SummaryBackgroundAsthma exacerbations contribute to significant morbidity, mortality and healthcare utilization. Furthermore, viral infections are associated with asthma exacerbations by mechanisms that are not fully understood.ObjectiveThe aim of this analysis was to determine whether cytokine patterns in patients with colds could identify risks for subsequent asthma exacerbations.MethodsWe 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‐β, IFN‐γ, IFN‐λ1, IP‐10, TRAIL), cell recruiting (G‐CSF, IL‐1β, 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).ResultsThe 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‐1β 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 RelevanceAlthough 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.

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.

The nucleoticle receptor P2X(7) is expressed by most leukocytes and initiates signaling events that amplify numerous LPS responses. We tested the hypothesis that loss-of-function polymorphisms in the human P2X(7) gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X7 pore assay in whole blood that magnifies the activity from wild-type alleles and preserves the gene dosage effect for the 1513 C polymorphism (AA, 69 +/- 4; AC, 42 +/- 4; and CC, 6 +/- 1-fold stimulation). Thirty of 200 healthy individuals were identified as having low P2X, pore activity. Seven low pore subjects were 1513 CC, 3 and 11 participants had the other known variants 946 GA and 1729 TA respectively; the remaining 9 volunteers likely have novel polymorphisms. Because platelets are a large source of extracellular ATP during inflammation, whole blood was treated ex vivo with Salmonella typhimarium LPS in the absence of exogenous nucleotides. LPS-stimulated whole blood from individuals in the low pore activity group generated reduced plasma levels of TNF-alpha (p = 0.036) and higher amounts of IL-10 (P < 0.001) relative to the high pore controls. This reduction in the TNF-alpha to IL-10 ratio persisted to at least 24 h and is further decreased by cotreatment with 2-methylthio-ATP. The ability of P2X7 polymorphisms to regulate the LPS-induced TNF-alpha to IL-10 ratio suggests that 15% of healthy adults may exhibit anti-inflammatory mediator responses during major infectious perturbations of the immune system, which can be predicted by P2X7 pore activity.

Macrophages express several lipopolysaccharide (LPS) binding proteins and are potently activated by LPS to produce inflammatory mediators. Recent studies have shown that receptors for exogenous nucleotides (P2X and P2Y purinergic receptors) can modulate macrophage production of TNF-α , IL-1β and nitric oxide (NO) following LPS exposure. Macrophages and LPS-stimulated monocytes express elevated levels of P2Y 1 , P2Y 2 and P2X 7 mRNA, suggesting that both P2Y and P2X receptors can contribute to LPS-induced pathophysiology. In addition, oxidized-ATP treatment (which inhibits P2X 7 ) of macrophages blocks LPS-induced NO production, NF-κB and ERK-1/2 activation. Also, an LPS-binding domain located in the P2X 7 C-terminus appears important for receptor trafficking/function. Moreover, the purinergic receptor ligand 2-MeS-ATP attenuates LPS-induced cytokine and NO production in vivo and ex vivo. These data suggest that P2X 7 and certain P2Ys are linked to LPS effects, although their relative contribution in vivo is unclear. Accordingly, we tested the capacity of several adenine nucleotides to modulate LPS-induced mortality in mice. We found that the P2X 7 -directed ligand BzATP was unable to prevent LPS-induced death, whereas 2-MeS-ATP and 2-Cl-ATP, which bind to multiple P2X and P2Y receptors were able to protect mice from LPS-induced death. These data suggest that the co-ordinate action of P2Y and P2X 7 receptors are critical for controlling LPS responses in vivo and that agents directed against both receptor classes may provide the greatest therapeutic advantage.

The expression of certain COOH-terminal truncation mutants of the epidermal growth factor receptor (EGFR) can lead to cell transformation, and with ligand stimulation, a broader spectrum of phosphorylated proteins appears compared with EGF-treated cells expressing wild-type EGFR. Accordingly, it has been proposed that elements within the COOH terminus may determine substrate specificity of the EGFR tyrosine kinase (Decker, S. J., Alexander, C., and Habib, T. (1992) J. Biol. Chem. 267, 1104-1108; Walton, G. M., Chen, W. S., Rosenfeld, M. G., and Gill, G. N. (1990) J. Biol. Chem. 265, 1750-1754). To address this hypothesis, we analyzed in vitro the steady-state kinetic parameters for phosphorylation of several substrates by both wild-type EGFR and an oncogenic EGFR mutant (the ct1022 mutant) truncated at residue 1022. The substrates included: (i) a phospholipase C-gamma fragment (residues 530-850); (ii) the 46-kDa isoform of the Shc adapter protein; (iii) a 13-residue peptide mimic for the region around the major autophosphorylation tyrosine and the Shc binding site (the Y1173 peptide); (iv) a poly(Glu,Tyr) 4:1 copolymer; and (v) the 8-residue peptide, angiotensin II. Our data demonstrate that the steady-state kinetic parameters for the ct1022 mutant differ from those of the wild-type enzyme, and the differences are substrate-dependent. These results support the concept that this oncogenic truncation/mutation alters EGFR substrate specificity, rather than causing a general alteration of activity. We performed the experiments using a non-radioactive fluorescence polarization assay that quantifies the degree of phosphorylation of peptide as well as natural substrates. The results are consistent with those from the traditional [gamma-32P]ATP/filtration assay.

Previous reports about the nucleotide receptor P2X(7), which exhibits ion channel and pore-forming activity and is known to promote IL-1beta processing, have centered largely on its role in macrophage function, whereas its participation in monocyte activity has been unclear. However, because extracellular ATP has been shown to affect monocytes with respect to IL-1beta release, we hypothesized that the P2X(7) receptor is also present and functional in a subpopulation of blood monocytes. Flow cytometric analysis revealed that about 70% of monocytes isolated from normal human donors expressed the P2X(7) receptor. Activation of P2X(7) receptor-associated pore formation by the agonist BzATP resulted in a 9- to 15-fold increase in the uptake of the membrane-impermeant fluorescent dye YO-PRO, and this dye uptake is markedly inhibited by the P2X(7) receptor antagonists KN-62 and oATP. Evidence supporting the presence of the functional P2X(7) receptor in monocytes also includes the observation that BzATP exposure results in a dose-dependent increase in the activation of mitogen-activated 2protein kinases and the nuclear translocation of the transcription factor NF-kappaB in human monocytes and in THP-1 human monocytic cells. Furthermore, treatment of monocytes with BzATP induced the expression of cyclooxygenase-2 (COX-2) and tissue factor, which are two important endpoints that have not been previously shown to be regulated by nucleotide receptor action in monocytes. Together, these data indicate that a subpopulation of human monocytes express P2X(7) receptors that are functional with respect to pore formation, signal transduction, and mediator production, further supporting a key role for this nucleotide receptor in host immune responses.

Caveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nM EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor.

To evaluate the mechanisms by which epidermal growth factor (EGF) regulates actin-based cellular processes such as cell migration, we first examined the effects of EGF on cell adhesion, which is essential for cell migration. In mouse B82L fibroblasts transfected with the full-length EGF receptor, EGF promotes cell Pounding and attenuates cell spreading on fibronectin, laminin, and vitronectin, and thus appears to reduce the strength of cell adhesion. Moreover, EGF synergizes with multiple extracellular matrix (ECM) components in the promotion of integrin-mediated cell migration of several different cell types, including fibroblasts and various carcinoma and osteosarcoma cell lilies. Interestingly, co-presentation (co-positioning) of EGF with laminin or fibronectin is essential for EGF-stimulated migration. When EGF is mixed with the cells instead of the ECM components, it has little effect on cell migration. These results suggest that co-presentation of EGF with ECM components can enhance the polarization events required for directional cell movement. To identify the EGF receptor elements critical for the EGF stimulation of cell migration, B82L fibroblasts were transfected with either mutated or wild-type EGF receptors, Surprisingly, we found that B82L-Parental cells that lack the EGF receptor are not able to migrate to fibronectin, even though they can adhere to fibronectin. However, the introduction of wild-type EGF receptors into these fibroblasts enables them to migrate toward fibronectin even in the absence of EGF, The requirement of the EGF receptor for cell migration does not appear to result from the secretion of EGF or TGF-alpha by the cells transfected with the EGF receptor. Furthermore, cells expressing EGF receptors that are kinase-inactive, or C-terminally truncated, exhibit little migration toward fibronectin, indicating that an intact EGF receptor kinase is required for fibronectin-induced cell migration. In addition, neutralizing anti-EGF receptor antibodies attenuate cell migration in the presence of EGF, and inhibit migration to fibronectin or laminin alone. These results further suggest that the EGF receptor is downstream of integrin activation in the signal transduction pathways leading to fibroblast migration.

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.

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.