Complex association patterns for inflammatory mediators in induced sputum from subjects with asthma.

BACKGROUND: The release of various inflammatory mediators into the bronchial lumen is thought to reflect both the type and degree of airway inflammation, eosinophilic Th2, and Th9, or neutrophilic Th1, and Th17, in patients with asthma. AIMS: We investigated whether cytokines and chemokines differed in sputum from subjects with more severe compared with milder asthma and whether unbiased factor analysis of cytokine and chemokine groupings indicates specific inflammatory pathways. METHODS: Cell-free supernatants from induced sputum were obtained from subjects with a broad range of asthma severity (n = 158) and assessed using Milliplex® Cytokines/Chemokine kits I, II and III, measuring 75 individual proteins. Each cytokine, chemokine or growth factor concentration was examined for differences between asthma severity groups, for association with leucocyte counts, and by factor analysis. RESULTS: Severe asthma subjects had 9 increased and 4 decreased proteins compared to mild asthma subjects and fewer differences compared to moderate asthma. Twenty-six mediators were significantly associated with an increasing single leucocyte type: 16 with neutrophils (3 interleukins [IL], 3 CC chemokines, 4 CXC chemokines, 4 growth factors, TNF-α and CX3CL1/Fractalkine); 5 with lymphocytes (IL-7, IL-16, IL-23, IFN-α2 and CCL4/MIP1ß); IL-15 and CCL15/MIP1δ with macrophages; IL-5 with eosinophils; and IL-4 and TNFSF10/TRAIL with airway epithelial cells. Factor analysis grouped 43 cytokines, chemokines and growth factors which had no missing data onto the first 10 factors, containing mixes of Th1, Th2, Th9 and Th17 inflammatory and anti-inflammatory proteins. CONCLUSIONS: Sputum cytokines, chemokines and growth factors were increased in severe asthma, primarily with increased neutrophils. Factor analysis identified complex inflammatory protein interactions, suggesting airway inflammation in asthma is characterized by overlapping immune pathways. Thus, focus on a single specific inflammatory mediator or pathway may limit understanding the complexity of inflammation underlying airway changes in asthma and selection of appropriate therapy.

Differential requirement for P2X7R function in IL-17 dependent vs. IL-17 independent cellular immune responses.

IL17-dependent autoimmunity to collagen type V (Col V) has been associated with lung transplant obliterative bronchiolitis. Unlike the T helper 1 (Th1)-dependent immune responses to Tetanus Toxoid (TT), the Th17 response to Col V in lung transplant patients and its Th1/17 variant observed in coronary artery disease patients requires IL-1ß, tumor necrosis factor α and CD14(+) cells. Given the involvement of the P2X7 receptor (P2X7R) in monocyte IL-1ß responses, we investigated its role in Th17-, Th1/17- and Th1-mediated proinflammatory responses. Transfer of antigen-pulsed peripheral blood mononucleated cells (PBMCs) from Col V-reactive patients into SCID mouse footpads along with P2X7R antagonists revealed a selective inhibition of Col V-, but not TT-specific swelling responses. P2X7R inhibitors blocked IL-1ß induction from monocytes, including both Col V-α1 peptide-induced (T-dependent), as well as native Col V-induced (T-independent) responses. Significantly higher P2X7R expression was found on CXCR3(neg) CCR4(+)/6(+) CD4(+) [Th17] versus CXCR3(+)CCR4/6(neg) CD4(+) [Th1] subsets in PBMCs, suggesting that the paradigm of selective dependence on P2X7R might extend beyond Col V autoimmunity. Indeed, P2X7R inhibitors suppressed not only anti-Col V, but also Th1/17-mediated alloimmunity, in a heart transplant patient without affecting anti-viral Epstein-Barr virus responses. These results suggest that agents targeting the P2X7R might effectively treat Th17-related transplant pathologies, while maintaining Th1-immunity to infection.

Nasal lavage VEGF and TNF-α levels during a natural cold predict asthma exacerbations.

BACKGROUND: Asthma exacerbations contribute to significant morbidity, mortality and healthcare utilization. Furthermore, viral infections are associated with asthma exacerbations by mechanisms that are not fully understood. OBJECTIVE: The aim of this analysis was to determine whether cytokine patterns in patients with colds could identify risks for subsequent asthma exacerbations. METHODS: We analysed cytokine levels in nasal lavage fluid (NLF) in 59 subjects (46 with asthma) with acute upper respiratory symptoms and after symptomatic resolution. Analyte choice was based on potential relevance to asthma exacerbations: antiviral (IFN-α, IFN-ß, IFN-γ, IFN-λ1, IP-10, TRAIL), cell recruiting (G-CSF, IL-1ß, IL-8, MCP-1, MCP-3, TNF-α), polarizing (CXCL13, IL-10, IL-13, IL-17, TSLP), and injury remodelling (fibronectin, IL-33, MMP-9, VEGF). RESULTS: The overall cytokine response induced during viral infections was not different between asthmatic and non-asthmatic individuals for a wide array of cytokines. However, mean levels of VEGF, TNF-α and IL-1ß were 1.7-, 5.1- and 4.7-fold higher in samples from asthma subjects who exacerbated in the first 3 weeks of the cold compared with those who did not exacerbate (P = 0.006, 0.01, 0.048, respectively). Using receiver operating characteristic curve-defined thresholds, high VEGF and TNF-α levels predicted a shorter time-to-exacerbation after NLF sampling (25% exacerbation rate: 3 vs. 45 days, and 3 vs. 26 days; P = 0.03, 0.04, respectively). CONCLUSION AND CLINICAL RELEVANCE: Although they produce similar cytokine responses to viral infection as non-asthmatics, asthmatics with higher levels of VEGF and TNF-α in NLF obtained during acute cold phases predicted subsequent asthma exacerbations in this cohort of patients with mild-to-moderate disease. In the future, stratifying the risk of an asthma exacerbation by cytokine profile may aid the targeting of personalized treatment and intervention strategies.

Interferon gene expression in sputum cells correlates with the Asthma Index Score during virus-induced exacerbations.

BACKGROUND: The majority of asthma exacerbations are related to viral respiratory infections. Some, but not all, previous studies have reported that low interferon responses in patients with asthma increase the risk for virus-induced exacerbations. OBJECTIVE: We sought to determine the relationship between lower airway inflammatory biomarkers, specifically interferon gene expression, and the severity or presence of an exacerbation in asthmatics experiencing a naturally occurring viral infection. METHODS: Sputum samples were analysed from subjects in an asthma exacerbation study who experienced a confirmed viral infection. Subjects were monitored for daily symptoms, medication use and peak expiratory flow rate until baseline. Sputum samples were assessed for cell counts and gene expression. RESULTS: Interferon gamma expression was significantly greater in patients with asthma exacerbations compared to non-exacerbating patients (P = 0.002). IFN-α1, IFN-ß1 and IFN-γ mRNA levels correlated with the peak Asthma Index (r = 0.58, P < 0.001; r = 0.57, P = 0.001; and r = 0.51, P = 0.004, respectively). Additionally, IL-13, IL-10 and eosinophil major basic protein mRNA levels were greater in patients with asthma exacerbations compared to non-exacerbating patients (P = 0.03, P = 0.06 and P = 0.02, respectively), and IL-13 mRNA correlated with the peak Asthma Index (P = 0.006). CONCLUSIONS: Our findings indicate that asthma exacerbations are associated with increased rather than decreased expression of interferons early in the course of infection. These findings raise the possibility that excessive virus-induced interferon production during acute infections can contribute to airway inflammation and exacerbations of asthma.

Anti-IL-5 attenuates activation and surface density of ß(2) -integrins on circulating eosinophils after segmental antigen challenge.

BACKGROUND: IL-5 activates α(M) ß(2) integrin on blood eosinophils in vitro. Eosinophils in bronchoalveolar lavage (BAL) following segmental antigen challenge have activated ß(2) -integrins. OBJECTIVE: To identify roles for IL-5 in regulating human eosinophil integrins in vivo. METHODS: Blood and BAL eosinophils were analysed by flow cytometry in ten subjects with allergic asthma who underwent a segmental antigen challenge protocol before and after anti-IL-5 administration. RESULTS: Blood eosinophil reactivity with monoclonal antibody (mAb) KIM-127, which recognizes partially activated ß(2) -integrins, was decreased after anti-IL-5. Before anti-IL-5, surface densities of blood eosinophil ß(2) , α(M) and α(L) integrin subunits increased modestly post challenge. After anti-IL-5, such increases did not occur. Before or after anti-IL-5, surface densities of ß(2) , α(M) , α(L) and α(D) and reactivity with KIM-127 and mAb CBRM1/5, which recognizes high-activity α(M) ß(2) , were similarly high on BAL eosinophils 48 h post-challenge. Density and activation state of ß(1) -integrins on blood and BAL eosinophils were not impacted by anti-IL-5, even though anti-IL-5 ablated a modest post-challenge increase on blood or BAL eosinophils of P-selectin glycoprotein ligand-1 (PSGL-1), a receptor for P-selectin that causes activation of ß(1) -integrins. Forward scatter of blood eosinophils post-challenge was less heterogeneous and on the average decreased after anti-IL-5; however, anti-IL-5 had no effect on the decreased forward scatter of eosinophils in post-challenge BAL compared with eosinophils in blood. Blood eosinophil KIM-127 reactivity at the time of challenge correlated with the percentage of eosinophils in BAL post-challenge. CONCLUSION AND CLINICAL RELEVANCE: IL-5 supports a heterogeneous population of circulating eosinophils with partially activated ß(2) -integrins and is responsible for up-regulation of ß(2) -integrins and PSGL-1 on circulating eosinophils following segmental antigen challenge but has minimal effects on properties of eosinophils in BAL. Dampening of ß(2) -integrin function of eosinophils in transit to inflamed airway may contribute to the decrease in lung inflammation caused by anti-IL-5.

Predicting worsening asthma control following the common cold.

The asthmatic response to the common cold is highly variable, and early characteristics that predict worsening of asthma control following a cold have not been identified. In this prospective multicentric cohort study of 413 adult subjects with asthma, the mini-Asthma Control Questionnaire (mini-ACQ) was used to quantify changes in asthma control and the Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21) to measure cold severity. Univariate and multivariable models were used to examine demographic, physiological, serological and cold-related characteristics for their relationship to changes in asthma control following a cold. Clinically significant worsening of asthma control was observed following a cold (mean+/-SD increase in mini-ACQ score of 0.69+/-0.93). Univariate analysis demonstrated that season, centre location, cold duration and cold severity measurements were all associated with a change in asthma control. Multivariable analysis of the covariates available within the first 2 days of cold onset revealed that the day 2 and cumulative sum of day 1 and 2 WURSS-21 scores were significant predictors of the subsequent changes in asthma control. In asthmatic subjects, cold severity within the first 2 days can be used to predict subsequent changes in asthma control. This information may help clinicians prevent deterioration in asthma control following a cold.

Nucleotide receptor P2RX7 stimulation enhances LPS-induced interferon-ß production in murine macrophages.

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-ß 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-ß expression following P2RX(7) stimulation is likely transcriptionally regulated, as this enhancement is observed at the IFN-ß promoter level. Furthermore, P2RX(7) stimulation is able to increase the phosphorylation and subsequent IFN-ß promoter occupancy of IRF-3, a transcription factor that is critical for IFN-ß 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.

Standardized method to minimize variability in a functional P2X(7) flow cytometric assay for a multi-center clinical trial.

BACKGROUND: Flow cytometric analysis of human P2X(7) pore activity segregates variant from common P2RX7 genotypes and may serve as a biomarker for cancer, pain, inflammation, and immune responses to infection. Standardization is needed to accommodate variable sample age and instrumentation differences in a multicenter clinical trial. METHODS: CD14-PE-stained whole blood samples were treated with YO-PRO-1 combined with a P2X(7) agonist (BzATP) or control, followed by the addition of PI after closure of the P2X(7) pore. Recalled instrument settings from previous publications were used to adapt a standardized fluorescent particle-adjusted set-up method. Experiments were performed to compare the two methods while evaluating components of systematic variability and facilitating reliable processing of samples with varied ages. RESULTS: The median YO-PRO-1 fluorescence of BzATP-treated samples had less variability when collected by the bead-adjusted method and was less influenced by the compensation strategy used. The average day-to-day coefficient of variance for assessments of P2X(7) pore activity by this method was 0.11 +/- 0.04, and the exclusion of nonviable cells was found to accommodate samples aged up to 4 days after phlebotomy. The bead-adjusted set-up method produced measurements differing by only 2.0% +/- 1.5% on two analog cytometers, and within similar decades when comparing analog to digital instruments. CONCLUSIONS: These results provide a standardized method for quantitative flow cytometric analysis of P2X(7) receptor phenotypes in blood monocytes with minimal intralaboratory variation and potential for interlaboratory comparisons that can greatly facilitate multicenter functional genomic clinical studies.

Nuclear translocation of NF-kappaB in lipopolysaccharide-treated macrophages fails to correspond to endotoxicity: evidence suggesting a requirement for a gamma interferon-like signal.

Elucidation of a signal transduction pathway essential to lipopolysaccharide (LPS)-induced macrophage activation has the capacity to provide new targets for the treatment of septic shock. In this regard, activation of the transcription factor NF-kappaB is commonly thought to be critical to LPS-stimulated macrophage inflammatory mediator production, although certain immunological, genetic, and molecular evidence suggests that other factors are involved. To address this issue, we hypothesized that the degree of LPS-induced NF-kappaB mobilization should correlate with the murine endotoxicity of the species of LPS used for in vitro study. Therefore, using D-galactosamine-sensitized mice, we assessed the lethal potencies of eight LPS preparations from Escherichia, Salmonella, Klebsiella, Bacteroides, Pseudomonas, Neisseria, and Rhodobacter species as well as that of the endotoxin substructure lipid X. The lethal potencies of these LPS preparations varied by > 160-fold. Treatment of RAW 264.7 cells with the same LPS preparations induced levels of tumor necrosis factor alpha (TNF-alpha) and NO production that correlated with the LPS 50% lethal dose. The combined analysis of the levels of these two mediators produced in response to LPS in RAW cells was found to be a strong predictor of murine endotoxic lethality. Interestingly, while relatively nontoxic in mice, Rhodobacter capsulatus LPS stimulated RAW cell NF-kappaB-like DNA binding protein mobilization and TNF-alpha production to levels comparable to those of more toxic species of LPS but was unable to induce NO generation in RAW cells. These data indicate that neither NF-kappaB activation nor TNF-alpha production alone is a dependable predictor of LPS lethality. Additionally, cotreatment of RAW cells with the potent inflammatory mediator ADP had no effect on the ability of R. capsulatus LPS to stimulate NO production but significantly enhanced induction of NO production by the toxic species of LPS. In contrast, cotreatment of RAW cells or peritoneal macrophages with gamma interferon (IFN-gamma) normalized the abilities of both toxic and nontoxic LPS preparations to induce NO production, suggesting that selected preparations of LPS may preferentially generate an IFN-gamma-like signal that accounts for enhanced toxicity. In sum, the activation of NF-kappaB does not correspond to LPS lethality, thereby complicating models of macrophage activation that highlight NF-kappaB alone as a signal transduction factor necessary for LPS-mediated toxicity.

The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SK-N-SH neuroblastoma cells. A role for Gp in receptor compartmentation.

The relationship between muscarinic receptor activation of phosphoinositide hydrolysis and the sequestration of cell surface muscarinic receptors has been examined for both intact and digitonin-permeabilized human SK-N-SH neuroblastoma cells. Addition of the aminosteroid 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) to intact cells resulted in the inhibition of oxotremorine-M-stimulated inositol phosphate release and of Ca2+ signaling by greater than 75%. In contrast, when phospholipase C was directly activated by the addition of the calcium ionophore ionomycin, inclusion of U-73122 had little inhibitory effect. Addition of U-73122 to intact cells also inhibited the agonist-induced sequestration of cell surface muscarinic receptors and their subsequent down-regulation with an IC50 value (4.1 microM) similar to that observed for inhibition of inositol phosphate release (3.7 microM). In contrast, when oxotremorine-M-stimulated phosphoinositide hydrolysis was inhibited by depletion of extracellular Ca2+, no reduction in the extent of receptor sequestration was observed. When introduced into digitonin-permeabilized cells, U-73122 more markedly inhibited inositol phosphate release elicited by either oxotremorine-M or guanosine-5'-O-(3-thiotriphosphate) than that induced by added Ca2+. Addition of oxotremorine-M to permeabilized cells resulted in muscarinic receptor sequestration and down-regulation. Both the loss of muscarinic acetylcholine receptors and activation of phosphoinositide hydrolysis in permeabilized cells were inhibited by the inclusion of guanosine-5'-O-(2-thiodiphosphate). The results indicate that the agonist-induced sequestration of muscarinic acetylcholine receptor in SK-N-SH cells requires the involvement of a GTP-binding protein but not the production of phosphoinositide-derived second messenger molecules.

Protection of mice from endotoxic death by 2-methylthio-ATP.

The lethal effects of endotoxin, a bacterial product shed into the blood during bacteremia, are thought to be due to macrophage release of mediators such as tumor necrosis factor alpha and interleukin 1. Although much is known about the pathophysiology of endotoxemia, relatively little is known about the cellular signaling mechanisms that are involved. The data in this study suggest that extracellular adenine nucleotides can influence the development of endotoxin shock. An adenine nucleotide analog, 2-methylthio-ATP, inhibited the endotoxin-stimulated release of toxic mediators (i.e., tumor necrosis factor alpha and interleukin 1), and it protected mice from endotoxin-induced death. These studies suggest a fundamental and unusual role for adenine nucleotides on endotoxin action, and they provide a potentially new therapeutic approach for the control of the pathophysiology of Gram-negative septicemia.

Purinergic receptor modulation of lipopolysaccharide signaling and inducible nitric-oxide synthase expression in RAW 264.7 macrophages.

Previous studies have suggested that the P2Z/P2X7 purinergic receptor can participate in nucleotide-induced modulation of lipopolysaccharide (LPS) stimulated inflammatory mediator production. To test this hypothesis, we evaluated whether antagonism of the P2Z/P2X7 receptor can influence LPS signaling and expression of the inducible form of nitric-oxide synthase (iNOS) in RAW 264.7 macrophages. In the present study, we demonstrate that pretreatment of RAW 264.7 macrophages with a P2Z/P2X7 receptor antagonist, periodate oxidized adenosine 5'-triphosphate (o-ATP), substantially inhibits LPS-stimulated NO production and iNOS expression without altering cell viability. This effect on LPS-induced iNOS expression is mimicked by a pyridoxal-phosphate-based antagonist (pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid) of the P2Z/P2X7 purinergic receptor, indicating that these results are not unique to o-ATP. Additionally, o-ATP prevents cell death induced by P2Z/P2X7 receptor agonists. To ascertain how P2Z/P2X7 receptor antagonists influence LPS signaling, we evaluated the capacity of o-ATP to regulate LPS-mediated activation of the transcription factor, nuclear factor-kappaB, and the mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2. These experiments reveal that pretreatment of RAW 264.7 cells with o-ATP attenuates the LPS stimulation of a nuclear factor-kappaB-like binding activity. Moreover, the activation of ERK1 and ERK2 by LPS, but not by the phorbol ester, phorbol 12-myristate 13-acetate, is also blocked in RAW 264.7 cells by o-ATP pretreatment. In summary, these data suggest that the P2Z/P2X7 receptor modulates LPS-induced macrophage activation as assessed by iNOS expression and NO production. This report implicates the P2Z/P2X7 receptor in the control of protein kinase cascades and transcriptional processes, and these observations are likely to be important for the development of selective purinergic receptor antagonists for the treatment of septic shock.

Cutting edge: the nucleotide receptor P2X7 contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide.

The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of kappaB-alpha isoform (IkappaB-alpha) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.

Regulation of inducible nitric oxide synthase expression by macrophage purinoreceptors and calcium.

Macrophage activation is central to the progression of multiple diseases via the release of inflammatory mediators such as cytokines and nitric oxide. Despite the recognized overlap in the regulatory mechanisms involved in mediator production, little formation exists regarding receptor-initiated signaling pathways that coordinately control multiple end points, such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide production. In this study, the expression of inducible nitric oxide synthase (iNOS) in macrophages is shown to be regulated by calcium and by a purinoreceptor signaling system. The P2Y purinoreceptor partial agonist, 2-methylthio-ATP (2-MeS-ATP), inhibits the expression of iNOS induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) in primary macrophages. Additionally, 2-MeS-ATP attenuates the expression of iNOS in macrophages isolated from CD-1 mice challenged with LPS, and it inhibits LPS-induced TNF-alpha and interleukin-1 alpha (IL-1 alpha) release, thereby preventing endotoxic death. Thus, purinoreceptors and calcium are likely to be critical for macrophage activation and the production of inflammatory mediators stimulated by LPS.