Concerted expression of eotaxin-1, eotaxin-2, and eotaxin-3 in human bronchial epithelial cells

Eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 bind specifically and exclusively to CC chemokine receptor (CCR) 3, which is a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Bronchial epithelial cells represent an important source of chemokines, and thus we investigated in vitro and in vivo expression of eotaxin-2 and eotaxin-3 in bronchial epithelial cells in comparison with that of eotaxin-1. Immunohistochemistry showed increased expression of both eotaxin-2 and eotaxin-3 in addition to eotaxin-1 in asthmatics. Considerable amounts of eotaxins were secreted by bronchial epithelial lineage. As with eotaxin-1 production, generation of eotaxin-2 and eotaxin-3 by bronchial epithelial cells was up-regulated by IL-4 and IL-13, and attenuated by IFN-gamma and glucocorticoids. In addition to eotaxin-1 expression, but also eotaxin-2 and eotaxin-3 expression in the bronchial epithelium should be taken into consideration when developing the therapeutic strategies to treat eosinophilic airway diseases.     

The eotaxin chemokines and CCR3 are fundamental regulators of allergen-induced pulmonary eosinophilia

The eotaxin chemokines have been implicated in allergen-induced eosinophil responses in the lung. However, the individual and combined contribution of each of the individual eotaxins is not well defined. We aimed to examine the consequences of genetically ablating eotaxin-1 or eotaxin-2 alone, eotaxin-1 and eotaxin-2 together, and CCR3. Mice carrying targeted deletions of these individual or combined genes were subjected to an OVA-induced experimental asthma model. Analysis of airway (luminal) eosinophilia revealed a dominant role for eotaxin-2 and a synergistic reduction in eotaxin-1/2 double-deficient (DKO) and CCR3-deficient mice. Examination of pulmonary tissue eosinophilia revealed a modest role for individually ablated eotaxin-1 or eotaxin-2. However, eotaxin-1/2 DKO mice had a marked decrease in tissue eosinophilia approaching the low levels seen in CCR3-deficient mice. Notably, the organized accumulation of eosinophils in the peribronchial and perivascular regions of allergen-challenged wild-type mice was lost in eotaxin-1/2 DKO and CCR3-deficient mice. Mechanistic analysis revealed distinct expression of eotaxin-2 in bronchoalveolar lavage fluid cells consistent with macrophages. Taken together, these results provide definitive evidence for a fundamental role of the eotaxin/CCR3 pathway in eosinophil recruitment in experimental asthma. These results imply that successful blockade of Ag-induced pulmonary eosinophilia will require antagonism of multiple CCR3 ligands.     

Effect of phoshpodiesterase 4 (PDE4) inhibibtors on eotaxin expression in humen bronchial epithelial cells

The increasing number of eosinophils into bronchoaelvolar space is observed during noninfectious inflammatory lung diseases. Eotaxins (eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26) are the strongest chemotactic agents for eosinophils. Inhibitors of phosphodiesterase 4 (PDE4), the enzyme decomposing cAMP, are anti-inflammatory agents which act through cAMP elevation and inhibit numerous steps of allergic inflammation. The effect of PDE4 inhibitors on eotaxin expression is not known in details. The aim of our study was to evaluate the influence of PDE4 inhibitors: rolipram and RO-20-1724 on expression of eotaxins in bronchial epithelial cell line BEAS-2B. Cells were preincubated with PDE4 inhibitors or dexamethasone for 1 hour and then stimulated with IL-4 or IL-13 alone or in combination with TNF-α. After 48 hours eotaxin protein level was measured by ELISA and mRNA level by real time PCR. Results: PDE4 inhibitors decreased CCL11 and CCL26 expression only in cultures co-stimulated with TNF-α. In cultures stimulated with IL-4 and TNF-α rolipram and RO-20-1724 diminished CCL11 mRNA expression by 34 and 37%, respectively, and CCL26 by 43 and 47%. In cultures stimulated with IL-13 and TNF-α rolipram and RO-20-1724 decreased expression of both eotaxins by about 50%. These results were confirmed at the protein level. The effect of PDE4 inhibitors on eotaxin expression in BEAS-2B cells, in our experimental conditions, depends on TNF-α contribution.     

Eotaxin-3/CCL26 is a natural antagonist for CC chemokine receptors 1 and 5. A human chemokine with a regulatory role

Eotaxin-3 (CCL26), like eotaxin (CCL11) and eotaxin-2 (CCL24), has long been considered a specific agonist for CC chemokine receptor 3 (CCR3), attracting and activating eosinophils, basophils, and Th2 type T lymphocytes. Although not characterized extensively yet, its expression profile coincides with a potential role in allergic inflammation. We recently reported that eotaxin-3 is an antagonist for CCR2 (Ogilvie, P., Paoletti, S., Clark-Lewis, I., and Uguccioni, M. (2003) Blood 102, 789-784). In the present report, we provide evidence that eotaxin-3 acts as a natural antagonist on CCR1 and -5 as well. Eotaxin-3 bound to cells transfected with either CCR1 or -5 as well as to monocytes expressing both receptors. Further, it inhibited chemotaxis, the release of free intracellular calcium, and actin polymerization when cells were stimulated with known agonists of CCR1 and -5. An analysis of its three-dimensional structure indicated the presence of two distinct epitopes that may be involved in specific binding to CCR1, -2, -3, and -5. Taken together, our data thus indicate eotaxin-3 to be the first human chemokine that features broadband antagonistic activities, suggesting that it may have a modulatory rather than an inflammatory function. Further, eotaxin-3 may play an unrecognized role in the polarization of cellular recruitment by attracting Th2 lymphocytes as well as eosinophils and basophils via CCR3, while concomitantly blocking the recruitment of Th1 lymphocytes and monocytes via CCR1, -2, and -5.     

Unusual chemokine receptor antagonism involving a mitogen-activated protein kinase pathway

Antagonism of chemokines on chemokine receptors constitutes a new regulatory principle in inflammation. Eotaxin (CCL11), an agonist for CCR3 and an attractant of eosinophils, basophils, and Th2 lymphocytes, was shown to act as an antagonist for CCR2, which is widely expressed on leukocytes and is essential for inflammatory responses. In this report we provide direct evidence for a novel mechanism how chemokine receptor function can be arrested by endogenous ligands. We show that binding of eotaxin to CCR2 stimulates the mitogen-activated protein kinases extracellular signal-regulated kinase 1/2 (ERK1/2). Activation of the mitogen-activated protein kinase kinase 1/2-ERK pathway is indispensable for eotaxin-mediated attenuation of CCR2 function, as inhibition of ERK phosphorylation abolishes the arresting effect. ERK is also activated by CCR2 agonists, e.g., monocyte chemoattractant protein-1 (CCL2). However, the involved pathways are different, although in either case coupling of CCR2 to pertussis toxin-sensitive heterotrimeric G proteins is necessary. The results are in agreement with the view that CCR2 could assume different activation states depending on the ligand it encounters. With respect to actin polymerization and calcium mobilization, the different activation states lead to agonistic and antagonistic responses. It is conceivable that the intracellular signal transduction pathway that is activated by eotaxin could cause an attenuation of proinflammatory responses mediated by CCR2.     

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.     

CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.     

CCL21/CCR7 prevents apoptosis via the ERK pathway in human non-small cell lung cancer cells

Previously, we confirmed that C-C chemokine receptor 7 (CCR7) promotes cell proliferation via the extracellular signal-regulated kinase (ERK) pathway, but its role in apoptosis of non-small cell lung cancer (NSCLC) cell lines remains unknown. A549 and H460 cells of NSCLC were used to examine the effect of CCL21/CCR7 on apoptosis using flow cytometry. The results showed that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant decline in the percent of apoptosis. Western blot and real-time PCR assays indicated that activation of CCR7 significantly caused upregulation of anti-apoptotic bcl-2 and downregulation of pro-apoptotic bax and caspase-3, but not p53, at both protein and mRNA levels. CCR7 small interfering RNA significantly attenuated these effects of exogenous CCL21. Besides, PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished these effects of CCL21/CCR7. Coimmunoprecipitation further confirmed that there was an interaction between p-ERK and bcl-2, bax, or caspase-3, particularly in the presence of CCL21. These results strongly suggest that CCL21/CCR7 prevents apoptosis by upregulating the expression of bcl-2 and by downregulating the expression of bax and caspase-3 potentially via the ERK pathway in A549 and H460 cells of NSCLC.     

CCL21/CCR7 promotes G2/M phase progression via the ERK pathway in human non-small cell lung cancer cells

C-C chemokine receptor 7 (CCR7) contributes to the survival of certain cancer cell lines, but its role in the proliferation of human non-small cell lung cancer (NSCLC) cells remains vague. Proliferation assays performed on A549 and H460 NSCLC cells using Cell Counting Kit-8 indicated that activation of CCR7 by its specific ligand, exogenous chemokine ligand 21 (CCL21), was associated with a significant linear increase in cell proliferation with duration of exposure to CCL21. The CCL21/CCR7 interaction significantly increased the fraction of cells in the G(2)/M phase of the cell cycle as measured by flow cytometry. In contrast, CCL21/CCR7 had no significant influence on the G(0)/G(1) and S phases. Western blot and real-time PCR indicated that CCL21/CCR7 significantly upregulated expression of cyclin A, cyclin B1, and cyclin-dependent kinase 1 (CDK1), which are related to the G(2)/M phase transition. The expression of cyclin D1 and cyclin E, which are related to the G(0)/G(1) and G(1)/S transitions, was not altered. The CCL21/CCR7 interaction significantly enhanced phosphorylation of extracellular signal-regulated kinase (P-ERK) but not Akt, as measured by Western blot. LY294002, a selective inhibitor of PI3K that prevents activation of the downstream Akt, did not weaken the effect of CCL21/CCR7 on P-ERK. Coimmunoprecipitation further confirmed that there was an interaction between P-ERK and cyclin A, cyclin B1, or CDK1, particularly in the presence of CCL21. CCR7 small interfering RNA or PD98059, a selective inhibitor of MEK that disrupts the activation of downstream ERK, significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 contributes to the time-dependent proliferation of human NSCLC cells by upregulating cyclin A, cyclin B1, and CDK1 potentially via the ERK pathway.     

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1α (CCL3) whose expression was induced by the Th1 cytokines IL-1β and IFN-γ. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.     

Modulation of eotaxin-3 (CCL26) in alveolar type II epithelial cells

Airway epithelial inflammation associated with emphysema, chronic bronchitis, chronic obstructive pulmonary disease (COPD) and asthma is regulated in part by alveolar type II cell chemokine signaling. Data suggest that resident lung cells use CCR3, CCR5 and CCR2 chemokine receptor/ligand systems to regulate the profile of leukocytes recruited in disease-associated inflammatory conditions. Thus studies were designed to test whether alveolar type II cells possess a Th1-activated CCR5-ligand system that modulates the Th2-activated CCR3/eotaxin-2 (CCL24), eotaxin-3 (CCL26) chemokine systems. The A549 alveolar type II epithelial-like cell culture model was used to demonstrate that alveolar type II cells constitutively express CCR5 which may be upregulated by MIP-1alpha (CCL3) whose expression was induced by the Th1 cytokines IL-1beta and IFN-gamma. Selective down-regulation of CCL26, but not CCL24, was observed in CCL3 and IL-4/CCL3 stimulated cells. Down-regulation was reversed by anti-CCR5 neutralizing antibody treatment. Thus, one mechanism through which Th1-activated CCCR5/ligand pathways modulate Th2-activated CCR3/ligand pathways is the differential down-regulation of CCL26 expression. Results suggest that the CCR3 and CCR5 receptor/ligand signaling pathways may be important targets for development of novel mechanism-based adjunctive therapies designed to abrogate the chronic inflammation associated with airway diseases.     

Migration of eosinophils across endothelial cell monolayers: interactions among IL-5, endothelial-activating cytokines, and C-C chemokines

Eosinophils are the predominant cell type recruited in inflammatory reactions in response to allergen challenge. The mechanisms of selective eosinophil recruitment in allergic reactions are not fully elucidated. In this study, the ability of several C-C chemokines to induce transendothelial migration (TEM) of eosinophils in vitro was assessed. Eotaxin, eotaxin-2, monocyte chemotactic protein (MCP)-4, and RANTES induced eosinophil TEM across unstimulated human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner with the following rank order of potency: eotaxin approximately eotaxin-2 > MCP-4 approximately RANTES. The maximal response induced by eotaxin or eotaxin-2 exceeded that of RANTES or MCP-4. Preincubation of eosinophils with anti-CCR3 Ab (7B11) completely blocked eosinophil TEM induced by eotaxin, MCP-4, and RANTES. Activation of endothelial cells with IL-1beta or TNF-alpha induced concentration-dependent migration of eosinophils, which was enhanced synergistically in the presence of eotaxin and RANTES. Anti-CCR3 also inhibited eotaxin-induced eosinophil TEM across TNF-alpha-stimulated HUVEC. The ability of eosinophil-active cytokines to potentiate eosinophil TEM was assessed by investigating eotaxin or RANTES-induced eosinophil TEM across resting and IL-1beta-stimulated HUVEC in the presence or absence of IL-5. The results showed synergy between IL-5 and the chemokines but not between IL-5 and the endothelial activator IL-1beta. Our data suggest that eotaxin, eotaxin-2, MCP-4, and RANTES induce eosinophil TEM via CCR3 with varied potency and efficacy. Activation of HUVEC by IL-1beta or TNF-alpha or priming of eosinophils by IL-5 both promote CCR3-dependent migration of eosinophils from the vasculature in conjunction with CCR3-active chemokines.     

Small molecule receptor agonists and antagonists of CCR3 provide insight into mechanisms of chemokine receptor activation

Chemokine receptor CCR3 is highly expressed by eosinophils and signals in response to binding of the eotaxin family of chemokines, which are up-regulated in allergic disorders. Consequently, CCR3 blockade is of interest as a possible therapeutic approach for the treatment of allergic disease. We have described previously a bispecific antagonist of CCR1 and CCR3 named UCB35625 that was proposed to interact with the transmembrane residues Tyr-41, Tyr-113, and Glu-287 of CCR1, all of which are conserved in CCR3. Here, we show that cells expressing the CCR3 constructs Y113A and E287Q are insensitive to antagonism by UCB35625 and also exhibit impaired chemotaxis in response to CCL11/eotaxin, suggesting that these residues are important for antagonist binding and also receptor activation. Furthermore, mutation of the residue Tyr-113 to alanine was found to turn the antagonist UCB35625 into a CCR3 agonist. Screens of small molecule libraries identified a novel specific agonist of CCR3 named CH0076989. This was able to activate eosinophils and transfectants expressing both wild-type CCR3 and a CCR1-CCR3 chimeric receptor lacking the CCR3 amino terminus, indicating that this region of CCR3 is not required for CH0076989 binding. A direct interaction with the transmembrane helices of CCR3 was supported by mutation of the residues Tyr-41, Tyr-113, and Glu-287 that resulted in complete loss of CH0076989 activity, suggesting that the compound mimics activation by CCL11. We conclude that both agonists and antagonists of CCR3 appear to occupy overlapping sites within the transmembrane helical bundle, suggesting a fine line between agonism and antagonism of chemokine receptors.     

Production of CCL20 from lung cancer cells induces the cell migration and proliferation through PI3K pathway

Tumour inflammatory microenvironment is considered to play a role in the sensitivity of tumour cells to therapies and prognosis of patients with lung cancer. The expression of CCL20, one of the critical chemoattractants responsible for inflammation cells recruitment, has been shown overexpressed in variety of tumours. This study aimed at investigating potential mechanisms of CCL20 function and production in human non‐small cell lung cancer (NSCLC). Expression of CCL20 gene and protein in lung tissues of patients with NSCLC and NSCLC cells (A549) were determined. The interleukin (IL)‐1β‐induced signal pathways in A549 and the effect of CCL20‐induced A549 cell migration and proliferation were determined using migration assays and cell‐alive monitoring system. Mechanisms of signal pathways involved in the migration of CCL20 were also studied. We initially found that NSCLC tumour tissues markedly overexpressed CCL20 in comparison with normal lung samples. In addition, IL‐1β could directly promote CCL20 production in lung cancer cells, which was inhibited by extracellular signal‐regulated kinase (ERK)1/2 inhibitor, p38 mitogen‐activated protein kinase (p38 MARP) inhibitor or PI3K inhibitors. CCL20 promoted lung cancer cells migration and proliferation in an autocrine manner via activation of ERK1/2‐MAPK and PI3K pathways. Our data indicated that IL‐1β could stimulate CCL20 production from lung cancer cells through the activation of MAPKs and PI3K signal pathways, and the auto‐secretion of CCL20 could promote lung cancer cell migration and proliferation through the activation of ERK and PI3K signal pathways. Our results may provide a novel evidence that CCL20 could be a new therapeutic target for lung cancer.