPES + 0.1% HSA) in a 6 well plate and incubated for 30 minutes at 37 C. The samples were preincubated with varying concentrations of JAK inhibitor I (0-1 µm), AG490 (0-300 µm), or DMSO for 6 hours and then stimulated with 300 pm concentration of IL-5 for 30 minutes. Stimulated eosinophils were diluted in STOP buffer (20 mm HEPES, 137 mm NaCl, 1 mm EDTA, 1 mm sodium orthovanadate, 10 mm sodium fluoride, 20 mm - glycerophosphate, and mammalian protease inhibitor cocktail (MPI)(1.04 mm AEBSF, mm aprotinin, 0.02 mm leupeptin, 0.04 mm bestatin, mm pepstatin A, and mm F-64). Cell viability was determined via trypan blue exclusion. Cells were centrifuged for 3 minutes at 520 x g at 4 C. Supernatant was removed and pellet was lysed in RIPA buffer. Lysates were sonicated, and insoluble material was removed by centrifugation at 15,800 x g at 4 C for 10 minutes. A minimum of 40 µg of total protein was resolved on a SDSpolyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with anti-phospho-stat5 to detect phosphorylation of STAT5. The membrane was stripped and reprobed with a STAT5b antibody as a loading control. Images were analyzed using UVP Bioimaging Systems Labworks (Upland, CA) and Image J 1.33 u (National Institutes of Health, USA). 22

3 Results Effect of JAK Inhibitors, AG490 and JAK Inhibitor I on Phosphorylation of STAT5 following IL-5 Stimulation To determine the effect of inhibition of JAK2 on processes downstream of the IL-5 receptor we first wanted to determine if inhibition of JAK2 would lead to inhibition of STAT5 phosphorylation. Cells were preincubated for 6 hr with DMSO control or increasing concentrations of inhibitor, AG490, then stimulated with 300 pm of IL-5 for 30 min. We then assessed the phosphorylation state of STAT5 via immunoblotting. The experiment was done twice and this is a representative blot of the two experiments (Figure 2A). If you look at the blot closely you are able to see that the inhibition of phosphorylation begins to occur around 30 mm. The membranes were stripped and reprobed for total STAT5 to insure equal protein loading. Beneath the blot is a graph that depicts ranges and averages of the two experiments performed (Figure 2B). This data supports our hypothesis that inhibition of JAK2 will inhibit STAT5 phosphorylation. stimulated with 300 pm IL-5 for 30 min. We then assessed the phosphorylation state of STAT5 via immunoblotting. The experiment was done twice and this is a representative blot of the two experiments (Figure 3A). If you look at the blot closely you are able to see that the inhibition of phosphorylation begins to occur around 300 nm and total inhibition occurs at 1 mm. The membranes were stripped and reprobed for total STAT5 to ensure equal protein loading. Beneath the blot is a graph that depicts ranges and averages of the two experiments performed (Figure 3B). Figure 2. AG490 Inhibition of STAT5 Phosphorylation A, Representative immunoblot (n=2) is shown of the phosphorylation state of STAT5 following inhibition of JAK2 with AG490 and subsequent stimulation with IL-5. Peripheral blood eosinophils were preincubated with increasing concentrations of AG490 (+) or DMSO control (-) for 6 hr and then stimulated with IL-5 (300 pm) for 30 min. Protein ( 40 mg) from resulting whole cell lysates was separated by SDS-PAGE and immunoblotted for phosphorylated STAT5. Membranes were stripped and reprobed for total STAT5 as a loading control. B, Densitometry analysis of immunoblots showing the average values and ranges of values in the experiments. Another inhibitor approach was used to determine the effect of JAK2 inhibition on STAT5 phosphorylation. Cells were preincubated for 3 hr with DMSO control or increasing concentrations of inhibitor, JAK Inhibitor I, and then 23 Figure 3. JAK Inhibitor I Inhibition of STAT5 Phosphorylation A, Representative immunoblot (n=2) is shown of the phosphorylation state of STAT5 following inhibition of JAK2 with JAK Inhibitor I and subsequent stimulation with IL-5. Peripheral blood eosinophils were preincubated with increasing concentrations of JAK Inhibitor I (+) or DMSO control (-) for 3 hr and then stimulated with IL-5 (300 pm) for 30 min. Protein ( 40 mg) from resulting whole cell lysates was separated by SDS-PAGE and immunoblotted for phosphorylated STAT5. Membranes were stripped and reprobed for total STAT5 as a loading control. B, Densitometry analysis of immunoblots showing the average values and ranges of values in the experiments. Effect of JAK Inhibitors, AG490 and JAK Inhibitor I on IL-5-dependent Eosinophil Survival. To connect a physiological outcome with inhibition of JAK2 we performed experiments using AG490 to inhibit JAK2 and assess the affect of JAK2 inhibition on IL-5-dependent eosinophil survival. Here we incubated the cells for 40 hr with DMSO control or increasing concentrations of inhibitor, AG490, as well as 100 pm IL-5. Cell viability was assessed by propidium iodide incorporation (uptake) and Annexin V binding. The following graph represents results from three experiments showing the percentage of cells that took up propidium iodide. This indicates that with increasing concentrations of AG490, there appears to be a reduction of IL-5-dependent eosinophil survival (Figure 4A).

4 Again to connect a physiological outcome with inhibition of JAK2 we performed experiments using JAK Inhibitor I to inhibit JAK2 and assess the affect of JAK2 inhibition on IL-5- dependent eosinophil survival. Here we incubated the cells for 40 hr with DMSO control or increasing concentrations of inhibitor, JAK Inhibitor I, as well as 100 pm of IL-5. Cell viability was assessed by propidium iodide incorporation (uptake) and Annexin V binding. The following graph represents results from two experiments showing the percentage of cells that took up propidium iodide. Results indicate that JAK Inhibitor I appears to have little to no affect on IL-5-dependent eosinophil survival (Figure 4B). Discussion The cytokine IL-5 is important to many aspects of eosinophil biology, including potentiation of cell survival. Mechanisms by which IL-5 mediates this process are poorly understood. Therefore, the goal of these studies is to examine the importance of JAK2 and Pim-1 mechanisms underlying IL-5- dependent eosinophil survival. These studies utilized pharmacological inhibitors of JAK2, AG490 and JAK Inhibitor I, to assess the role of JAK2 in STAT5 phosphorylation and IL-5-dependent eosinophil survival. Immunoblotting data demonstrated that inhibition of JAK2 using both AG490 and JAK Inhibitor I resulted in inhibition of STAT5 phosphorylation. Inhibition of JAK2 using AG490 resulted in a decrease in cell survival of eosinophils cultured in the presence of both inhibitor and IL-5. However, these results were not consistent with results obtained from experiments in which eosinophils were cultured with both JAK Inhibitor I and IL-5. These data show little to no effect of JAK Inhibitor I on IL-5-dependent eosinophil survival. A possible reason for the discrepancies between the experiments performed with the AG490 or JAK Inhibitor I could be patient to patient variation in the effectiveness of JAK Inhibitor I. Figure 4. JAK2 Inhibition on IL-5-dependent Eosinophil Survival Peripheral blood eosinophils were incubated for 40 hr with A, AG490 (+), B, JAK Inhibitor I (+) or DMSO control (-) and 100 pm IL-5. Cells were stained with Annexin V-FITC and Propidium Iodide for 15 min before collection on FacScan. Analysis of the data was performed using WinMDI software. The percentage of propidium iodide positive cells is expressed as the averages of experiments, AG490 (n=3) and JAK Inhibitor I (n=2) and the range of values obtained from these experiments. JAK inhibitors, AG490 and JAK Inhibitor I appear to have an effect on the phosphorylation of STAT5 following IL-5 stimulation. Differing results were attained when assessing the effect of the JAK inhibitors on IL-5-dependent eosinophil survival. AG490 appears to have an effect beginning to occur at 30 mm, whereas JAK Inhibitor I appears not to have an effect on IL-5-dependent survival. 24 Collectively, these data support our hypotheses that inhibition of JAK2 will affect phosphorylation of STAT5 and IL-5- dependent eosinophil survival. However, additional replicates of the experiments need to be performed to make a solid interpretation of the findings. Currently, studies are underway to interfere with the endogeneous function of Pim-1 to determine the role it plays in IL-5-dependent eosinophil survival. These studies will further our understanding of mechanisms by which eosinophil survival is potentiated in vivo. Acknowledgements I would like to first thank the Center for Biological Education for the opportunity to gain research experience in the area of Biology. I would also like to thank Janet Branchaw, director of the Integrated Biological Sciences-Summer Research Program (IBS-SRP) for making the journey at Madison an easy one. Thanks to Paul J. Bertics for accepting me into his lab. Lastly, I would like to thank Cynthia Koziol and the other lab members of the Bertics lab for introducing me to new techniques and working with me. This research was supported by: National Science Foundation ( ) University of Wisconsin-Madison Graduate School References 1. Busse, W.W., and Lemanske, R.F Asthma. N. Engl. J. Med. 344:

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