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.

Stimulation of P2RX(7) with extracellular ATP potentiates numerous LPS-induced proinflammatory events, including cytokine induction in macrophages, but the molecular mechanisms underlying this process are not well defined. Although P2RX(7) ligation has been proposed to activate several transcription factors, many of the LPS-induced mediators affected by P2RX(7) activation are not induced by P2RX(7) agonists alone, suggesting a complementary role for P2RX(7) in transcriptional regulation. Type I IFN production, whose expression is tightly controlled by multiple transcription factors that form an enhanceosome, is critical for resistance against LPS-containing bacteria. The effect of purinergic receptor signaling on LPS-dependent type I IFN is unknown and would be of great relevance to a diverse array of inflammatory conditions. The present study demonstrates that stimulation of macrophages with P2RX(7) agonists substantially enhances LPS-induced IFN-β expression, and this enhancement is ablated in macrophages that do not express functional P2RX(7) or when the MAPK MEK1/2 pathways are inhibited. Potentiation of LPS-induced IFN-β expression following P2RX(7) stimulation is likely transcriptionally regulated, as this enhancement is observed at the IFN-β promoter level. Furthermore, P2RX(7) stimulation is able to increase the phosphorylation and subsequent IFN-β promoter occupancy of IRF-3, a transcription factor that is critical for IFN-β transcription by TLR agonists. This newly discovered role for P2RX(7) in IFN regulation may have implications in antimicrobial defense, which has been linked to P2RX(7) activation in other studies.

SummaryBackgroundAllergic airway inflammation contributes to the airway remodelling that has been linked to increased obstruction and morbidity in asthma. However, the mechanisms by which allergens contribute to airway remodelling in humans are not fully established. CCL18, chitotriosidase (CHIT1) and YKL‐40 are readily detectable in the lungs and contribute to remodelling in other fibrotic diseases, but their involvement in allergic asthma is unclear.ObjectiveWe hypothesized that CCL18, YKL‐40 and CHIT1 bioactivity are enhanced in allergic asthma subjects after segmental allergen challenge and are related to increased pro‐fibrotic and Th2‐associated mediators in the lungs.MethodsLevels of CCL18 and YKL‐40 protein and chitotriosidase (CHIT1) bioactivity in bronchoalveolar lavage (BAL) fluid, as well as CCL18, YKL‐40 and CHIT1 mRNA levels in BAL cells were evaluated in patients with asthma at baseline and 48 h after segmental allergen challenge. We also examined the correlation between CCL18 and YKL‐40 levels and CHIT1 activity with the levels of other pro‐fibrotic factors and chemokines previously shown to be up‐regulated after allergen challenge.ResultsChitotriosidase activity and YKL‐40 and CCL18 levels were elevated after segmental allergen challenge and these levels correlated with those of other pro‐fibrotic factors, T cell chemokines, and inflammatory cells after allergen challenge. CCL18 and YKL‐40 mRNA levels also increased in BAL cells after allergen challenge.Conclusions and Clinical RelevanceOur results suggest that CCL18 and YKL‐40 levels and CHIT1 activity are enhanced in allergic airway inflammation and thus may contribute to airway remodelling in asthma.

Eosinophils contribute to allergic asthma exacerbation and can undergo excessive recruitment to the lungs where their activation leads to tissue damage and fibrosis. Interleukin-5 (IL-5) family cytokine receptors, which are critical for eosinophil recruitment/activation, are proposed to exist in membrane microdomains. Because cholesterol-rich microdomains are linked to signal regulation in diverse receptor systems, and because hypercholesterolemia is an asthma risk-factor, we tested the hypothesis that cholesterol-rich plasma membrane microdomains are central to IL-5-family cytokine action in eosinophils. Purified human blood eosinophils were incubated (1 hr) with the cholesterol-chelating agent methyl-β-cyclodextrin (MβCD) or soluble cholesterol (MβCD pre-loaded with cholesterol), followed by cholesterol/membrane microdomain analyses via flow cytometry and confocal microscopy or stimulation with IL-5. Eosinophil activation was determined via immunoblotting for activated p38 MAPK, activated transcriptional regulator STAT5, cyclin D3 (MAPK-dependent), or Pim1 (STAT-dependent). MβCD decreases, and soluble cholesterol increases, membrane cholesterol content in a dose-dependent manner as assessed by flow cytometry. Likewise, confocal microscopy confirmed MβCD disrupts membrane microdomains. Furthermore, MβCD attenuates IL-5-induced p38 phosphorylation compared to control (p<0.001, N=10), whereas soluble cholesterol restores IL-5-induced p38 phosphorylation and significantly elevates basal phosphorylation (p<0.05, N=10). Cyclin D3 up-regulation is blocked by MβCD treatment but unaffected by soluble cholesterol addition (N=3). Neither MβCD nor soluble cholesterol appears to alter IL-5-induced STAT5 phosphorylation (N=3) or Pim1 up-regulation, suggesting a selective action of these agents (N=2). These studies reveal that disturbances in eosinophil membrane cholesterol content selectively affect eosinophil signaling, suggesting that in vivo cholesterol levels may direct eosinophilic function and inflammatory capacity.

RATIONALE: Eosinophils have been proposed to play an important role in the exacerbation of allergic asthma, as these cells can undergo excessive recruitment to the lungs where unregulated activation leads to tissue damage and fibrosis. Furthermore, interleukin-5 (IL-5) family cytokine receptors, critical for eosinophil recruitment and activation, are proposed to exist in membrane microdomains. Given that cholesterol-rich microdomains are linked to signal transduction regulation in numerous receptor systems, and because hypercholesterolemia is a risk factor for asthma, our current studies test the hypothesis that cholesterol-rich plasma membrane microdomains are important for IL-5-family cytokine stimulation of eosinophils. METHODS: Purified human peripheral blood eosinophils were incubated 1 hour with the cholesterol-chelating agent methyl-β-cyclodextrin (MβCD) or soluble cholesterol (MβCD pre-loaded with cholesterol), followed by stimulation with 100 pM IL-5. Eosinophil activation was determined via immunoblotting for activated MAP Kinase family members (phospho-ERK1/2, phospho-p38) or the transcriptional regulator phospho-STAT5. RESULTS: Addition of MβCD significantly decreases IL-5-induced ERK1/2 phosphorylation as compared to media control (p<0.001, N=8). Soluble cholesterol enhances basal ERK1/2 phosphorylation and restores the IL-5-induced ERK1/2 phosphorylation that was blocked by treatment with MβCD alone (p<0.05, N=8). Neither MβCD nor soluble cholesterol alters IL-5-induced STAT5 phosphorylation (N=8) suggesting a selective action of these agents. Also, trends indicate MβCD does not affect IL-5-induced p38 phosphorylation whereas soluble cholesterol augments both basal and IL-5-induced p38 phosphorylation. CONCLUSIONS: These studies suggest that disturbances in eosinophil cellular membrane cholesterol may affect only certain aspects of eosinophilic function and inflammatory activity. Thus, in vivo cholesterol levels may influence eosinophilic function/inflammatory/capacity.

RATIONALE: HRV infections are a major cause of exacerbations in chronic respiratory conditions, and previous studies suggest that airway macrophages are central to the innate immune response to HRV. Interestingly, striking similarities exist between features of severe asthma and the response to respiratory infections in asthma, including intensity of airflow obstruction, neutrophilic inflammation, and diminished response to corticosteroids. Therefore, we are examining the idea that macrophage phenotype can contribute to the cytokine dysregulation and airway dysfunction associated with severe asthma. METHODS: BAL fluid macrophages from 37 patients, including non-asthmatics and asthmatic individuals with a wide range of disease severity, were evaluated for pulmonary function and underwent bronchoalveolar lavage (BAL). BAL cells were treated ex vivo with a major group (HRV-16) or minor group (HRV-1a) rhinovirus for 72h and cytokine release was assessed via ELISA. The amounts of cytokines released were compared to other pulmonary parameters (including asthma severity, FEV1 % predicted, exhaled nitric oxide (eNO), IgE and methacholine PC20) for statistical significance. RESULTS: Overall, the release of cytokines from macrophages of severe asthmatics was generally reduced as compared to cytokine release seen in normal or non-severe asthmatics. The release of many of the tested cytokines (IL-1Ra, CXCL10, CCL2, CCL8, CCL-18) in response to HRV correlated significantly with both airway hyperreactivity (p = 0.003-0.02) and eNO levels (p = 0.009-0.05). CONCLUSIONS: These data suggest that severe asthmatics may have an attenuated ability to mount a robust immune response after HRV exposure, which may impair viral clearance, and thus contribute to exacerbations.

RATIONALE: Given the presence of thymic stromal lymphopoietin (TSLP) and eosinophils as prominent components of allergic inflammation, we sought to determine whether eosinophils express the TSLP receptor a-chain (TSLPR) and respond directly to TSLP stimulation. METHODS: Eosinophil mRNA expression for TSLPR was examined by real-time quantitative PCR of purified human peripheral blood eosinophils treated with cytokines in various combinations, including IL-3, IL-5, GM-CSF, and TNFa. Immunoblotting for TSLPR protein expression was performed on eosinophil lysates with a goat anti-TSLPR antibody. Flow cytometry with a goat anti-TSLPR antibody was used to detect surface TSLPR expression. Eosinophils were stimulated with human recombinant TSLP at various concentrations for 4 hours and supernatants were analyzed by ELISA for degranulation of eosinophil derived neurotoxin (EDN). RESULTS: Expression of mRNA and cell surface TSLPR was upregulated by stimulation with TNFa and IL-5 family cytokines. TSLPR protein was also detectable by Western blotting of eosinophil lysates. Stimulation of eosinophils with TSLP resulted in phosphorylation of the STAT5 transcription factor. In addition, eosinophil stimulation with TSLP resulted in degranulation of eosinophil derived neurotoxin (EDN) at levels comparable to IL-5. Furthermore, the TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to the TSLPR. CONCLUSIONS: This study demonstrates a role for eosinophils as a target of the allergic inflammatory cytokine, TSLP. Thus, in allergic inflammation, multiple pathways can serve to activate eosinophils, perhaps in a synergistic manner.

Summary Background Human rhinovirus (HRV) infections are a major cause of exacerbations in chronic respiratory conditions such as asthma and chronic obstructive pulmonary disease, but HRV‐induced immune responses of the lower airway are poorly understood. Earlier work examining cytokine release following HRV infection has focused on epithelial cells because they serve as the principal site of viral replication, and internalization and replication of viral RNA appear necessary for epithelial cell mediator release. However, during HRV infection, only a small proportion of epithelial cells become infected. As HRV‐induced cytokine levels in vivo are markedly elevated, this observation suggests that other mechanisms independent of direct viral infection may induce epithelial cell cytokine release. Objective Our aim was to test for the importance of interactions between human bronchial epithelial cells (HBECs) and monocytic cells in the control of mediator release during HRV exposure. Methods In vitro models of HRV serotype‐16 (HRV16) infection of primary HBECs and human monocytic cells, in mono or co‐culture, were used. We assessed HRV16‐induced CXCL10 and CCL2 protein release via ELISA. Results Co‐culture of human monocytic and bronchial epithelial cells promoted a synergistic augmentation of CXCL10 and CCL2 protein release following HRV16 challenge. Transfer of conditioned media from HRV16‐treated monocytic cells to epithelial cultures induced a robust release of CXCL10 by the epithelial cells. This effect was greatly attenuated by type I IFN receptor blocking antibodies, and could be recapitulated by IFN‐α addition. Conclusions Our data indicate that epithelial CXCL10 release during HRV infection is augmented by a monocytic cell‐dependent mechanism involving type I IFN(s). Our findings support a key role for monocytic cells in the amplification of epithelial cell chemokine production during HRV infection, and help to explain how an inflammatory milieu is created in the lower airways even in the absence of extensive viral replication and epithelial infection. Cite this as : N. L. Korpi‐Steiner, S. M. Valkenaar, M. E. Bates, M. D. Evans, J. E. Gern and P. J. Bertics, Clinical & Experimental Allergy , 2010 (40) 1203–1213.

BACKGROUND AND OBJECTIVE:Human gingival fibroblasts exhibit proliferative responses following epidermal growth factor exposure, which are thought to enhance periodontal regeneration in the absence of bacterial products such as lipopolysacharide. However, lipopolysaccharide challenge activates human gingival fibroblasts to release several inflammatory mediators that contribute to the immune response associated with periodontitis and attenuate wound repair. We tested the hypothesis that Porphyromonas gingivalis lipopolysaccharide-activated signaling pathways down-regulate epidermal growth factor receptor-dependent events.MATERIAL AND METHODS:To study lipopolysaccharide/epidermal growth factor interactions in human gingival fibroblasts, we introduced the catalytic subunit of human telomerase into human gingival fibroblasts, thereby generating a more long-lived cellular model. These cells were characterized and evaluated for lipopolysaccharide/epidermal growth factor responsiveness and regulation of epidermal growth factor-dependent pathways.RESULTS:Comparison of human telomerase-transduced gingival fibroblasts with human gingival fibroblasts revealed that both cell lines exhibit a spindle-like morphology and express similar levels of epidermal growth factor receptor, CD14 and Toll-like receptors 2 and 4. Importantly, human telomerase-transduced gingival fibroblasts proliferation rates are increased 5-9 fold over human gingival fibroblasts and exhibit a longer life span in culture. In addition, human telomerase-transduced gingival fibroblasts and human gingival fibroblasts exhibit comparable profiles of mitogen-activated protein kinase kinase (extracellular signal-regulated kinase 1/2) activation upon epidermal growth factor or P. gingivalis lipopolysaccharide administration. Interestingly, treatment with P. gingivalis lipopolysaccharide leads to a down-regulation of epidermal growth factor-dependent extracellular signal-regulated kinase 1/2, p38 and cyclic-AMP response element binding protein phosphorylation in both cell types.CONCLUSION:These studies demonstrate that human telomerase-transduced gingival fibroblasts exhibit an extended life span and recapitulate human gingival fibroblasts biology. Moreover, this system has allowed for the first demonstration of lipopolysaccharide down-regulation of epidermal growth factor activated pathways in human gingival fibroblasts and should facilitate the analysis of signaling events relevant to the pathogenesis and treatment of periodontitis.

Activation of the nucleotide receptor P2RX7 serves to modulate monocyte/macrophage-induced inflammatory events, but the molecular mechanisms behind this modulation are poorly understood. Because P2RX7 stimulation promotes inflammatory mediator production, and the promoter region of many of these inflammatory genes have activating protein-1 (AP-1) consensus sequences, the involvement of P2RX7 in the activation of members of the AP-1 family were investigated. Human embryonic kidney (HEK) cells that heterologously express human P2RX7 (HEK-P2RX7), murine RAW 264.7 macrophages and primary human monocytes isolated from blood draws were cultured in complete media, and incubated with the P2RX7 agonist, BzATP, for various times. Cell lysates were examined for expression of AP-1 proteins (cFos, FosB and JunB) through immunoblotting. Stimulation of HEK-P2RX7 cells, RAW 264.7 macrophages and primary human monocytes with BzATP induces FosB expression within 1 h of treatment (n > 3). Conversely, BzATP-treatment of RAW 264.7 macrophages expressing non-functional P2RX7 or HEK cells not expressing P2RX7 fail to induce FosB expression (n = 3). Treatment of RAW 264.7 macrophages and primary human monocytes with BzATP also induces JunB, but not cFos, expression (n > 3). Induction of FosB/JunB expression in RAW 264.7 macrophages is attenuated by the MEK antagonist, UO126 (n > 3, p < 0.05). Taken together, these data support that P2RX7 stimulation can induce formation of the AP-1 proteins FosB and JunB, which likely play a role in modulating monocytic gene expression during inflammation. This is the first report where P2RX7 activation leads to protein synthesis without priming with other inflammatory stimuli.

Viral respiratory infections are a major cause of asthma exacerbations and can contribute to the pathogenesis of asthma. Major group human rhinovirus enters cells by binding to the cell surface molecule ICAM-1 that is present on epithelial and monocytic lineage cells. The focus of the resulting viral infection is in bronchial epithelia. However, previous studies of the cytokine dysregulation that follows rhinovirus infection have implicated monocytic lineage cells in establishing the inflammatory environment even though productive infection is not a result. We have determined that human alveolar macrophages and human peripheral blood monocytes release MCP-1 upon exposure to human rhinovirus 16 (HRV16). Indeed, we have found p38 MAPK activation in human alveolar macrophages within 15 min of exposure to HRV16, and this activation lasts up to I h. The targets of p38 MAPK activation include transcriptional activators of the MCP-1 promoter. The transcription factor ATF-2, a p38 MAPK substrate, is phosphorylated 45 min after HRV16 exposure. Furthermore, I kappa B alpha, the inhibitor of the transcription factor NF-kappa B, is degraded. Prevention of HRV16 binding was effective in blocking p38 MAPK activation, ATF-2 phosphorylation, and MCP-1 release. This is the first report of a relationship between HRV16 exposure, MCP-1 release and monocytic-lineage cells suggesting that MCP-1 plays a role in establishing the inflammatory microenvironment initiated in the human airway upon exposure to rhinovirus.

RATIONALE: Airway eosinophils are phenotypically different from peripheral blood eosinophils, and these differences likely result from cell exposure to multiple factors during transmigration and pulmonary localization. It is unclear how these events affect eosinophil signaling capacity. Therefore we tested the hypothesis that blood and airway eosinophils, acquired from bronchoalveolar lavage fluid following segmental antigen challenge, exhibit an altered profile of cytokine (IL-5) and chemoattractant (FMLP)-induced activation/expression of key signaling molecules such as Ras, STAT5 and formyl-peptide receptors (FPRL1). METHODS: Purified blood and airway eosinophils were stimulated in vitro with IL-5 and/or FMLP. Active, GTP-bound Ras was captured from cell lysates by binding to an immobilized peptide encompassing the Ras-binding-domain of Raf. Intracellular proteins were visualized by immunoblotting and FPRL1 was quantified by flow cytometry. RESULTS: a) Airway eosinophils exhibit enhanced FMLP-induced Ras activation when compared to blood eosinophils (N=5), although IL-5 priming of blood eosinophils for 1h induces the cells to have an FMLP-responsiveness similar to airway cells (N = 5). b) Surface expression of FPRL1 is greater in airway cells (N=3), whereas IL-5-stimulated STAT5 phosphorylation is markedly greater in blood cells (N=4), perhaps due to the reduced expression of IL-5Ra subunit in airway cells. c) Expression of the IL-5-responsive kinase Pim-1 is greater in airway cells (N = 8). CONCLUSIONS: The signaling capacity of blood eosinophils is substantially altered as they transmigrate to the airway and this likely plays an important role in controlling eosinophil responsiveness to chemoattractants, cytokines and other factors in the inflammatory milieu.

Because interleukin (IL)-5 family cytokines are critical regulators of eosinophil development, recruitment, and activation, this study was initiated to identify proteins induced by these cytokines in eosinophils. Using oligonucleotide microarrays, numerous transcripts were identified as responsive to both IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF), but no transcripts were markedly affected by one cytokine and not the other. Expression of several gene products were seen to be increased following in vitro stimulation of human blood eosinophils, including the IL-3 receptor alpha subunit, lymphotoxin beta, Pim-1, and cyclin D3. Given that eosinophils recovered from the bronchoalveolar lavage fluid of allergic patients after antigen challenge are exposed to IL-5 or GM-CSF in the airway prior to isolation, the hypothesis was tested that selected IL-5- and GM-CSF-responsive genes are upregulated in airway eosinophils relative to the expression in blood cells. Airway eosinophils displayed greater cell surface expression of the IL-3 receptor alpha subunit, CD44, CD25, and CD66e, suggesting that these proteins may be markers of eosinophil activation by IL-5 family cytokines in airway eosinophils. Other genes that were induced by both IL-5 and GM-CSF showed protein expression at similar or decreased levels in airway eosinophils relative to their circulating counterparts (i.e., lymphotoxin beta and CD24). These studies have identified several transcriptional targets of IL-5 and GM-CSF in human eosinophils and suggest that a number of protein products are critical to the responsiveness of airway eosinophils.

RATIONALE: Respiratory infections with rhinovirus (RV) are a major cause of asthma exacerbations and may contribute to the overall pathogenesis of asthma.Alveolar macrophages, the predominant immune cell in the airway lumen, have been implicated in promoting cytokine dysregulation within the inflammatory microenvironment in response to RV respiratory infections.Given the importance of RV in the pathobiology of asthma and the paucity of information on the intracellular mechanisms by which RV activates macrophages, this study evaluated the activity of MAP Kinases and CREB, a transcription factor critical for the induction of several proinflammatory cytokines, following monocyte or macrophage exposure to RV. METHODS: Primary human monocytes or alveolar macrophages were incubated with purified human RV-16 (MOI=0.01-100)for 5-120min.Cell lysates were immunoblotted with antibodies for activated (phosphorylated) MAP Kinases or CREB.RESULTS: Exposure of human monocytic cells to RV results in the activation of both JNK and p38 MAP Kinases in a time-and dose-dependent manner (N=3).However ERK1 and ERK2 activation following RV stimulation is not discernable.Furthermore, RV-induces phosphorylation of the CREB transcription factor.Lastly, pretreatment of human monocytic cells with SB202190, a selective inhibitor of p38 MAP Kinase, diminishes RV-induced CREB activation (N=3).CONCLUSIONS: These studies demonstrate that RV interaction with monocytic cells stimulates the phosphorylation and activation of MAP Kinase pathways, and CREB and suggest that this is an important mechanism regulating macrophage cytokine elaboration during virus-induced exacerbations of asthma.

Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Ralpha on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.