The effects of a CCR3 inhibitor, AXP1275, on allergen-induced airway responses in adults with mild-to-moderate atopic asthma.

BACKGROUND: CCR3 is the cognate receptor for major human eosinophil chemoattractants from the eotaxin family of proteins that are elevated in asthma and correlate with disease severity. OBJECTIVE: This proof-of-mechanism study examined the effect of AXP1275, an oral, small-molecule inhibitor of CCR3, on airway responses to inhaled allergen challenge. METHODS: Twenty-one subjects with mild atopic asthma and documented early and late asthmatic responses to an inhaled aeroallergen completed a randomized double-blind cross-over study to compare early and late allergen-induced asthmatic responses, methacholine PC20 , blood and sputum eosinophils and exhaled nitric oxide after 2 weeks of treatment with once-daily doses of AXP1275 (50 mg) or placebo. RESULTS: There was a significant increase in methacholine PC20 after 12 days of AXP1275 treatment compared to placebo (increase of 0.92 doubling doses versus 0.17 doubling doses, P = .01), but this protection was lost post-allergen challenge. There was no effect of AXP1275 on allergen-induced late asthmatic responses, or eosinophils in blood and sputum. The early asthmatic response and exhaled nitric oxide levels were slightly lower with AXP1275, but this did not reach statistical significance. The number of subjects who experienced treatment-emergent adverse events while receiving AXP1275 was comparable placebo. CONCLUSIONS & CLINICAL RELEVANCE: AXP1275 50 mg administered daily was safe and well tolerated, and there was no difference in the type, severity or frequency of treatment-emergent adverse events in subjects while receiving AXP1275 compared to placebo. AXP1275 increased the methacholine PC20 ; however, the low and variable exposure to APX1275 over a short treatment period may have contributed to poor efficacy on other outcomes.

Corrigendum: Palaeo leaf economics reveal a shift in ecosystem function associated with the end-Triassic mass extinction event.

This corrects the article DOI: 10.1038/nplants.2017.104.

Palaeo leaf economics reveal a shift in ecosystem function associated with the end-Triassic mass extinction event.

Climate change is likely to have altered the ecological functioning of past ecosystems, and is likely to alter functioning in the future; however, the magnitude and direction of such changes are difficult to predict. Here we use a deep-time case study to evaluate the impact of a well-constrained CO2-induced global warming event on the ecological functioning of dominant plant communities. We use leaf mass per area (LMA), a widely used trait in modern plant ecology, to infer the palaeoecological strategy of fossil plant taxa. We show that palaeo-LMA can be inferred from fossil leaf cuticles based on a tight relationship between LMA and cuticle thickness observed among extant gymnosperms. Application of this new palaeo-LMA proxy to fossil gymnosperms from East Greenland reveals significant shifts in the dominant ecological strategies of vegetation found across the Triassic-Jurassic transition. Late Triassic forests, dominated by low-LMA taxa with inferred high transpiration rates and short leaf lifespans, were replaced in the Early Jurassic by forests dominated by high-LMA taxa that were likely to have slower metabolic rates. We suggest that extreme CO2-induced global warming selected for taxa with high LMA associated with a stress-tolerant strategy and that adaptive plasticity in leaf functional traits such as LMA contributed to post-warming ecological success.

A role for mitogen kinase kinase 3 in pulmonary inflammation validated from a proteomic approach.

Proteomics is a powerful tool to ascertain which proteins are differentially expressed in the context of disease. We have used this approach on inflammatory cells obtained from patients with asthma to ascertain whether novel drugs targets could be illuminated and to investigate the role of any such target in a range of in vitro and in vivo models of inflammation. A proteomic study was undertaken using peripheral blood mononuclear cells from mild asthmatic subjects compared with healthy subjects. The analysis revealed an increased expression of the intracellular kinase, mitogen activated protein kinase (MKK3), and the function of this protein was investigated further in preclinical models of inflammation using MKK3 knockout mice. We describe a 3.65 fold increase in the expression of MKK3 in CD8(+) T lymphocytes obtained from subjects with asthma compared with healthy subjects using a proteomic approach which we have confirmed in CD8(+), but not in CD4(+) T lymphocytes or human bronchial epithelial cells from asthmatic patients using a Western blot technique. In wild type mice, bacterial lipopolysaccharide (LPS) caused a significant increase in MKK3 expression and significantly reduced airway neutrophilia in MKK3(-/-) mice (median, 25, 75% percentile; wild/LPS; 5.3 (0.7-9.9) × 10(5) cells/mL vs MKK3(-/-)/LPS; 0 (0-1.9) × 10(5) cells/mL, P < 0.05). In contrast, eosinophilia in sensitized wild type mice challenged with allergen (0.5 (0.16-0.65) × 10(5) cells/mL) was significantly increased in MKK3(-/-) mice (2.2 (0.9-3.5) × 10(5) cells/mL, P < 0.05). Our results suggest that asthma is associated with MKK3 over-expression in CD8(+) cells. We have also demonstrated that MKK3 may be critical for airway neutrophilia, but not eosinophilia, suggesting that this may be a target worthy of further consideration in the context of diseases associated with neutrophil activation such as severe asthma and COPD.

Modification of surface antigens in blood CD8+ T-lymphocytes in COPD: effects of smoking.

In contrast to the effects of cigarette smoke on T-lymphocyte subsets in the airways, it has not yet been determined whether smoking has immunomodulatory effects on surface antigens of peripheral blood T-lymphocytes and, if that is the case, whether these effects differ in smokers with and without chronic obstructive pulmonary disease (COPD). The present authors have, therefore, examined the expression of the surface activation marker CD28, the levels of cytotoxic effector lymphocytes (CD27-/CD45RA+) and the expression of the lung type (Tc)1-specific chemokine receptor CXCR(3)+ on peripheral blood CD8+ T-lymphocytes. The present authors have also studied the chemotactic activity of CD8+ T-lymphocytes on monocyte chemotactic protein (MCP)-1 and compared 13 nonsmoking controls, 12 smokers with COPD and 14 smokers without airflow limitation. There was a decrease in the total count of CD8+ T-cells and an increase in the CD4+/CD8+ ratio in smokers with COPD compared with smokers without COPD and controls. Expression of the Tc1-specific chemokine receptor CXCR(3)+ by CD8+ T-cells was increased in smokers with COPD compared with smokers without COPD and controls. The expression of activated and of cytotoxic effector CD8+ T-cells in smokers with and without COPD showed an increase compared with controls. CD8+ T-cells from smokers with and without COPD showed a decrease in chemotactic activity to MCP-1 compared with controls. In conclusion, chronic obstructive pulmonary disease may be a systemic immunomodulatory disease associated with the modification of surface antigens in blood CD8+ T-lymphocytes.

Cockroach allergen extract stimulates protease-activated receptor-2 (PAR-2) expressed in mouse lung fibroblast.

OBJECTIVE: To investigate whether cockroach allergen extract can stimulate Protease-activated receptor 2 (PAR-2) expressed in mouse lung fibroblast. MATERIALS: We established an immortalized lung fibroblast cell line, DM5, from PAR-2 deficient mice. By stable transfection with either an empty vector (DM5/EV) or an expression vector encoding mouse PAR-2 cDNA (DM5/Par2), a pair of lung fibroblast cell lines with or without functional PAR-2 expression were prepared. TREATMENT: The cells were exposed to cockroach allergen extract [up to 800 protein nitrogen unit (PNU)/ml], trypsin (up to 100 nM), SLIGRL agonist peptide (up to 500 microM), and trans-cinnamoyl-LIGRO agonist peptide (up to 400 microM). METHODS: The cells were loaded with Fluo-3 calcium indicator and mobilization of intracellular calcium with the stimuli was monitored by a fluorometric plate reader. Extracellular signal-regulated kinase (ERK) phosphorylation was examined by Western blot analysis using an anti-phospho ERK antibody. RESULTS: The cockroach extract induced intracellular calcium transients in a concentration dependent manner in DM5/Par2 but not in DM5/EV. The activity was abolished when the cockroach extract was heat denatured or pre-incubated with PMSF (phenylmethanesulfonyl fluoride) prior to the assay. Concomitantly, ERK phosphorylation was seen in DM5/Par2 with the cockroach extract but not with a heat-denatured extract. The responses were sensitive to an inositol-1,4,5 triphosphate antagonist (2-APB) indicating that calcium was mobilized from intracellular store. CONCLUSIONS: Cockroach allergen extract can activate PAR-2 and thereby stimulate mouse lung fibroblasts likely through protease(s). The present study proposes a potential mechanism for cockroach antigens, similar to house dust mite antigens, in the etiology of respiratory diseases.

Differential modulation of human basophil functions through prostaglandin D2 receptors DP and chemoattractant receptor-homologous molecule expressed on Th2 cells/DP2.

BACKGROUND: Both prostaglandin (PG) D receptor (DP) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells)/DP2 are high-affinity receptors for PGD2. Previous studies have demonstrated that PGD2 enhances releasability and induces CRTH2/DP2-mediated migration in human basophils, but the precise effects of PGD2 on basophils as well as receptor usage have not been fully clarified. OBJECTIVE: We comprehensively explored the roles of DP and CRTH2/DP2 in basophil functions by using selective agonists and antagonists for each receptor. METHODS: DP and CRTH2/DP2 transcripts were quantified by real-time PCR. We studied the effects of selective agonists (DP: BW245C; CRTH2/DP2: 13,14-dihydro-15-keto (DK)-PGD2) and/or antagonists (DP: BWA868C; CRTH2/DP2: ramatroban) on Ca2+ mobilization, migration, degranulation, CD11b expression and survival of human basophils. RESULTS: Basophils expressed transcripts of both DP and CRTH2/DP2, but the levels of CRTH2/DP2 transcripts were ca. 100-fold higher compared with DP transcripts. Ca2+ influx was induced in basophils by either PGD2 or DK-PGD2/CRTH2 agonist but not by BW245C/DP agonist. Basophils treated with PGD2 were completely desensitized to subsequent stimulation with DK-PGD2, but not vice versa. DK-PGD2 as well as PGD2 up-regulated CD11b expression, induced migration and enhanced degranulation, and those effects were completely antagonized by ramatroban/CRTH2 antagonist. In contrast, BW245C/DP agonist exhibited an inhibitory effect on basophil migration and IgE-mediated degranulation, and the migration inhibitory effect was effectively antagonized by BWA868C/DP antagonist. On the other hand, while PGD2 significantly shortened the basophil life-span, neither DK-PGD2/CRTH2 agonist nor BW245C/DP agonist did. CONCLUSION: CRTH2/DP2 is primarily responsible for the pro-inflammatory effects of PGD2 on human basophils, while DP introduces negative signals capable of antagonizing the effects of CRTH2/DP2 in these cells. The effects of PGD2 on longevity imply a mechanism(s) other than via DP or CRTH2/DP2. CRTH2/DP2 on basophils may afford opportunities for therapeutic targeting in allergic inflammation.

Molecular cloning and functional characterization of Cynomolgus monkey (Macaca fascicularis) CC chemokine receptor, CCR3.

We have cloned and performed the first functional characterization of the chemokine receptor, CCR3, of Cynomolgus monkey (Macaca fascicularis). The deduced amino acid sequence of the cloned Cynomolgus CCR3 was found to be more similar to that of a previously-reported Rhesus (Macaca mulatta) CCR3 (99.4%) than that of a reported Cynomolgus CCR3 (98.0%). Stably-transfected Cynomolgus CCR3 bound human eotaxin (CCL11) with similar kinetics (Kd 240 pM) and was responsive to human CCR3 ligands (eotaxin [CCL11], eotaxin-2 [CCL24], and MCP4 [CCL13]) in Ca(2+) mobilization and chemotaxis assays, thus provides a useful alternative species model system for the analysis of modulators of eotaxin--CCR3 induced signaling and activation.

Discovery and evaluation of piperidinyl carboxylic acid derivatives as potent alpha(4)beta(1) integrin antagonists.

Piperidinyl carboxylic acid-based derivatives were prepared as antagonists of the leukocyte cell adhesion process that is mediated through the interaction of the alpha(4)beta(1) integrin (VLA-4, very late antigen 4) and the vascular cell adhesion molecule 1 (VCAM-1). Compounds 2a-h inhibited the adhesion in a cell-based assay in the low and sub micromolar range, a pharmacokinetic study of 2d is reported.

Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.

The neuronal repellent Slit inhibits leukocyte chemotaxis induced by chemotactic factors.

Migration is a basic feature of many cell types in a wide range of species. Since the 1800s, cell migration has been proposed to occur in the nervous and immune systems, and distinct molecular cues for mammalian neurons and leukocytes have been identified. Here we report that Slit, a secreted protein previously known for its role of repulsion in axon guidance and neuronal migration, can also inhibit leukocyte chemotaxis induced by chemotactic factors. Slit inhibition of the chemokine-induced chemotaxis can be reconstituted by the co-expression of a chemokine receptor containing seven transmembrane domains and Roundabout (Robo), a Slit receptor containing a single transmembrane domain. Thus, there is a functional interaction between single and seven transmembrane receptors. Our results reveal the activity of a neuronal guidance cue in regulating leukocyte migration and indicate that there may be a general conservation of guidance mechanisms underlying metazoan cell migration. In addition, we have uncovered an inhibitor of leukocyte chemotaxis, and propose a new therapeutic approach to treat diseases involving leukocyte migration and chemotactic factors.

Chemokine receptor antagonism as a new therapy for multiple sclerosis.

New information about the role of tissue inflammation in the pathogenesis of multiple sclerosis (MS) has driven a search for effective and specific therapeutics that address leukocyte trafficking. These developments in understanding MS are complemented by advances in clarifying the molecular mechanisms of leukocyte extravasation and providing the knowledge base needed to modulate tissue inflammation. Of particular interest are the chemokines and their receptors. Chemokines constitute a large family of chemoattractant peptides that regulate the vast spectrum of leukocyte migration events. This review discusses MS and proposes that identifying the chemokines and receptors involved in the inflammation associated with this disorder may lead to therapeutic intervention.

The chemokine fractalkine inhibits Fas-mediated cell death of brain microglia.

Fractalkine is a CX3C-family chemokine, highly and constitutively expressed on the neuronal cell surface, for which a clear CNS physiological function has yet to be determined. Its cognate receptor, CX3CR-1, is constitutively expressed on microglia, the brain-resident macrophages; however, these cells do not express fractalkine. We now show that treatment of microglia with fractalkine maintains cell survival and inhibits Fas ligand-induced cell death in vitro. Biochemical characterization indicates that this occurs via mechanisms that may include 1) activation of the phosphatidylinositol-3 kinase/protein kinase B pathway, resulting in phosphorylation and blockade of the proapoptotic functions of BAD; 2) up-regulation of the antiapoptotic protein Bcl-xL; and 3) inhibition of the cleavage of BH3-interacting domain death agonist (BID). The observation that fractalkine serves as a survival factor for primary microglia in part by modulating the protein levels and the phosphorylation status of Bcl-2 family proteins reveals a novel physiological role for chemokines. These results, therefore, suggest that the interaction between fractalkine and CX3CR-1 may play an important role in promoting and preserving microglial cell survival in the CNS.

Alternative antigen receptor (TCR) signaling in T cells derived from ZAP-70-deficient patients expressing high levels of Syk.

ZAP-70-deficient patients present with nonfunctional CD4+ T cells in the periphery. We find that a subset of primary ZAP-70-deficient T cells, expressing high levels of the related protein-tyrosine kinase Syk, can proliferate in vitro. These cells (denoted herein as Syk(hi)/ZAP-70(-) T cells) provide a unique model in which the contribution of Syk to TCR-mediated responses can be explored in a nontransformed background. Importantly, CD3-induced responses, such as tyrosine phosphorylation of cellular substrates (LAT, SLP76, and PLC-gamma1), as well as calcium mobilization, which are defective in T cells expressing neither ZAP-70 nor Syk, are observed in Syk(hi)/ZAP-70(-) T cells. However, Syk(hi)/ZAP-70(-) T cells differ from control T cells with respect to the T cell antigen receptor (TCR)-mediated activation of the MAPK cascades: extracellular signal-regulated kinase activity and recruitment of the JNK and p38 stress-related MAPK pathways are diminished. This distinct phenotype of Syk(hi)/ZAP-70(-) T cells is associated with a profound decrease in CD3-mediated interleukin 2 secretion and proliferation relative to control T cells. Thus, ZAP-70 and Syk appear to play distinct roles in transducing a TCR-mediated signal.

NF-kappaB-dependent fractalkine induction in rat aortic endothelial cells stimulated by IL-1beta, TNF-alpha, and LPS.

Fractalkine is an endothelial cell-derived CX3C chemokine that is chemotactic mainly to mononuclear cells. Fractalkine was induced in rat aortic endothelial cells (RAEC) by interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS) transcriptionally and translationally. This induction correlated with increased NF-kappaB DNA binding activity as determined by gel mobility shift assay. Supershift assays revealed that the NF-kappaB subunits p50 and p65 were responsible for kappaB binding. Accordingly, we examined the role of NF-kappaB in fractalkine induction in RAEC through the use of an adenovirus-mediated mutant IkappaB as a specific inhibitor. Delivery of a dominant-negative form of IkappaBalpha in RAEC dramatically reduced the induction of fractalkine by these stimuli, suggesting a role for NF-kappaB activation in fractalkine induction. The inhibition of fractalkine expression by two potent NF-kappaB inhibitors, sulfasalazine and sanguinarine, further supported the central role of NF-kappaB in fractalkine transcription regulation and suggested a novel therapeutic target aimed at modulating leukocyte endothelial cell interaction.

Chemokines and their receptors in neurobiology: perspectives in physiology and homeostasis.

Chemokines are a large family of small secreted proteins (8-14 kDa) associated with the trafficking of leukocytes in physiological immunosurveillance as well as inflammatory cell recruitment in different disease processes. A limited repertoire of chemokines and their specific cognate receptors are detectable in cells of the CNS such as microglia, astrocytes and neurons under physiological conditions. Coupled with distinct patterns of ligand and receptor expression in various pathologies including multiple sclerosis, trauma, neuro-AIDS, Alzheimer's disease, stroke, neuro- and glioblastomas, such phenomena have fueled the strong belief that chemokines must fulfill significant and potentially diverse functional roles in the CNS.