Human airway eosinophils exhibit preferential reduction in STAT signaling capacity and increased CISH expression.

Allergic asthma, a chronic respiratory disorder marked by inflammation and recurrent airflow obstruction, is associated with elevated levels of IL-5 family cytokines and elevated numbers of eosinophils (EOS). IL-5 family cytokines elongate peripheral blood EOS (EOS(PB)) viability, recruit EOS(PB) to the airways, and, at higher concentrations, induce degranulation and reactive oxygen species generation. Although airway EOS (EOS(A)) remain signal ready in that GM-CSF treatment induces degranulation, treatment of EOS(A) with IL-5 family cytokines no longer confers a survival advantage. Because the IL-5 family receptors have common signaling capacity, but are uncoupled from EOS(A) survival, whereas other IL-5 family induced endpoints remain functional, we tested the hypothesis that EOS(A) possess a JAK/STAT-specific regulatory mechanism (because JAK/STAT signaling is critical to EOS survival). We found that IL-5 family-induced STAT3 and STAT5 phosphorylation is attenuated in EOS(A) relative to blood EOS from airway allergen-challenged donors. However, IL-5 family-induced ERK1/2 phosphorylation is not altered between EOS(A) and EOS from airway allergen-challenged donors. These observations suggest EOS(A) possess a regulatory mechanism for suppressing STAT signaling distinct from ERK1/2 activation. Furthermore, we found, in EOS(PB), IL-5 family cytokines induce members of the suppressors of cytokine signaling (SOCS) genes, CISH and SOCS1. Additionally, following allergen challenge, EOS(A) express significantly more CISH and SOCS1 mRNA and CISH protein than EOS(PB) counterparts. In EOS(PB), long-term pretreatment with IL-5 family cytokines, to varying degrees, attenuates IL-5 family-induced STAT5 phosphorylation. These data support a model in which IL-5 family cytokines trigger a selective downregulation mechanism in EOS(A) for JAK/STAT pathways.

Rhinoviruses, allergic inflammation, and asthma.

Viral infections affect wheezing and asthma in children and adults of all ages. In infancy, wheezing illnesses are usually viral in origin, and children with more severe wheezing episodes are more likely to develop recurrent episodes of asthma and to develop asthma later in childhood. Children who develop allergen-specific immunoglobulin E (allergic sensitization) and those who wheeze with human rhinoviruses (HRV) are at especially high risk for asthma. In older children and adults, HRV infections generally cause relatively mild respiratory illnesses and yet contribute to acute and potentially severe exacerbations in patients with asthma. These findings underline the importance of understanding the synergistic nature of allergic sensitization and infections with HRV in infants relative to the onset of asthma and in children and adults with respect to exacerbations of asthma. This review discusses clinical and experimental evidence of virus-allergen interactions and evaluates theories which relate immunologic responses to respiratory viruses and allergens to the pathogenesis and disease activity of asthma. Greater understanding of the relationship between viral respiratory infections, allergic inflammation, and asthma is likely to suggest new strategies for the prevention and treatment of asthma.

LPS modulates rhinovirus-induced chemokine secretion in monocytes and macrophages.

Recent studies suggest that both bacteria and rhinoviruses (RVs) contribute to asthma exacerbations. We hypothesized that bacteria might alter antiviral responses early in the course of infection by modifying monocyte-lineage chemokine responses to RV infection. To test this hypothesis, human blood monocytes or bronchoalveolar lavage (BAL) macrophages were treated with RV types A016, B014, A001, and/or A002 in the presence or absence of LPS, and secretion of chemokines (CXCL10, CXCL11, CCL2, and CCL8) and IFN-α was measured by ELISA. Treatment with RV alone induced blood monocytes and BAL macrophages to secrete CXCL10, CXCL11, CCL2, and CCL8. Pretreatment with LPS significantly attenuated RV-induced CXCL10, CXCL11, and CCL8 secretion by 68-99.9% on average (P < 0.0001, P < 0.004, and P < 0.002, respectively), but did not inhibit RV-induced CCL2 from blood monocytes. Similarly, LPS inhibited RV-induced CXCL10 and CXCL11 secretion by over 88% on average from BAL macrophages (P < 0.002 and P < 0.0001, respectively). Furthermore, LPS inhibited RV-induced signal transducer and activator of transcription 1 phosphorylation (P < 0.05), as determined by immunoblotting, yet augmented RV-induced IFN-α secretion (P < 0.05), and did not diminish expression of RV target receptors, as measured by flow cytometry. In summary, major and minor group RVs strongly induce chemokine expression and IFN-α from monocytic cells. The bacterial product, LPS, specifically inhibits monocyte and macrophage secretion of RV-induced CXCL10 and CXCL11, but not other highly induced chemokines or IFN-α. These effects suggest that airway bacteria could modulate the pattern of virus-induced cell recruitment and inflammation in the airways.

Chemoattractant-induced signaling via the Ras-ERK and PI3K-Akt networks, along with leukotriene C4 release, is dependent on the tyrosine kinase Lyn in IL-5- and IL-3-primed human blood eosinophils.

Human blood eosinophils exhibit a hyperactive phenotype in response to chemotactic factors after cell "priming" with IL-5 family cytokines. Earlier work has identified ERK1/2 as molecular markers for IL-5 priming, and in this article, we show that IL-3, a member of the IL-5 family, also augments fMLP-stimulated ERK1/2 phosphorylation in primary eosinophils. Besides ERK1/2, we also observed an enhancement of chemotactic factor-induced Akt phosphorylation after IL-5 priming of human blood eosinophils. Administration of a peptide antagonist that targets the Src family member Lyn before cytokine (IL-5/IL-3) priming of blood eosinophils inhibited the synergistic increase of fMLP-induced activation of Ras, ERK1/2 and Akt, as well as the release of the proinflammatory factor leukotriene C(4). In this study, we also examined a human eosinophil-like cell line HL-60 clone-15 and observed that these cells exhibited significant surface expression of IL-3Rs and GM-CSFRs, as well as ERK1/2 phosphorylation in response to the addition of IL-5 family cytokines or the chemotactic factors fMLP, CCL5, and CCL11. Consistent with the surface profile of IL-5 family receptors, HL-60 clone-15 recapitulated the enhanced fMLP-induced ERK1/2 phosphorylation observed in primary blood eosinophils after priming with IL-3/GM-CSF, and small interfering RNA-mediated knockdown of Lyn expression completely abolished the synergistic effects of IL-3 priming on fMLP-induced ERK1/2 phosphorylation. Altogether, our data demonstrate a central role for Lyn in the mechanisms of IL-5 family priming and suggest that Lyn contributes to the upregulation of the Ras-ERK1/2 and PI3K-Akt cascades, as well as the increased leukotriene C(4) release observed in response to fMLP in "primed" eosinophils.

GM-CSF production by glioblastoma cells has a functional role in eosinophil survival, activation, and growth factor production for enhanced tumor cell proliferation.

Medicinal interventions of limited efficacy are currently available for the treatment of glioblastoma multiforme (GBM), the most common and lethal primary brain tumor in adults. The eosinophil is a pivotal immune cell in the pathobiology of atopic disease that is also found to accumulate in certain tumor tissues. Inverse associations between atopy and GBM risk suggest that the eosinophil may play a functional role in certain tumor immune responses. To assess the potential interactions between eosinophils and GBM, we cultured human primary blood eosinophils with two separate human GBM-derived cell lines (A172, U87-MG) or conditioned media generated in the presence or absence of TNF-α. Results demonstrated differential eosinophil adhesion and increased survival in response to coculture with GBM cell lines. Eosinophil responses to GBM cell line-conditioned media included increased survival, activation, CD11b expression, and S100A9 release. Addition of GM-CSF neutralizing Abs to GBM cell cultures or conditioned media reduced eosinophil adhesion, survival, and activation, linking tumor cell-derived GM-CSF to the functions of eosinophils in the tumor microenvironment. Dexamethasone, which has been reported to inhibit eosinophil recruitment and shrink GBM lesions on contrast-enhanced scans, reduced the production of tumor cell-derived GM-CSF. Furthermore, culture of GBM cells in eosinophil-conditioned media increased tumor cell viability, and generation of eosinophil-conditioned media in the presence of GM-CSF enhanced the effect. These data support the idea of a paracrine loop between GM-CSF-producing tumors and eosinophil-derived growth factors in tumor promotion/progression.

Eosinophils in glioblastoma biology.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The development of this malignant glial lesion involves a multi-faceted process that results in a loss of genetic or epigenetic gene control, un-regulated cell growth, and immune tolerance. Of interest, atopic diseases are characterized by a lack of immune tolerance and are inversely associated with glioma risk. One cell type that is an established effector cell in the pathobiology of atopic disease is the eosinophil. In response to various stimuli, the eosinophil is able to produce cytotoxic granules, neuromediators, and pro-inflammatory cytokines as well as pro-fibrotic and angiogenic factors involved in pathogen clearance and tissue remodeling and repair. These various biological properties reveal that the eosinophil is a key immunoregulatory cell capable of influencing the activity of both innate and adaptive immune responses. Of central importance to this report is the observation that eosinophil migration to the brain occurs in response to traumatic brain injury and following certain immunotherapeutic treatments for GBM. Although eosinophils have been identified in various central nervous system pathologies, and are known to operate in wound/repair and tumorstatic models, the potential roles of eosinophils in GBM development and the tumor immunological response are only beginning to be recognized and are therefore the subject of the present review.

Human eosinophils express RAGE, produce RAGE ligands, exhibit PKC-delta phosphorylation and enhanced viability in response to the RAGE ligand, S100B.

This study tested the hypothesis that human eosinophils produce ligands for the receptor for advanced glycation end-products (RAGE), express RAGE and exhibit RAGE-mediated responses. In examining our microarray data, we identified the presence of RAGE and RAGE ligand (S100A4, S100A6, S100A8, S100A9, S100A11, S100P, HMGB1) transcripts. Expression of eosinophil RAGE mRNA was also compared with a known positive control and further assessed via bioinformatics and sequence analysis of RAGE cDNA. Positive and negative controls were used to identify RAGE, S100A8 and S100A9 protein in human primary eosinophils. Immunoblot assessment of eosinophils treated with cytokines (IL-5 or granulocyte macrophage colony-stimulating factor) indicated an up-regulation of S100A8 and S100A9 production, whereas co-treatment of eosinophils with a RAGE ligand and cytokines displayed a down-regulation in the levels of RAGE. Analysis of eosinophil-conditioned media revealed that eosinophils are capable of releasing RAGE, S100A8 and S100A9. To test the eosinophil response to RAGE activation, the most well-characterized RAGE ligand, S100B, was examined. Treatment of eosinophils with S100B resulted in RAGE-mediated PKC-delta phosphorylation, a 3-fold dose-dependent increase in cell survival and an increase in the level of cellular RAGE. Combined, these studies reveal eosinophil expression of RAGE, RAGE ligands and RAGE-mediated responses. The expression of eosinophil RAGE, soluble RAGE and RAGE ligands may be pivotal to the functions of eosinophils in various human diseases involving RAGE and S100 ligands.

Extracellular ATP may contribute to tissue repair by rapidly stimulating purinergic receptor X7-dependent vascular endothelial growth factor release from primary human monocytes.

Extracellular ATP has been proposed to act as a danger signal to alert the immune system of cell damage. Release of high local concentrations of ATP activates the nucleotide receptor, purinergic receptor X7 (P2RX7), on monocytic cells, which promotes the processing/release of proinflammatory mediators. Although the proinflammatory actions of P2RX7 are well recognized, little is known regarding the potential function of P2RX7 in repair responses. Because the resolution of inflammation is characterized by monocytic cell-dependent production of proangiogenic factors, we evaluated the contribution of P2RX7 to this process. We observed that both short-term and long-term P2RX7 activation promotes the robust release of vascular endothelial growth factor from primary human monocytes. This vascular endothelial growth factor release is calcium dependent and associated with reactive oxygen species production. This previously unrecognized action of P2RX7 suggests that it may not only participate in inflammation and cell death, but that it is also likely to be important in the control of angiogenesis and wound repair.

Human airway eosinophils respond to chemoattractants with greater eosinophil-derived neurotoxin release, adherence to fibronectin, and activation of the Ras-ERK pathway when compared with blood eosinophils.

Human blood eosinophils exposed ex vivo to hematopoietic cytokines (e.g., IL-5 or GM-CSF) subsequently display enhanced responsiveness to numerous chemoattractants, such as chemokines, platelet-activating factor, or FMLP, through a process known as priming. Airway eosinophils, obtained by bronchoalveolar lavage after segmental Ag challenge, also exhibit enhanced responsiveness to selected chemoattractants, suggesting that they are primed during cell trafficking from the blood to the airway. Earlier work has shown that chemoattractants stimulate greater activation of ERK1 and ERK2 following IL-5 priming in vitro, thus revealing that ERK1/ERK2 activity can be a molecular readout of priming under these circumstances. Because few studies have examined the intracellular mechanisms regulating priming as it relates to human airway eosinophils, we evaluated the responsiveness of blood and airway eosinophils to chemoattractants (FMLP, platelet-activating factor, CCL11, CCL5, CXCL8) with respect to degranulation, adherence to fibronectin, or Ras-ERK signaling cascade activation. When compared with blood eosinophils, airway eosinophils exhibited greater FMLP-stimulated eosinophil-derived neurotoxin release as well as augmented FMLP- and CCL11-stimulated adherence to fibronectin. In airway eosinophils, FMLP, CCL11, and CCL5 stimulated greater activation of Ras or ERK1/ERK2 when compared with baseline. Ras activation by FMLP in blood eosinophils was also enhanced following IL-5 priming. These studies are consistent with a model of in vivo priming of eosinophils by IL-5 or related cytokines following allergen challenge, and further demonstrate the key role of priming in the chemoattractant-stimulated responses of eosinophils. These data also demonstrate the importance of the Ras-ERK signaling pathway in the regulation of eosinophil responses to chemoattractants in the airway. Human airway eosinophils respond to several chemoattractants with increased activation of the Ras-ERK cascade, eosinophil-derived neurotoxin release, and adherence to fibronectin relative to blood eosinophils.

Retargeted clostridial neurotoxins as novel agents for treating chronic diseases.

Botulinum neurotoxin (BoNT) A and B are used to treat neuropathic disorders; if retargeted, these agents could be used to treat medical conditions that involve secretion from nonneuronal cells. Here, we report novel strategies for successfully retargeting BoNTs, and also tetanus neurotoxin (TeNT), to primary human blood monocyte-derived macrophages where BoNT/B inhibited the release of tumor necrosis factor-α, a cytokine that plays a key role in inflammation. Furthermore, mice treated with retargeted BoNT/B exhibited a significant reduction in macrophage (MΦ) recruitment, indicating that these toxins can be used to treat chronic inflammation.

Activation of the transcription factor FosB/activating protein-1 (AP-1) is a prominent downstream signal of the extracellular nucleotide receptor P2RX7 in monocytic and osteoblastic cells.

Activation of the ionotropic P2RX7 nucleotide receptor by extracellular ATP has been implicated in modulating inflammatory disease progression. Continuous exposure of P2RX7 to ligand can result in apoptosis in many cell types, including monocytic cells, whereas transient activation of P2RX7 is linked to inflammatory mediator production and the promotion of cell growth. Given the rapid hydrolysis of ATP in the circulation and interstitial space, transient activation of P2RX7 appears critically important for its action, yet its effects on gene expression are unclear. The present study demonstrates that short-term stimulation of human and mouse monocytic cells as well as mouse osteoblasts with P2RX7 agonists substantially induces the expression of several activating protein-1 (AP-1) members, particularly FosB. The potent activation of FosB after P2RX7 stimulation is especially noteworthy considering that little is known concerning the role of FosB in immunological regulation. Interestingly, the magnitude of FosB activation induced by P2RX7 stimulation appears greater than that observed with other known inducers of FosB expression. In addition, we have identified a previously unrecognized role for FosB in osteoblasts with respect to nucleotide-induced expression of cyclooxygenase-2 (COX-2), which is the rate-limiting enzyme in prostaglandin biosynthesis from arachidonic acid and is critical for osteoblastic differentiation and immune behavior. The present studies are the first to link P2RX7 action to FosB/AP-1 regulation in multiple cell types, including a role in nucleotide-induced COX-2 expression, and support a role for FosB in the control of immune and osteogenic function by P2RX7.

Ordering transitions in nematic liquid crystals induced by vesicles captured through ligand-receptor interactions.

We report that phospholipid vesicles incorporating ligands, when captured from solution onto surfaces presenting receptors for these ligands, can trigger surface-induced orientational ordering transitions in nematic phases of 4'-pentyl-4-cyanobiphenyl (5CB). Specifically, whereas avidin-functionalized surfaces incubated against vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were observed to cause the liquid crystal (LC) to adopt a parallel orientation at the surface, the same surfaces incubated against biotinylated vesicles (DOPC and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotin-DOPE)) caused the homeotropic (perpendicular) ordering of the LC. The use of a combination of atomic force microscopy (AFM), ellipsometry and quantitative fluorimetry, performed as a function of vesicle composition and vesicle concentration in solution, revealed the capture of intact vesicles containing 1% biotin-DOPE from buffer at the avidin-functionalized surfaces. Subsequent exposure to water prior to contact with the LC, however, resulted in the rupture of the majority of vesicles into interfacial multilayer assemblies with a maximum phospholipid loading set by random close packing of the intact vesicles initially captured on the surface (5.1 ± 0.2 phospholipid molecules/nm(2)). At high concentrations of biotinylated lipid (>10% biotin-DOPE) in the vesicles, the limiting lipid loading was measured to be 4.0 ± 0.3 phospholipid molecules/nm(2), consistent with the maximum phospholipid loading set by the spontaneous formation of a bilayer during incubation with the biotinylated vesicles. We measured the homeotropic ordering of the LC on the surfaces independently of the initial morphology of the phospholipid assembly captured on the surface (intact vesicle, planar multilayer). We interpret this result to infer the reorganization of the phospholipid bilayers either prior to or upon contact with the LCs such that interactions of the acyl chains of the phospholipid and the LC dominate the ordering of the LC, a conclusion that is further supported by quantitative measurements of the orientation of the LC as a function of the phospholipid surface density (>1.8 molecules/nm(2) is required to cause the homeotropic ordering of the LC). These results and others presented herein provide fundamental insights into the interactions of phospholipid-decorated interfaces with LCs and thereby provide guidance for the design of surfaces on which phospholipid assemblies captured through ligand-receptor recognition can be reported via ordering transitions in LCs.

Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.

The primary function of IL-7 is to promote maturation and survival of T cells. Through microarray expression analysis, we previously observed that human blood eosinophils express mRNA for IL-7R alpha (CD127) and its common gamma chain (CD132). The purpose of this study was to determine whether eosinophils have functional IL-7 receptors and to assess the potential contribution of IL-7 to eosinophilic airway inflammation by evaluating its presence in bronchoalveolar lavage (BAL) fluid of subjects with atopic asthma before and after segmental bronchoprovocation with allergen. Immunoblot analysis revealed that CD127 is present in highly purified human blood eosinophils. Furthermore, eosinophils responded to IL-7 with phosphorylation of STAT5, up-regulation of the activation marker CD69, and prolonged survival. Neutralization of GM-CSF but not IL-5 significantly blunted these functional responses, suggesting that IL-7 mediates its effects by promoting eosinophil release of autologous GM-CSF. Notably, the suppressive effect of anti-GM-CSF on STAT5 phosphorylation occurred within 10 min of eosinophil exposure to IL-7. Thus, IL-7 likely activates eosinophil release of preformed rather than newly synthesized GM-CSF. The biological relevance of IL-7 to eosinophilia in vivo was implicated in a study of airway allergen challenge in patients with allergic asthma. IL-7 concentrations in BAL fluid increased significantly 48 h after segmental allergen challenge and were highly correlated with BAL eosinophils (r = 0.7, p < 0.001). In conclusion, the airway response to allergen is associated with the generation of IL-7, which may contribute to airway inflammation by promoting enhanced eosinophil activation and survival. Activation of eosinophils is a novel function for IL-7.

Mutation of putative N-linked glycosylation sites on the human nucleotide receptor P2X7 reveals a key residue important for receptor function.

The nucleotide receptor P2X(7) is an immunomodulatory cation channel and a potential therapeutic target. P2X(7) is expressed in immune cells such as monocytes and macrophages and is activated by extracellular ATP following tissue injury or infection. Ligand binding to P2X(7) can stimulate ERK1/2, the transcription factor CREB, enzymes linked to the production of reactive oxygen species and interleukin-1 isoforms, and the formation of a nonspecific pore. However, little is known about the biochemistry of P2X(7), including whether the receptor is N-linked glycosylated and if this modification affects receptor function. Here we provide evidence that P2X(7) is sensitive to the glycosidases EndoH and PNGase F and that the human receptor appears glycosylated at N187, N202, N213, N241, and N284. Mutation of N187 results in weakened P2X(7) agonist-induced phosphorylation of ERK1/2, CREB, and p90 ribosomal S6 kinase, as well as a decreased level of pore formation. In further support of a role for glycosylation in receptor function, treatment of RAW 264.7 macrophages with the N-linked glycosylation synthesis inhibitor tunicamycin attenuates P2X(7) agonist-induced, but not phorbol ester-induced, ERK1/2 phosphorylation. Interestingly, residue N187 belongs to an N-linked glycosylation consensus sequence found in six of the seven P2X family members, suggesting this site is fundamentally important to P2X receptor function. To address the mechanism whereby N187 mutation attenuates receptor activity, we developed a live cell proteinase K digestion assay that demonstrated altered cell surface expression of P2X(7) N187A. This is the first report to map human P2X(7) glycosylation sites and reveal residue N187 is critical for receptor trafficking and function.

Adenoviral transduction of EGFR into pregnancy-adapted uterine artery endothelial cells remaps growth factor induction of endothelial dysfunction.

During pregnancy, uterine vascular vasodilation is enhanced through adapted Ca2+ signaling, facilitated through increased endothelial connexin 43 (Cx43) gap junctional communication (GJC). In preeclampsia (PE), this adaptive response is missing. Of note, the angiogenic factor VEGF can also act via Src and ERK to close Cx43 gap junctions. While VEGFR2 is necessary for such closure, a role VEGFR1 is less clear. We reasoned if VEGFR2 is acting alone, then substituting another growth factor receptor with VEGFR2-like signaling should have the same effect. In uterine artery endothelial cells derived from pregnant sheep (P-UAEC), endogenous EGFR expression is very low. When we used adenovirus to raise EGFR, we also dose-dependently induced EGF-sensitive Cx43 phosphorylation mainly via ERK, and corresponding loss of Ca2+ bursts, but eliminated VEGF effects on phosphorylation of Cx43 or loss of Ca2+ bursting. This surprising observation suggests that while activated EGFR may indeed substitute for VEGFR2, it also sequesters a limited pool of effector molecules needed for VEGFR2 to phosphorylate Cx43. Thus, low endogenous EGFR expression in P-UAEC may be a necessary strategy to allow VEGFR-2 control of GJC, a first step in initiating angiogenesis in healthy pregnancy. Of further note, trophoblasts are rich in EGFR, and we have demonstrated shed PLAP+/EGFR + extracellular vesicles in maternal circulation in first trimester plasma samples using nanoscale high resolution flow cytometry. Collectively our data suggest that placenta derived exosomes positive for EGFR should be further considered as a possible cause of endothelial dysfunction in women with PE.

The nucleotide receptor P2RX7 mediates ATP-induced CREB activation in human and murine monocytic cells.

Nucleotide receptors serve as sensors of extracellular ATP and are important for immune function. The nucleotide receptor P2RX7 is a cell-surface, ligand-gated cation channel that has been implicated in many diseases, including arthritis, granuloma formation, sepsis, and tuberculosis. These disorders are often exacerbated by excessive mediator release from activated macrophages in the inflammatory microenvironment. Although P2RX7 activation can modulate monocyte/macrophage-induced inflammatory events, the relevant molecular mechanisms are poorly understood. Previous studies suggest that MAPK cascades and transcriptional control via CREB-linked pathways regulate the inflammatory capacity of monocytic cells. As P2RX7 promotes MAPK activation and inflammatory mediator production, we examined the involvement MAPK-induced CREB activation in P2RX7 action. Our data reveal that stimulation of multiple monocytic cell lines with P2RX7 agonists induces rapid CREB phosphorylation. In addition, we observed a lack of nucleotide-induced CREB phosphorylation in RAW 264.7 cells expressing nonfunctional P2RX7 and a gain of nucleotide-induced CREB phosphorylation in human embryonic kidney-293 cells that heterologously express human P2RX7. Furthermore, our results indicate that P2RX7 agonist-induced CREB phosphorylation is partly mediated via Ca(2+) fluxes and the MEK/ERK system. Mechanistic analyses revealed that macrophage stimulation with a P2RX7 agonist induces CREB/CREB-binding protein complex formation, which is necessary for CREB transcriptional activation. Also, we demonstrate that P2RX7 activation induces a known CREB-dependent gene (c-fos) and that dominant-negative CREB constructs attenuate this response. These studies support the idea that P2RX7 stimulation can directly regulate protein expression that is not dependent on costimulation with other immune modulators such as LPS.

Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: epidermal growth factor receptor as a model system.

Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was (32)P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (clones H11 and 111.6) and one phosphospecific EGF receptor antibody (clone pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82 : 1, exceeding the signal-to-background measured on the ELISA plate (<48 : 1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the transmembrane protein from the PDMS surface was found to be 75-81%. However, the transfer of non-specifically bound protein was substantially less than 75%, thus leading to the important finding that mechanical transfer of the EGF receptor leads to an approximately four-fold increase in signal-to-background from 20 : 1 to 88 : 1. The signal-to-background obtained following mechanical transfer is also better than that obtained using ELISA plates and stripping buffer (<48 : 1). The EGF receptor is a clinically important protein and the target of numerous anticancer agents and thus these results, when combined, provide guidance for the design of PDMS-based microanalytical systems for the capture and isolation of complex and clinically important transmembrane proteins.

Quantitative methods based on twisted nematic liquid crystals for mapping surfaces patterned with bio/chemical functionality relevant to bioanalytical assays.

We report methods for the acquisition and analysis of optical images formed by thin films of twisted nematic liquid crystals (LCs) placed into contact with surfaces patterned with bio/chemical functionality relevant to surface-based assays. The methods are simple to implement and are shown to provide easily interpreted maps of chemical transformations on surfaces that are widely exploited in the preparation of analytic devices. The methods involve acquisition of multiple images of the LC as a function of the orientation of a polarizer; data analysis condenses the information present in the stack of images into a spatial map of the twist angle of the LC on the analytic surface. The potential utility of the methods is illustrated by mapping (i) the displacement of a monolayer formed from one alkanethiol on a gold film by a second thiol in solution, (ii) coadsorption of mixtures of amine-terminated and ethylene glycol-terminated alkanethiols on gold films, which leads to a type of mixed monolayer that is widely exploited for immobilization of proteins on analytic surfaces, and (iii) patterns of antibodies printed onto surfaces. These results show that maps of the twist angle of the LC constructed from families of optical images can be used to reveal surface features that are not apparent in a single image of the LC film. Furthermore, the twist angles of the LC can be used to quantify the energy of interaction of the LC with the surface with a spatial resolution of <10 microm. When combined, the results described in this paper suggest nondestructive methods to monitor and validate chemical transformations on surfaces of the type that are routinely employed in the preparation of surface-based analytic technologies.

Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation.

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X7 agonists ATP and 3'-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X7-mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ERK2, p38, and JNK1/JNK2) phosphorylation and that the antioxidants N-acetylcysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.