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.

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.

The C-terminus of human nucleotide receptor P2X7 is critical for receptor oligomerization and N-linked glycosylation.

BACKGROUND: The P2X7 receptor binds extracellular ATP to mediate numerous inflammatory responses and is considered a potential biomarker and therapeutic target for diverse inflammatory and neurological diseases. P2X7 contains many single nucleotide polymorphisms, including several mutations located within its intracellular C-terminal trafficking domain. Mutations within the trafficking domain result in attenuated receptor activity and cell surface presentation, but the mechanisms by which amino acid changes within this region promote altered P2X7 function have not been elucidated. METHODS AND RESULTS: We analyzed the amino acid sequence of P2X7 for any potential trafficking signals and found that P2X7 contains putative Arg-X-Arg ER retention sequences. Alanine substitutions near or within these sequences were constructed, and we determined that single mutation of R574 and R578 but not R576 or K579 attenuates P2X7-stimulated activation of ERK1/2 and induction of the transcription factors FosB and ΔFosB. We found that mutation of R578 within the trafficking domain to the naturally occurring Gln substitution disrupts P2X7 localization at the plasma membrane and results in R578Q displaying a higher apparent molecular weight in comparison to wild-type receptor. We used the glycosidase endoglycosidase H to determine that this difference in mass is due in part to the R578Q mutant possessing a larger mass of oligosaccharides, indicative of improper N-linked glycosylation addition and/or trimming. Chemical cross-linking experiments were also performed and suggest that the R578Q variant also does not form trimers as well as wild-type receptor, a function required for its full activity. CONCLUSIONS: These data demonstrate the distal C-terminus of P2X7 is important for oligomerization and post-translational modification of the receptor, providing a mechanism by which mutations in the trafficking domain disrupt P2X7 activity and localization at the plasma membrane.

Lactoferrin regulates an axis involving CD11b and CD49d integrins and the chemokines MIP-1α and MCP-1 in GM-CSF-treated human primary eosinophils.

Eosinophils are multifunctional immune cells that contribute to innate and adaptive immune/repair responses. Lactoferrin (LF) is an iron-binding protein indicated to alter cell adhesion and immune function by receptor-mediated interactions or by participating in redox mechanisms. The eosinophil adhesion molecules, αMß2 and α4ß1, are differentially expressed following exposure to the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and various redox agents. We hypothesized that LF can alter the function and production of proteins involved in adhesion/migration. Utilizing eosinophil peroxidase activity or fluorescent labeling adhesion assays, LF reduced GM-CSF-induced eosinophil adhesion in the presence of fibronectin or vascular adhesion molecule-1 compared with GM-CSF treatment alone. Flow cytometric analysis of eosinophil αM (CD11b) and α4 (CD49d) integrins revealed that cotreatments (24 h) with LF plus GM-CSF induced a significant increase in CD11b compared with control and GM-CSF treatments but a significant decrease in CD49d compared with control and GM-CSF treatments. These changes in CD11b and CD49d levels were significantly correlated with the increased production of chemokines (macrophage inflammatory Protein-1α, monocyte chemotactic protein-1) and an identified increase in S100A9 production. Thus, LF release at sites of inflammation may alter eosinophil recruitment/activation and possibly the progression of diseases such as cancer and asthma where significant eosinophil influx has been described.

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.

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.

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.

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.

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.

Transcriptional control mechanisms associated with the nucleotide receptor P2X7, a critical regulator of immunologic, osteogenic, and neurologic functions.

The nucleotide receptor P2X(7) is an attractive therapeutic target and potential biomarker for multiple inflammatory and neurologic disorders, and it is expressed in several immune, osteogenic, and neurologic cell types. Aside from its role in the nervous system, it is activated by ATP released at sites of tissue damage, inflammation, and infection. Ligand binding to P2X(7) stimulates many cell responses, including calcium fluxes, MAPK activation, inflammatory mediator release, and apoptosis. Much work has centered on P2X(7) action in cell death and mediator processing (e.g., pro-interleukin-1 cleavage by the inflammasome), but the contribution of P2X(7) to transcriptional regulation is less well defined. This review will focus on the growing evidence for the importance of nucleotide-mediated gene expression, highlight several animal models, human genetic, and clinical studies that support P2X(7) as a therapeutic target, and discuss the latest developments in anti-P2X(7) clinical trials.

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.

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.

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.

Regulation of keratinocyte signaling and function via changes in epidermal growth factor presentation.

Motivated by the need for bioactive materials that can accelerate dermal wound healing, this work describes the responses of keratinocytes to covalently immobilized epidermal growth factor (EGF) and how differences in the physical presentation of this growth factor affect cell function. Specifically, human keratinocytes were cultured with EGF delivered in soluble form, immobilized in a homogeneous pattern or immobilized in a gradient pattern, followed by analysis of cellular signaling, proliferation and migration. By changing the manner in which EGF was presented, keratinocyte behavior was dramatically altered. Keratinocytes responded to immobilized EGF patterns with high EGF receptor (EGFR) but low ERK1/2 and Akt phosphorylation, accompanied by low proliferation, high migratory activity and coordinated cell alignment. In contrast, keratinocytes treated with soluble EGF experienced lower EGFR but higher ERK1/2 and Akt phosphorylation and displayed a highly proliferative, rather than migratory, phenotype. Keratinocytes also responded to differences in immobilized EGF patterns, as migration was fastest upon immobilized gradients of EGF. A better understanding the interaction of cells with soluble vs. immobilized growth factors can help elucidate native healing events and achieve greater control over cell function, which may be useful in the development of wound repair treatments for many types of tissues.

Design of surfaces for liquid crystal-based bioanalytical assays.

Surface-induced ordering of liquid crystals (LCs) offers the basis of a label-free analytical technique for the detection of surface-bound biomolecules. The orientation-dependent energy of interaction of a LC with a surface (anchoring energy of LC), in particular, is both sensitive to the presence of surface-bound molecules and easily quantified. Herein, we report a study that analyzes a simple model of twisted nematic LC systems and thereby identifies surfaces with LC anchoring energies in the range of 0.5 microJ/m(2) to 2.0 microJ/m(2) to be optimal for use with LC-based analytical methods. Guided by these predictions, we demonstrate that analytic surfaces possessing anchoring energies within this range can be fabricated with a high level of precision (< 0.1 microJ/m(2)) through formation of monolayers of organothiols (with omega-functional groups corresponding to oligoethyleneglycols and amines) on gold films deposited by physical vapor deposition at oblique angles of incidence. Finally, by using the human epidermal growth factor receptor (EGFR) as a model protein analyte, we have characterized the influence of the anchoring energies of the surfaces on the response of the LC to the presence of surface-bound EGFR. These results, when combined with (32)P-radiolabeling of the EGFR to independently quantify the surface concentration of EGFR, permit identification of surfaces that allow use of LCs to report surface densities of EGFR of 30-40 pg/mm(2). Overall, the results reported in this paper guide the design of surfaces for use in LC-based analytical systems.

Cell signaling via the P2X(7) nucleotide receptor: linkage to ROS production, gene transcription, and receptor trafficking.

Extracellular nucleotides can act as important intercellular signals in diverse biological processes, including the enhanced production of factors that are key to immune response regulation. One receptor that binds extracellular adenosine triphosphate released at sites of infection and injury is P2X(7), which is an ionotrophic receptor that can also lead to the formation of a non-specific pore, activate multiple mitogen-activated protein kinases (MAPKs), and stimulate the production of immune mediators including interleukin family members and reactive oxygen species (ROS). In the present report, we have investigated the signaling mechanisms by which P2X(7) promotes monocytic cell mediator production and induces transcription factor expression/phosphorylation, as well as how receptor-associated pore activity is regulated by intracellular trafficking. We report that P2X(7) stimulates ROS production in macrophages through the MAPKs ERK1/2 and the nicotinamide adenine dinucleotide phosphate oxidase complex, activates several transcription factors including cyclic-AMP response element-binding protein and components of the activating protein-1 complex, and contains specific sequences within its intracellular C-terminus that appear critical for its activity. Altogether, these data further implicate P2X(7) activation and signaling as a fundamental modulator of macrophage immune responses.

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.

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.