Konrad Pazdrak

The lnterleukin-5/Receptor Interaction Activates Lyn and Jak2 Tyrosine Kinases and Propagates Signals via the Ras-Raf-1-MAP Kinase and the Jak-STAT Pathways in Eosinophils

We have shown that the interaction of interleukin (IL)-5 with the receptor activates Lyn tyrosine kinase within 1 min and Jak2 tyrosine kinase within 1-3 min. IL-5 also stimulates GTP binding to p21ras. The signal is subsequently propagated through the activation of Raf-1, MEK, and MAP kinases as shown by their increased autophosphorylation in vitro and phosphorylation in situ. Jak2 kinase has been shown to phosphorylate STAT nuclear proteins. The activation of STAT nuclear factors was studied by electrophoretic mobility shift assay using a gamma activation site (GAS) probe. We found that IL-5 induces two GAS-binding proteins in eosinophils, one of which is STAT1. We conclude that IL-5 induced signals are propagated through two distinct pathways: (1) Lyn-->Ras-->Raf-1-->MEK-->MAP kinase and (2) Jak2-->STAT1.

Systems biology approaches to understanding Epithelial Mesenchymal Transition (EMT) in mucosal remodeling and signaling in asthma

A pathological hallmark of asthma is chronic injury and repair, producing dysfunction of the epithelial barrier function. In this setting, increased oxidative stress, growth factor- and cytokine stimulation, together with extracellular matrix contact produces transcriptional reprogramming of the epithelial cell. This process results in epithelial-mesenchymal transition (EMT), a cellular state associated with loss of epithelial polarity, expression of mesenchymal markers, enhanced mobility and extracellular matrix remodeling. As a result, the cellular biology of the EMT state produces characteristic changes seen in severe, refractory asthma: myofibroblast expansion, epithelial trans-differentiation and subepithelial fibrosis. EMT also induces profound changes in epithelial responsiveness that affects innate immune signaling that may have impact on the adaptive immune response and effectiveness of glucocorticoid therapy in severe asthma. We discuss how this complex phenotype is beginning to be understood using systems biology-level approaches through perturbations coupled with high throughput profiling and computational modeling. Understanding the distinct changes induced by EMT at the systems level may provide translational strategies to reverse the altered signaling and physiology of refractory asthma.

Priming of Eosinophils by GM-CSF Is Mediated by Protein Kinase CβII-Phosphorylated L-Plastin

The priming of eosinophils by cytokines leading to augmented response to chemoattractants and degranulating stimuli is a characteristic feature of eosinophils in the course of allergic inflammation and asthma. Actin reorganization and integrin activation are implicated in eosinophil priming by GM-CSF, but their molecular mechanism of action is unknown. In this regard, we investigated the role of L-plastin, an eosinophil phosphoprotein that we identified from eosinophil proteome analysis. Phosphoproteomic analysis demonstrated the upregulation of phosphorylated L-plastin after eosinophil stimulation with GM-CSF. Additionally, coimmunoprecipitation studies demonstrated a complex formation of phosphorylated L-plastin with protein kinase CβII (PKCβII), GM-CSF receptor α-chain, and two actin-associated proteins, paxilin and cofilin. Inhibition of PKCβII with 4,5-bis(4-fluoroanilino)phtalimide or PKCβII-specific small interfering RNA blocked GM-CSF–induced phosphorylation of L-plastin. Furthermore, flow cytometric analysis also showed an upregulation of αMβ2 integrin, which was sensitive to PKCβII inhibition. In chemotaxis assay, GM-CSF treatment allowed eosinophils to respond to lower concentrations of eotaxin, which was abrogated by the above-mentioned PKCβII inhibitors. Similarly, inhibition of PKCβII blocked GM-CSF induced priming for degranulation as assessed by release of eosinophil cationic protein and eosinophil peroxidase in response to eotaxin. Importantly, eosinophil stimulation with a synthetic L-plastin peptide (residues 2–19) phosphorylated on Ser5 upregulated αMβ2 integrin expression and increased eosinophil migration in response to eotaxin independent of GM-CSF stimulation. Our results establish a causative role for PKCβII and L-plastin in linking GM-CSF–induced eosinophil priming for chemotaxis and degranulation to signaling events associated with integrin activation via induction of PKCβII-mediated L-plastin phosphorylation.

Lipoxin A4 Counterregulates GM-CSF Signaling in Eosinophilic Granulocytes

Eosinophils are granulated leukocytes that are involved in many inflammation-associated pathologies including airway inflammation in asthma. Resolution of eosinophilic inflammation and return to homeostasis is in part due to endogenous chemical mediators, for example, lipoxins, resolvins, and protectins. Lipoxins are endogenous eicosanoids that demonstrate antiinflammatory activity and are synthesized locally at sites of inflammation. In view of the importance of lipoxins (LXs) in resolving inflammation, we investigated the molecular basis of LXA4 action on eosinophilic granulocytes stimulated with GM-CSF employing the eosinophilic leukemia cell line EoL-1 as well as peripheral blood eosinophils. We report herein that LXA4 (1–100 nM) decreased protein tyrosine phosphorylation in EoL-1 cells stimulated with GM-CSF. Additionally, the expression of a number of GM-CSF-induced cytokines was inhibited by LXA4 in a dose-dependent manner. Furthermore, using a proteomics approach involving mass spectrometry and immunoblot analysis we identified 11 proteins that were tyrosine phosphorylated after GM-CSF stimulation and whose phosphorylation was significantly inhibited by LXA4 pretreatment. Included among these 11 proteins were α-fodrin (nonerythroid spectrin) and actin. Microscopic imaging showed that treatment of EoL-1 cells or blood eosinophils with GM-CSF resulted in the reorganization of actin and the translocation of α-fodrin from the cytoplasm to the plasma membrane. Importantly, α-fodrin translocation was prevented by LXA4 but actin reorganization was not. Thus, the mechanism of LXA4 action likely involves prevention of activation of eosinophilic granulocytes by GM-CSF through inhibition of protein tyrosine phosphorylation and modification of some cytoskeletal components.

Toward the proteome of the human peripheral blood eosinophil

Eosinophils (EOSs) are granular leukocytes that have significant roles in many inflammatory and immunoregulatory responses, especially asthma and allergic diseases. We have undertaken a fairly comprehensive proteomic analysis of purified peripheral blood EOSs from normal human donors primarily employing 2‐DE with protein spot identification by MALDI‐MS. Protein subfractionation methods employed included IEF (Zoom® Fractionator) and subcellular fractionation using differential protein solubilization. We have identified 3141 proteins, which had Mascot expectation scores of 10−3 or less. Of these 426 were unique and non‐redundant of which 231 were novel proteins not previously reported to occur in EOSs. Ingenuity Pathway Analysis showed that some 70% of the non‐redundant proteins could be subdivided into categories that are clearly related to currently known EOS biological activities. Cytoskeletal and associated proteins predominated among the proteins identified. Extensive protein posttranslational modifications were evident, many of which have not been previously reported that reflected the dynamic character of the EOS. This data set of eosinophilic proteins will prove valuable in comparative studies of disease versus normal states and for studies of gender differences and polymorphic variation among individuals.

Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3.

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs’ effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils’ response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs’ TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don’t upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.

Cytokine-Induced Glucocorticoid Resistance from Eosinophil Activation: Protein Phosphatase 5 Modulation of Glucocorticoid Receptor Phosphorylation and Signaling

The mechanisms contributing to persistent eosinophil activation and poor eosinopenic response to glucocorticoids in severe asthma are poorly defined. We examined the effect of cytokines typically overexpressed in the asthmatic airways on glucocorticoid signaling in in vitro activated eosinophils. An annexin V assay used to measure eosinophil apoptosis showed that cytokine combinations of IL-2 plus IL-4 as well as TNF-α plus IFN-γ, or IL-3, GM-CSF, and IL-5 alone significantly diminished the proapoptotic response to dexamethasone. We found that IL-2 plus IL-4 resulted in impaired phosphorylation and function of the nuclear glucocorticoid receptor (GCR). Proteomic analysis of steroid sensitive and resistant eosinophils identified several differentially expressed proteins, namely protein phosphatase 5 (PP5), formyl peptide receptor 2, and annexin 1. Furthermore, increased phosphatase activity of PP5 correlated with impaired phosphorylation of the GCR. Importantly, suppression of PP5 expression with small interfering RNA restored proper phosphorylation and the proapoptotic function of the GCR. We also examined the effect of lipoxin A4 on PP5 activation by IL-2 plus IL-4. Similar to PP5 small interfering RNA inhibition, pretreatment of eosinophils with lipoxin A4 restored GCR phosphorylation and the proaptoptotic function of GCs. Taken together, our results showed 1) a critical role for PP5 in cytokine-induced resistance to GC-mediated eosinophil death, 2) supported the dependence of GCR phosphorylation on PP5 activity, and 3) revealed that PP5 is a target of the lipoxin A4-induced pathway countering cytokine-induced resistance to GCs in eosinophils.

Activation of Human Peripheral Blood Eosinophils by Cytokines in a Comparative Time-Course Proteomic/Phosphoproteomic Study

Activated eosinophils contribute to airway dysfunction and tissue remodeling in asthma and thus are considered to be important factors in asthma pathology. We report here comparative proteomic and phosphoproteomic changes upon activation of eosinophils using eight cytokines individually and in selected cytokine combinations in time-course reactions. Differential protein and phosphoprotein expressions were determined by mass spectrometry after 2-dimensional gel electrophoresis (2DGE) and by LC-MS/MS. We found that each cytokine-stimulation produced significantly different changes in the eosinophil proteome and phosphoproteome, with phosphoproteomic changes being more pronounced and having an earlier onset. Furthermore, we observed that IL-5, GM-CSF, and IL-3 showed the greatest change in protein expression and phosphorylation, and this expression differed markedly from those of the other five cytokines evaluated. Comprehensive univariate and multivariate statistical analyses were employed to evaluate the comparative results. We also monitored eosinophil activation using flow cytometry (FC) analysis of CD69. In agreement with our proteomic studies, FC indicated that IL-5, GM-CSF, and IL-3 were more effective than the other five cytokines studied in stimulating a cell surface CD69 increase indicative of eosinophil activation. Moreover, selected combinations of cytokines revealed proteomic patterns with many proteins in common with single cytokine expression patterns but also showed a greater effect of the two cytokines employed, indicating a more complex signaling pathway that was reflective of a more typical inflammatory pathology.

Altered Eosinophil Proteome in a Patient with Hypereosinophilia from Acute Fascioliasis

ABSTRACT We used comparative proteomics to analyze eosinophils from a patient with hypereosinophilia due to fascioliasis. Using 2-dimensional electrophoresis and mass spectrometry, we demonstrated that the eosinophil proteome was significantly altered compared to those of healthy controls.

Functional Proteomics for the Characterization of Impaired Cellular Responses to Glucocorticoids in Asthma

In chronic airway inflammatory disorders, such as asthma, glucocorticoid (GC) insensitivity is a challenging clinical problem associated with life-threatening disease progression and the potential development of serious side effects. The mechanism of steroid resistance in asthma remains unclear and may be multifactorial. Excluding noncompliance with GC treatment, abnormal steroid pharmacokinetics, and rare genetic defects in the glucocorticoid receptor (GR), the majority of GC insensitivity in asthma can be attributed to secondary defects related to GR function. Airway inflammatory cells obtained from patients with GC-resistant asthma show a number of abnormalities in cell immune responses to GC, which suggests that there is a causative defect in GR signaling in GC-resistant cells that could be further elucidated by a functional and molecular proteomics approach. Since T cells, eosinophils, and monocytes play a major role in the pathogenesis of airway inflammation, most of the work published to date has focused on these cell types as the primary therapeutic targets in GC-insensitive asthma. We herein review several distinct techniques for the assessment of (1) the cellular response to GCs including the effect of GCs on cell viability, adhesion, and mediator release; (2) the functionality of GC receptors, including phosphorylation of the GR, nuclear translocation, and binding activities; and (3) the characterization of proteins differentially expressed in steroid-resistant cells by comparative 2DE-gel electrophoresis-based techniques and mass spectrometry. These comprehensive approaches are expected to reveal novel candidates for biomarkers of steroid insensitivity, which may lead to the development of effective therapeutic interventions for patients with chronic steroid-resistant asthma.