CCL11 exacerbates colitis and inflammation-associated colon tumorigenesis.

CCL11, also known as eotaxin-1, is described as an eosinophil chemoattractant, which has been implicated in allergic and Th2 inflammatory diseases. We have reported that CCL11 is significantly increased in the serum of inflammatory bowel disease (IBD) patients, colonic eosinophils are increased and correlate with tissue CCL11 levels in ulcerative colitis patients, and CCL11 is increased in dextran sulfate sodium (DSS)-induced murine colitis. Here, we show that CCL11 is involved in the pathogenesis of DSS-induced colitis and in colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinogenesis (CAC). Ccl11-/- mice exposed to DSS then allowed to recover had significantly less body weight loss and a decrease in histologic injury versus wild-type (WT) mice. In the AOM-DSS model, Ccl11-/- mice exhibited decreased colonic tumor number and burden, histologic injury, and colonic eosinophil infiltration versus WT mice. Ccl11 is expressed by both colonic epithelial and lamina propria immune cells. Studies in bone marrow chimera mice revealed that hematopoietic- and epithelial-cell-derived CCL11 were both important for tumorigenesis in the AOM-DSS model. These findings indicate that CCL11 is important in the regulation of colitis and associated carcinogenesis and thus anti-CCL11 antibodies may be useful for treatment and cancer chemoprevention in IBD.

An Overlook to the Characteristics and Roles Played by Eotaxin Network in the Pathophysiology of Food Allergies: Allergic Asthma and Atopic Dermatitis.

Investigations revealed substantial parts accomplished by chemokines specifically eotaxins and their specific receptors. They are functionally involved in the modulation of the pathologic state of tissue inflammation which is as a result of allergic reactions. Chemokines as small proteins with approximately 8-10 kDa molecular weight are considered and fit in the bigger family of cytokines, containing basic heparin-binding polypeptide mediators. Chemokines actively interfere in the processes of selective, oriented leukocyte (including eosinophil) recruitment. As eminent from their name, more specifically, eotaxins are specialized for eosinophils' oriented locomotion toward allergic inflamed regions. To date, three members are defined for eotaxin subfamily as follows: eotaxin-1 (CCL11), eotaxin-2 (CCL24), and eotaxin-3 (CCL26), all of them bind to and activate CCR3 but have a low level of homology and appear to exhibit different physiological potentials. Allergy is described as a clinical state in which a pathologic hypersensitivity reaction is always initiated throughout an immunologic mechanism; similar to other immunologic reactions, an allergic reaction could also either be antibody or cell mediated. This type of allergic reactions occurs in all age groups and damages several different organs, having a significant impact on the emotional and social health of patients and their families and relatives. Concerning introductory comments introduced above, the authors of the present review attempted to collect and provide the latest evidences and information regarding the correlation between expression of eotaxin family members and allergy, in a wider extent, in two important allergic disorders: atopic asthma (AA) and atopic dermatitis (AD). Overall, concerning the most recent articles published within the database in the life sciences literature regarding the fundamental role(s) played by eotaxins in the pathogenesis of AA and AD, the authors of the current article propose that eotaxins (CCL11, CCL24, and CCL26) play key role(s) during symptomatic inflammatory responses raised in response to allergic crisis of these two clinical states.

Eotaxin-1 is involved in parasite clearance during chronic filarial infection.

Eosinophil migration as key feature of helminth infection is increased during infection with filarial nematodes. In a mouse model of filariasis, we investigated the role of the eosinophil-attracting chemokine Eotaxin-1 on disease outcome. BALB/c and Eotaxin-1(-/-) mice were infected with the rodent filaria Litomosoides sigmodontis, and parasitic parameters, cellular migration to the site of infection, and cellular responsiveness were investigated. We found increased parasite survival but unaffected eosinophil migration to the site of infection in Eotaxin-1(-/-) mice. Expression of CD80 and CD86 was reduced on eosinophils from Eotaxin-1(-/-) mice after in vitro TLR2 stimulation and exposure to filarial antigen, respectively, suggesting a potential reduced activation state of eosinophils in Eotaxin-1 deficient mice. We further demonstrated that macrophages from Eotaxin-1(-/-) mice produce decreased amounts of IL-6 in vitro, a cytokine found to be associated with parasite containment, suggesting possible mechanisms by which Eotaxin-1 regulates activation of inflammatory cells and thus parasite survival.

CD14+CD33+ myeloid cell-CCL11-eosinophil signature in ulcerative colitis.

This study tested the hypothesis that eotaxins (CCL11, CCL24, and CCL26) and IL-5 contribute to eosinophil recruitment to the intestine in UC and that intestinal macrophages are important producers of CCL11 in this disease. Peripheral blood and rectal biopsy samples were obtained from patients with active (n=18) and quiescent UC (n=9), and control patients (n=7). Eosinophil and macrophage levels and activation were analyzed by flow cytometry. Rectal mRNA levels of CCL11, CCL24, CCL26, and IL-5 were determined by qRT-PCR. The cellular source of CCL11 was visualized by immunofluorescence analyses. Eosinophil numbers were elevated in the blood and rectum of active and quiescent UC patients compared with controls. Levels of activated eosinophils (CD66b(high)) correlated with disease severity. Rectal CCL11, CCL24, and CCL26 mRNA levels were increased in active UC, whereas only CCL11 was elevated in quiescent UC. Levels of CCL11, but not CCL24 and CCL26, positively correlated with eosinophil numbers. Numbers of CD14(+)CD33(+) cells correlated with CCL11 and eosinophil levels. Immunofluorescence analyses revealed the presence of CD14(+)CCL11(+) mononuclear cells in colonic biopsies in UC. These results support the hypothesis that CCL11 contributes to eosinophil recruitment in UC and that intestinal myeloid cells are a source of CCL11. Interestingly, rectal levels of CCL24, CCL26, and IL-5 only increase during active UC, coinciding with further elevation of eosinophil numbers and with the activation of rectal eosinophils. In conclusion, there is a link among CD14(+)CD33(+) myeloid cells, CCL11, and eosinophils in adult UC.

Control of extravillous trophoblast function by the eotaxins CCL11, CCL24 and CCL26.

STUDY QUESTION: What are the effects of the eotaxin group of chemokines (CCL11, CCL24 and CCL26) on extravillous trophoblast (EVT) functions important during uterine decidual vessel remodelling? SUMMARY ANSWER: CCL11, CCL24 and CCL26 can regulate EVT migration, invasion and adhesion, highlighting a potential regulatory role for these chemokines during uterine decidual spiral arteriole remodelling in the first trimester of human pregnancy. WHAT IS KNOWN ALREADY: A successful human pregnancy depends on adequate remodelling of the uterine decidual spiral arterioles, a process carried out by EVT which invade from the placenta. The invasion by EVT into the maternal uterine decidual vessels is regulated by the interaction of many factors including members of the chemokine subfamily of cytokines. STUDY DESIGN, SIZE, DURATION: This study used the HTR8/SVneo cell line as a model for invasive EVT. All experiments were repeated on at least three separate occasions. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effect of recombinant human CCL11, CCL24 and CCL26 on EVT migration and invasive potential was measured using the xCELLigence real-time system, wound-healing and Matrigel invasion assays, zymography to measure MMP activity and reverse zymography to measure TIMP activity. A commercially available adhesion assay was used to assess EVT adhesion to extracellular matrix proteins. MAIN RESULTS AND THE ROLE OF CHANCE: All the three eotaxins were found to significantly stimulate migration of the EVT-derived cell line HTR8/SVneo (P < 0.05) with no significant changes in cell number following treatment with each chemokine (P > 0.05). All the three eotaxins significantly increased HTR8/SVneo invasion (P < 0.05) and MMP2 activity (P < 0.05) without any effects on TIMP2 activity (P > 0.05). All the three eotaxins significantly increased HTR8/SVneo cell binding to collagen IV (P < 0.05) and fibronectin (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This work has been conducted in vitro with a commonly used cell line model of EVT, HTR8/SVneo. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first to comprehensively examine the effects of the eotaxin group of chemokines on EVT functions and demonstrates that all the three eotaxins have the ability to regulate EVT functions critical to their role in vessel remodelling. This identifies a new role for the eotaxin group of chemokines during placentation.

CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2.

CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5-oxo-eicosatetraenoate-induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.

IL-4 engagement of the type I IL-4 receptor complex enhances mouse eosinophil migration to eotaxin-1 in vitro.

BACKGROUND: Previous work from our laboratory demonstrated that IL-4Rα expression on a myeloid cell type was responsible for enhancement of Th2-driven eosinophilic inflammation in a mouse model of allergic lung inflammation. Subsequently, we have shown that IL-4 signaling through type I IL-4 receptors on monocytes/macrophages strongly induced activation of the IRS-2 pathway and a subset of genes characteristic of alternatively activated macrophages. The direct effect(s) of IL-4 and IL-13 on mouse eosinophils are not clear. The goal of this study was determine the effect of IL-4 and IL-13 on mouse eosinophil function. METHODS: Standard Transwell chemotaxis assay was used to assay migration of mouse eosinophils and signal transduction was assessed by Western blotting. RESULTS: Here we determined that (i) mouse eosinophils express both type I and type II IL-4 receptors, (ii) in contrast to human eosinophils, mouse eosinophils do not chemotax to IL-4 or IL-13 although (iii) pre-treatment with IL-4 but not IL-13 enhanced migration to eotaxin-1. This IL-4-mediated enhancement was dependent on type I IL-4 receptor expression: γC-deficient eosinophils did not show enhancement of migratory capacity when pre-treated with IL-4. In addition, mouse eosinophils responded to IL-4 with the robust tyrosine phosphorylation of STAT6 and IRS-2, while IL-13-induced responses were considerably weaker. CONCLUSIONS: The presence of IL-4 in combination with eotaxin-1 in the allergic inflammatory milieu could potentiate infiltration of eosinophils into the lungs. Therapies that block IL-4 and chemokine receptors on eosinophils might be more effective clinically in reducing eosinophilic lung inflammation.

CC and CXC chemokines induce airway smooth muscle proliferation and survival.

The increase in airway smooth muscle (ASM) mass is a major structural change in asthma. This increase has been attributed to ASM cell (ASMC) hyperplasia and hypertrophy. The distance between ASMC and the epithelium is reduced, suggesting migration of smooth muscle cells toward the epithelium. Recent studies have suggested a role of chemokines in ASMC migration toward the epithelium; however, chemokines have other biological effects. The objective of the current study is to test the hypothesis that chemokines (eotaxin, RANTES, IL-8, and MIP-1α) can directly influence ASMC mass by increasing the rate of proliferation or enhancing the survival of these cells. Human ASMCs were exposed to different concentrations of eotaxin, RANTES, IL-8, or MIP-1α. To test for proliferation, matched control and stimulated ASMC were pulsed with [(3)H]thymidine, or ASMCs were stained with BrdU and then analyzed with flow cytometry. Apoptosis was measured using Annexin V staining and flow cytometry. Expression of phosphorylated p42/p44 and MAPKs was assessed by Western blot. In a concentration-dependent manner, chemokines including eotaxin, RANTES, IL-8, and MIP-1α increased ASMC's [(3)H]thymidine incorporation and DNA synthesis. IL-8, eotaxin, and MIP-1α decreased the rate of apoptosis of ASMCs compared with the matched controls. A significant increase in phosphorylated p42/p44 MAPKs was seen after treating ASMCs with RANTES and eotaxin. Moreover, inhibition of p42/p44 MAPK phosphorylation reduced the level of chemokine-induced ASM proliferation. We conclude that chemokines might contribute to airway remodeling seen in asthma by enhancing the number and survival of ASMCs.

Phosphoinositide 3-kinase gamma mediates chemotactic responses of human eosinophils to platelet-activating factor.

BACKGROUND: Eosinophils are characteristic participants in allergic inflammation. The intracellular signalling mechanisms involved in the migration of eosinophils to sites of allergic inflammation are poorly understood. Chemotactic responses of eosinophils to platelet-activating factor (PAF), but not eotaxin, have been demonstrated to be dependent upon the activation of phosphoinositide 3-kinase (PI3K) but the specific isoform of PI3K involved has not been identified. OBJECTIVE: To determine the roles of the leukocyte-specific PI3K gamma and PI3K delta isoforms of PI3K in PAF-induced chemotaxis of human eosinophils. METHODS: Chemotactic responses of the EoL-1 eosinophilic cell line and human peripheral blood eosinophils were measured. The effects of a PI3K gamma-selective inhibitor (5-[2,2-difluorobenzo(1,3)dioxol-5-ylmethylene]-thiazolidine-2,4-dione; AS604850) and gene knock-down of PI3K gamma and PI3K delta on chemotactic responses were determined. RESULTS: AS604850 caused a concentration-dependent suppression of chemotactic responses of EoL-1 cells and blood eosinophils to PAF but not eotaxin. Specific siRNAs reduced the expression of PI3K gamma and PI3K delta in EoL-1 cells. Knock-down of PI3K gamma by siRNA resulted in a 75% inhibition of the chemotactic response to PAF but had no effect on the response to eotaxin. Knock-down of endogenous PI3K delta by siRNA resulted in a 38% inhibition of the chemotactic response to PAF but had no effect on the response to eotaxin. CONCLUSION: PI3K gamma plays a major role in the induction of chemotaxis in PAF-stimulated eosinophils, while PI3K delta plays a lesser role. Interventions which reduce the activity of PI3K gamma may have therapeutic potential in allergic diseases.

Targeting CCL11 in the treatment of ovarian cancer.

IMPORTANCE OF THE FIELD: The chemokine network, comprised of mediators of inflammation, has been implicated in the development of a number of human cancers. The eosinophil chemoattractant CCL11 was recently shown to play a role in the development of ovarian cancer. Here we review findings regarding CCL11 and discuss its use as a target in the treatment of ovarian cancer. AREAS COVERED IN THIS REVIEW: We review published findings related to the physiological actions of CCL11, its tumourigenic effects, the chemokine network and inflammatory response present in ovarian cancer, and the current state of therapeutics targeting CCL11 and its receptors. Findings published within the last 10 years receive particular attention. WHAT THE READER WILL GAIN: An overview of the emerging role of the chemokine network in malignancy and a review of the role of CCL11 in ovarian tumourigenesis. The reader will be presented with a description of the unique aspects of CCL11 action and the inflammatory environment in the setting of ovarian malignancy that make this chemokine an attractive target for intervention. TAKE HOME MESSAGE: Targeting CCL11 and its receptors through the use of monoclonal antibodies and small-molecule inhibitors may represent a beneficial new avenue of ovarian cancer treatment.

Interleukin-4 induces expression of eotaxin in endometriotic stromal cells.

OBJECTIVE: To study the relationship between eotaxin and interleukin-4 (IL-4) in the pathophysiology of endometriosis. DESIGN: Comparative and laboratory study. SETTING: University teaching hospital reproductive endocrinology and infertility practice. PATIENT(S): Ectopic endometrial tissues were collected from women with endometriosis. INTERVENTION(S): Ectopic endometrial stromal cells (ESCs) were isolated and cultured with IL-4. Ectopic endometriotic tissues were immunostained for eotaxin and IL-4. MAIN OUTCOME MEASURE(S): Gene expression of eotaxin was determined by standard and real-time reverse-transcriptase polymerase chain reaction. Secretion of eotaxin from ESC was measured using specific ELISA. The immunostained sections were examined. RESULT(S): Interleukin-4 (IL-4) increased mRNA expression and protein secretion of eotaxin from ESC in a dose-dependent manner. Immunohistochemical analysis showed that eotaxin-positive cells colocalized with IL-4-positive cells and accumulated around the blood vessels in the stroma of endometriotic tissue. CONCLUSION(S): IL-4 induces eotaxin in ESCs, which might promote angiogenesis and the subsequent development of endometriosis.

Eotaxin increases monolayer permeability of human coronary artery endothelial cells.

OBJECTIVE: The objective of this study was to determine the effects and molecular mechanisms of eotaxin, a newly discovered chemokine (CCL11), on endothelial permeability in the human coronary artery endothelial cells (HCAECs). METHODS AND RESULTS: Cells were treated with eotaxin, and the monolayer permeability was studied by using a costar transwell system with a Texas Red-labeled dextran tracer. Eotaxin significantly increased monolayer permeability in a concentration-dependent manner. In addition, eotaxin treatment significantly decreased the mRNA and protein levels of endothelial junction molecules including zonula occludens-1 (ZO-1), occludin, and claudin-1 in a concentration-dependent manner as determined by real-time RT-PCR and Western blot analysis, respectively. Increased oxidative stress was observed in eotaxin-treated HCAECs by analysis of cellular glutathione levels. Furthermore, eotaxin treatment substantially activated the phosphorylation of MAPK p38. HCAECs expressed CCR3. Consequently, antioxidants (ginkgolide B and MnTBAP), specific p38 inhibitor SB203580, and anti-CCR3 antibody effectively blocked the eotaxin-induced permeability increase in HCAECs. Eotaxin also increased the phosphorylation of Stat3 and nuclear translocation of NF-kappaB in HCAECs. CONCLUSIONS: Eotaxin increases vascular permeability through CCR3, the downregulation of tight junction proteins, increase of oxidative stress, and activation of MAPK p38, Stat3, and NF-kB pathways in HCAECs.

The roles of eotaxin and the STAT6 signalling pathway in eosinophil recruitment and host resistance to the nematodes Nippostrongylus brasiliensis and Heligmosomoides bakeri.

Expulsion of adult Nippostrongylus brasiliensis worms from the small intestine is profoundly impaired in signal transducer and activator of transcription (STAT)6-deficient mice. IL-5 transgenic (Tg) mice with constitutive eosinophilia show profound early resistance in the skin and/or later pre-lung phase of primary infections with N. brasiliensis. This study was designed to assess the importance of the eosinophil chemokine eotaxin and the STAT6/interleukin (IL)-4/IL-13 signalling pathway in early resistance to N. brasiliensis. Eosinophil recruitment into the skin following injection of N. brasiliensis larvae was reduced in STAT6- or eotaxin-deficient/IL-5 Tg double mutant mice. While ablation of eotaxin did not impair resistance in the pre-lung phase of N. brasiliensis infections in IL-5 Tg mice, elimination of STAT6 caused a modest reduction in resistance in both primary and secondary infections on this genetic background. STAT6(-/-)-, IL-13(-/-)- and IL-4Ralpha(-/-)-deficient single mutant and IL-13(-/-)/IL-4Ralpha(-/-) double mutant mice were more susceptible than WT mice during the pre-lung phase of secondary N. brasiliensis infections. In contrast, primary or secondary resistance were unaffected at either the pre-lung or gut stages of infection in eotaxin(-/-) single mutant mice. STAT6(-/-) and eotaxin(-/-) mice with or without the IL-5 transgene, were no more susceptible than WT or IL-5 Tg mice to protracted primary infections with Heligmosomoides bakeri, a parasitic nematode that is restricted to the gut. Our data suggest that parasitic nematodes that transit through the skin and lungs en route to the gut may be susceptible to early (pre-lung) innate and adaptive immune mechanisms that are dependent on the STAT6/IL-4/IL-13 signalling pathway, and this may be important for the development of effective therapies and vaccines.

Role of eotaxin-1 signaling in ovarian cancer.

PURPOSE: Tumor cell growth and migration can be directly regulated by chemokines. In the present study, the association of CCL11 with ovarian cancer has been investigated. EXPERIMENTAL DESIGN AND RESULTS: Circulating levels of CCL11 in sera of patients with ovarian cancer were significantly lower than those in healthy women or women with breast, lung, liver, pancreatic, or colon cancer. Cultured ovarian carcinoma cells absorbed soluble CCL11, indicating that absorption by tumor cells could be responsible for the observed reduction of serum level of CCL11 in ovarian cancer. Postoperative CCL11 levels in women with ovarian cancer negatively correlated with relapse-free survival. Ovarian tumors overexpressed three known cognate receptors of CCL11, CC chemokine receptors (CCR) 2, 3, and 5. Strong positive correlation was observed between expression of individual receptors and tumor grade. CCL11 potently stimulated proliferation and migration/invasion of ovarian carcinoma cell lines, and these effects were inhibited by neutralizing antibodies against CCR2, CCR3, and CCR5. The growth-stimulatory effects of CCL11 were likely associated with activation of extracellular signal-regulated kinase 1/2, MEK1, and STAT3 phosphoproteins and with increased production of multiple cytokines, growth factors, and angiogenic factors. Inhibition of CCL11 signaling by the combination of neutralizing antibodies against the ligand and its receptors significantly increased sensitivity to cisplatin in ovarian carcinoma cells. CONCLUSION: We conclude that CCL11 signaling plays an important role in proliferation and invasion of ovarian carcinoma cells and CCL11 pathway could be targeted for therapy in ovarian cancer. Furthermore, CCL11 could be used as a biomarker and a prognostic factor of relapse-free survival in ovarian cancer.

n-Nonanoyl-CCL14 (NNY-CCL14), a novel inhibitor of allergic airway inflammation is a partial agonist of human CCR2.

BACKGROUND: Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n-Nonanoyl-CC chemokine ligand 14 (NNY-CCL14), a modified analog of the naturally occurring chemokine CCL14(9-74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5. AIM OF THE STUDY: As CCL2 and its receptor CCR2 have been shown to play important roles in the development of Th2 inflammation, we further evaluated the effects of NNY-CCL14 treatment on CCL2-mediated activation of CCR2. METHODS: Effects of NNY-CCL14 treatment were studied on cell lines transfected with human CCR2 and primary leukocytes. Functional effects were assessed by calcium efflux assays, flow cytometry and chemotaxis. RESULTS: Prestimulation with NNY-CCL14 desensitized CCR2-mediated responses to further stimulation with its selective ligand CCL2. No significant internalization of CCR2 was observed when the cells were stimulated with NNY-CCL14, even at concentrations eliciting maximal [Ca(2+)]i mobilization. Above all, NNY-CCL14 pretreatment blocked CCL2-induced chemotaxis of monocytes. CONCLUSIONS: This study demonstrates that NNY-CCL14 is a partial agonist of CCR2, inhibiting responses of monocytes to the CCR2-selective ligand CCL2. NNY-CCL14 attenuates CCR2-mediated responses by rapidly desensitizing the receptor and preventing chemotaxis, although it is able to induce calcium mobilization but does not lead to CCR2 internalization. Hence this study provides further insights into the possible mechanisms of action of NNY-CCL14, which interacts with multiple chemokine receptors inhibiting the migration and activation of different cell populations involved, thus acting as a potential therapeutic compound to alleviate allergic inflammation.

CCL11 and GM-CSF differentially use the Rho GTPase pathway to regulate motility of human eosinophils in a three-dimensional microenvironment.

Asthma is a common disease that causes considerable morbidity. Increased numbers of airway eosinophils are a hallmark of asthma. Mechanisms controlling the entry of eosinophils into asthmatic lung have been intensively investigated, but factors regulating migration within the tissue microenvironment are less well understood. We modeled this by studying chemoattractant and growth factor-mediated human eosinophil migration within a three-dimensional collagen matrix. Stimulation with GM-CSF induced dose-dependent, random migration with a maximum of 77 +/- 4.7% of cells migrating. In contrast, CCL11 and C5a caused a more modest although significant degree of migration (19 +/- 1.8% and 20 +/- 2.6%, respectively). Migration to GM-CSF was partially dependent on Ca(2+) and alpha(M)beta(2) integrins. The Rho family of small GTPases regulates intracellular signaling of cell migration. GM-CSF-induced migration was only partially dependent on Rho kinase/Rho-associated kinase (ROCK) and was independent of RhoA activation. In contrast, CCL11-induced migration was fully dependent on both RhoA and ROCK. Activation of RhoA was therefore neither necessary nor sufficient to cause eosinophil migration in a three-dimensional collagen environment. This study suggests that eosinophil growth factors are likely to be required for eosinophil migration within the bronchial mucosa, and this involves signal transduction pathways distinct from those used by G protein-associated chemoattractants.

Pulmonary eosinophilia requires interleukin-5, eotaxin-1, and CD4+ T cells in mice immunized with respiratory syncytial virus G glycoprotein.

Severe illness, type 2 cytokine production, and pulmonary eosinophilia are adverse immune responses resulting from respiratory syncytial virus (RSV) challenge of vvGs-immunized mice. We have shown IL-4 and IL-13 activity must be simultaneously inhibited to reduce disease severity. We now address the contributions of IL-5, eotaxin-1, and CD4+ and CD8+ T cells to the induction of disease-enhancing immune responses. Depletion of CD4+ T cells during immunization prevented IL-4, IL-13, and eotaxin-1 production, diminished eosinophilia, and reduced weight loss. Conversely, CD8+ T cell depletion did not decrease eosinophilia, weight loss, or type 2 cytokines but did dramatically reduce mucus production and increase eotaxin production. Anti-IL-5 administration at immunization or challenge significantly decreased pulmonary eosinophilia. Strikingly, there were not concomitant decreases in weight loss. Following RSV challenge eotaxin-1-deficient mice immunized with vvGs exhibited significantly less eosinophilia without decreased weight loss or type 2 cytokine production. We conclude CD4+ T cell production of IL-5 and induction of eotaxin-1 are required for vvGs-induced eosinophilia following RSV challenge, while CD8+ T cells appear to down-regulate eotaxin-1 and mucus production. In summary, we demonstrate that pulmonary eosinophilia 1) is a by-product of memory CD4+ T cell activation, 2) does not necessarily correlate with mucus production, and, most importantly, 3) is not required for the RSV G-induced illness in mice. These findings have important implications for the evaluation of candidate RSV vaccines.

A phosphosite screen identifies autocrine TGF-beta-driven activation of protein kinase R as a survival-limiting factor for eosinophils.

The differential usage of signaling pathways by chemokines and cytokines in eosinophils is largely unresolved. In this study, we investigate signaling similarities and differences between CCL11 (eotaxin) and IL-5 in a phosphosite screen of human eosinophils. We confirm many previously known pathways of cytokine and chemokine signaling and elucidate novel phosphoregulation in eosinophils. The signaling molecules that were stimulated by both agents were members of the ERK1/2 and p38 MAPK pathways and their downstream effectors such as RSK and MSK1/2. Both agents inhibited S6 kinase, protein kinase Cepsilon, and glycogen synthase kinase 3 alpha and beta. The molecules that were differentially regulated include STATs and protein kinase R (PKR). One of the chief findings in this investigation was that PKR and eukaryotic initiation factor 2alpha are phosphorylated under basal conditions in eosinophils and neutrophils. This basal phosphorylation was linked to autocrine secretion of TGF-beta in eosinophils. TGF-beta directly activates PKR in eosinophils. Basal phosphorylation of PKR was inhibited by incubation of eosinophils with a neutralizing anti-TGF-beta Ab suggesting its physiological importance. We show that inhibition of PKR activity prolongs eosinophil survival. The eosinophil survival factor IL-5 strongly suppresses phosphorylation of PKR. The biological relevance of IL-5 inhibition of phospho-PKR was established by the observation that ex vivo bone marrow-derived eosinophils from OVA-immunized mice had no PKR phosphorylation in contrast to the high level of phosphorylation in sham-immunized mice. Together, our findings suggest that survival of eosinophils is in part controlled by basal activation of PKR through autocrine TGF-beta and that this could be modulated by a Th2 microenvironment in vivo.

Intravascular inactivation of CCR5 by n-Nonanoyl-CC chemokine ligand 14 and inhibition of allergic airway inflammation.

Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive, therapeutic strategy. Recently, we have shown that n-Nonanoyl (NNY)-CCL14 internalizes and desensitizes human (h)CCR3, resulting in the inactivation of eosinophils. In this study, we investigated the interaction of NNY-CCL14 with CCR1 and CCR5 and the relevance of these NNY-CCL14 receptors on its in vivo effects in allergic airway inflammation. NNY-CCL14 has inactivating properties on CCR1(+) and CCR5(+) cell lines and primary leukocytes. It desensitizes hCCR1- and hCCR5-mediated calcium release and internalizes these receptors from the cellular surface. Treatment of OVA-sensitized BALB/c mice with NNY-CCL14 resulted in reduced pulmonary inflammation. Above all, it is demonstrated that systemic treatment with NNY-CCL14 down-modulates CCR5 from the surface of lymphocytes in vivo. Although NNY-CCL14 acts on murine lymphocytes and internalizes CCR5, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. Therefore, the inhibitory effects of NNY-CCL14 in murine models of allergic airway inflammation can be assigned to its interaction with CCR5. The presented results substantiate the relevance of CCR5 as a target for allergic airway inflammation.