Potent Eosinophil Chemoattractant Research Articles

Two Potent OXE-R Antagonists: Assignment of Stereochemistry

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is formed by the oxidation of 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE), which is a major metabolite of enzymatic oxidation of arachidonic acid (AA). 5-Oxo-ETE is the most potent lipid chemoattractant for human eosinophils. Its actions are mediated by the selective OXE receptor, which is therefore an attractive target in eosinophilic diseases such as allergic rhinitis and asthma. Recently, we have reported two excellent OXE receptor antagonists that have IC50 values at low nanomolar concentrations. Each of these antagonists has a chiral center, and the isolation of the individual enantiomers by chiral high-performance liquid chromatography (HPLC) revealed that in each case one enantiomer is over 300 times more potent than the other. To unambiguously assign the stereochemistry of these enantiomers and to provide access to larger amounts of the active compounds for biological testing, we report here their total synthesis.

From airway inflammation to inflammatory bowel disease: Eotaxin-1, a key regulator of intestinal inflammation

Eotaxin-1 (CCL-11) is a potent eosinophil chemoattractant that is considered a major contributor to tissue eosinophilia. Elevated eotaxin-1 levels have been described in various pathologic conditions, ranging from airway inflammation, to Hodgkin lymphoma, obesity and coronary artery disease. The main receptor for eotaxin-1 is CCR3; however, recent evidence indicates that eotaxin-1 may also bind to other receptors expressed by various cell types, suggesting a more widespread regulatory role for eotaxin-1 beyond the recruitment of eosinophils. Eotaxin-1 is also strongly associated with various gastrointestinal (GI) disorders. Although the etiology of inflammatory bowel disease (IBD) is still unknown, eotaxin-1 may play a key role in the development of mucosal inflammation. In this review, we summarize the biological context and effects of eotaxin-1, as well as its potential role as a therapeutic target, with a special focus on gastrointestinal inflammation.

Fumaric acid attenuates the eotaxin-1 expression in TNF-α-stimulated fibroblasts by suppressing p38 MAPK-dependent NF-κB signaling

Eotaxin-1 is a potent chemoattractant for eosinophils and a critical mediator during the development of eosinophilic inflammation. Fumaric acid is an intermediate product of the citric acid cycle, which is source of intracellular energy. Although fumaric acid ameliorates psoriasis and multiple sclerosis, its involvement in eotaxin-1-mediated effects has not been assessed. In this study, we investigated the effects of fumaric acid on eotaxin-1 expression in a mouse fibroblast cell line. We found that fumaric acid significantly inhibited tumor necrosis factor-α (TNF-α-induced eotaxin-1 expression. This fumaric acid effect was mediated through the inhibition of p38 mitogen-activated protein kinase (MAPK)-dependent nuclear factor (NF)-κB signaling. We also found that fumaric acid operates downstream of MEKK3 during TNF-α-induced NF-κB signaling, which upregulated eotaxin-1 expression. In addition, fumaric acid attenuated expression of CC-chemokine receptor 3 (CCR3), an eotaxin-1 receptor, and adhesion molecules that play important roles in eosinophil binding to induce allergic inflammation. Taken together, these findings indicate that inhibiting TNF-α-induced eotaxin-1 expression by fumaric acid occurs primarily through suppression of NF-κB signaling, which is mediated by inhibiting p38 MAPK and suggest that fumaric acid may be used as a complementary treatment option for eotaxin-1-mediated diseases.

A role for Galectin‐9 in neutrophil trafficking

Galectin‐9 (Gal‐9) was initially characterized as a potent eosinophil chemoattractant but has subsequently been found to have immunosuppressive effects in models of collagen‐ induced arthritis and carageenan paw oedema. Its effects on neutrophil recruitment and behavior during inflammation have not been thoroughly investigated. Here we show that Gal‐9 binds readily to the surface of freshly isolated PMN and binding is not augmented upon activation with PAF but is sensitive to lactose addition. Gal‐9 binding to endothelial cells is low in comparison to PMN and is not modulated by endothelial activation by TNFα. Neutrophils were found to transmigrate to Gal‐9 under static conditions with peak recruitment observed at 10nM. This concentration was not however found to be directly chemotactic for neutrophils in a Boyden chamber assay. Pre‐incubation of PMN with Gal‐9 significantly increased neutrophil transmigration whilst pre‐incubation of HUVEC with Gal‐9 was without effect. At concentrations active in the flow chamber, Gal‐9 did not significantly alter PMN or endothelial adhesion molecule expression (active CD11b, L‐selectin shedding, E‐selectin, VCAM‐1, ICAM‐1). In conclusion, these data indicate that neutrophils rather than endothelial cells are target cells for Gal‐9 and that Gal‐9 promotes neutrophil transmigration during acute inflammation indicating a novel role for this protein.This work was supported by a Non‐Clinical Career Development Fellowship from Arthritis Research‐UK (18103).

Expression of galectin-9 by IFN-γ stimulated human nasal polyp fibroblasts through MAPK, PI3K, and JAK/STAT signaling pathways

Galectin-9 exhibited potent and selective eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo. Nasal polyposis is a chronic inflammatory disease of the upper airway characterized by the marked presence of inflammatory cells, particularly eosinophils. Thus, galectin-9 may be implicated in the pathogenesis of nasal polyposis. The study was designed to investigate whether interferon-gamma (IFN-γ) can induce the augmentation of galectin-9 expression and induce the expression of galectin-9 in nasal polyps. We examined the correlation between galectin-9 expression and eosinophil infiltration in nasal polyps. In addition, we identified the signaling pathways involved in the elevation of galectin-9 expression in response to IFN-γ. Our data demonstrate that the involvement of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3 phosphate kinase (PI3K), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) may play important roles in the selective recruitment of eosinophils in nasal polyp tissues through the production of galectin-9. These findings suggest that galectin-9 expression is associated with eosinophil infiltration in polyps of patients with nasal polyposis.

Differences in Eotaxin-3 Expression After Stimulation With IL-13 and IL-4 in Esophageal Squamous Cell Lines From Patients With Eosinophilic Esophagitis and GERD.

Introduction: In eosinophilic esophagitis (EoE), food and other allergens appear to activate Th2 cells to release IL-13 and IL-4, cytokines that can induce esophageal squamous epithelial cells to produce the potent eosinophil chemoattractant eotaxin-3. GERD also can cause an eosinophilic infiltration of the esophagus, usually milder than that seen in EoE, through mechanisms that are not clear. In earlier studies, we showed that IL-13 and IL-4 caused a marked increase in eotaxin-3 protein secretion in an esophageal squamous cell line derived from a patient with EoE, but only a minimal increase in eotaxin-3 protein secretion in esophageal squamous cell lines derived from GERD patients. To explore this mechanism further, we studied the effects of IL-13 and IL-4 on transcriptional activation of eotaxin-3 in esophageal squamous cell lines from patients with EoE and GERD. Methods: Two telomerase-immortalized, non-neoplastic esophageal squamous cell lines derived from patients with GERD (NES-B10T & NES-G4T), and one derived from a patient with EoE (EoE1-T) were stimulated with IL-13 (100 ng/ml) or IL-4 (1 ng/ml) for times ranging from 24-48 hours. Eotaxin-3 mRNA expression levels were determined by RT-PCR, and an eotaxin-3 promoter (-800bp) construct attached to a luciferase reporter was used to measure transcriptional activity. Results: In the GERD cells, IL-13 and IL-4 induced only slight increases in eotaxin-3 mRNA expression levels at 24 and 48 hours. In contrast, both cytokines caused a marked increase in eotaxin-3 mRNA expression levels at 24 and 48 hours in the EoE cells. Both IL-13 and IL-4 significantly increased eotaxin-3 promoter activity in all 3 cell lines. There was no significant difference in the degree of eotaxin-3 promoter induction by IL-13 and IL-4 between the two GERD cell lines (NES-G4T cells IL-13: 431 ± 42.5 SEM RLUs % control, IL-4: 279± 38; NES-B10T cells IL-13: 554 ± 109, IL-4: 463 ± 54). In contrast, the cytokine-stimulated increase in eotaxin-3 promoter activity in the EoE1 cells (IL-13: 1522 ± 225, p<0.01; IL-4: 1714 ± 385, p<0.01) was significantly greater than that in the two GERD cell lines. Conclusions: In an EoE esophageal squamous cell line, stimulation with IL-13 and IL-4 caused a far greater increase in eotaxin-3 mRNA expression and promoter transcriptional activity than in esophageal squamous cell lines from GERD patients. These observations suggests that the esophagus of patients with EoE produces more eotaxin3 in response to Th2 cytokine stimulation than the esophagus of patients with GERD, and that differences among patients in the degree of cytokine-stimulated production of eotaxin3 by esophageal squamous cells might underlie the degree of esophageal infiltration by activated eosinophils.

Markers of Tyrosine Kinase Activity for Diagnosis and Treatment Response in Eosinophilic Esophagitis: A Pilot Study of pERK 1/2 and pSTAT5

Introduction: In eosinophilic esophagitis (EoE), food and other allergens appear to activate Th2 cells to release IL-13 and IL-4, cytokines that can induce esophageal squamous epithelial cells to produce the potent eosinophil chemoattractant eotaxin-3. GERD also can cause an eosinophilic infiltration of the esophagus, usually milder than that seen in EoE, through mechanisms that are not clear. In earlier studies, we showed that IL-13 and IL-4 caused a marked increase in eotaxin-3 protein secretion in an esophageal squamous cell line derived from a patient with EoE, but only a minimal increase in eotaxin-3 protein secretion in esophageal squamous cell lines derived from GERD patients. To explore this mechanism further, we studied the effects of IL-13 and IL-4 on transcriptional activation of eotaxin-3 in esophageal squamous cell lines from patients with EoE and GERD. Methods: Two telomerase-immortalized, non-neoplastic esophageal squamous cell lines derived from patients with GERD (NES-B10T & NES-G4T), and one derived from a patient with EoE (EoE1-T) were stimulated with IL-13 (100 ng/ml) or IL-4 (1 ng/ml) for times ranging from 24-48 hours. Eotaxin-3 mRNA expression levels were determined by RT-PCR, and an eotaxin-3 promoter (-800bp) construct attached to a luciferase reporter was used to measure transcriptional activity. Results: In the GERD cells, IL-13 and IL-4 induced only slight increases in eotaxin-3 mRNA expression levels at 24 and 48 hours. In contrast, both cytokines caused a marked increase in eotaxin-3 mRNA expression levels at 24 and 48 hours in the EoE cells. Both IL-13 and IL-4 significantly increased eotaxin-3 promoter activity in all 3 cell lines. There was no significant difference in the degree of eotaxin-3 promoter induction by IL-13 and IL-4 between the two GERD cell lines (NES-G4T cells IL-13: 431 ± 42.5 SEM RLUs % control, IL-4: 279± 38; NES-B10T cells IL-13: 554 ± 109, IL-4: 463 ± 54). In contrast, the cytokine-stimulated increase in eotaxin-3 promoter activity in the EoE1 cells (IL-13: 1522 ± 225, p<0.01; IL-4: 1714 ± 385, p<0.01) was significantly greater than that in the two GERD cell lines. Conclusions: In an EoE esophageal squamous cell line, stimulation with IL-13 and IL-4 caused a far greater increase in eotaxin-3 mRNA expression and promoter transcriptional activity than in esophageal squamous cell lines from GERD patients. These observations suggests that the esophagus of patients with EoE produces more eotaxin3 in response to Th2 cytokine stimulation than the esophagus of patients with GERD, and that differences among patients in the degree of cytokine-stimulated production of eotaxin3 by esophageal squamous cells might underlie the degree of esophageal infiltration by activated eosinophils.

5-Oxo-ETE is a major oxidative stress-induced arachidonate metabolite in B lymphocytes

B lymphocytes convert arachidonic acid (AA) to the 5-lipoxygenase products leukotriene B4 (LTB4) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) when subjected to oxidative stress. 5-HETE has little biological activity, but can be oxidized by a selective dehydrogenase in some cells to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent eosinophil chemoattractant. We found that CESS cells, a B lymphocyte cell line, convert AA to 5-oxo-ETE and this is selectively stimulated by oxidative stress. In the presence of H2O2, 5-oxo-ETE is a major AA metabolite in these cells (5-oxo-ETE≈5-HETE>LTB4). The cyclooxygenase product 12-hydroxy-5,8,10-heptadecatrienoic acid is also formed, but is not affected by H2O2. Diamide had effects similar to those of H2O2 and both substances had similar effects on human tonsillar B cells. H2O2 also stimulated 5-oxo-ETE formation from its direct precursor 5-HETE in tonsillar B and CESS cells, and this was inhibited by the glutathione reductase inhibitor carmustine. H2O2 concomitantly induced rapid increases in GSSG and NADP+ and reductions in GSH and NADPH. We conclude that oxidative stress stimulates 5-oxo-ETE synthesis in B lymphocytes by two mechanisms: activation of 5-lipoxygenase and increased oxidation of 5-HETE by NADP+-dependent 5-hydroxyeicosanoid dehydrogenase. B lymphocyte-derived 5-oxo-ETE could contribute to eosinophilic inflammation in asthma and other allergic diseases.

Protective effect of eotaxin-2 inhibition in adjuvant-induced arthritis

Eotaxin-2 is a potent chemoattractant for eosinophils, basophils and T helper type 2 (Th2) lymphocytes. The eotaxin-2/CCL24 receptor CCR3 is expressed in human brain, skin, endothelium and macrophages. The aim of the current study was to evaluate the protective effect of a monoclonal anti-eotaxin-2 antibody on the development of adjuvant-induced arthritis in rats (AIA). Adjuvant arthritis was induced in Lewis rats by intradermal injection of incomplete Freund's adjuvant +Mycobacterium tuberculosis. Rats were treated by intraperitoneal (i.p.) injection with three monoclonal antibodies against eotaxin-2 (G7, G8, D8) three times per week. Controls were treated with total mouse immunoglobulin G (IgG), methotrexate (MTX) or phosphate-buffered saline (PBS). Arthritis severity was evaluated by measuring ankle swelling, arthritic score, whole animal mobility and body weight. Sample joints were obtained for pathological evaluation and postmortem X-ray of ankle joints was performed to document erosions. Significant inhibition of arthritis was observed in rats treated with anti-eotaxin-2 antibodies compared to those treated with immunoglobulin or PBS. Inhibition was manifest in ankle diameter, arthritic score and mobility score. The antibody marked D8 showed the greatest efficacy. The effect was observed both in animals treated before the appearance of arthritis and in those where treatment was begun after development of joint inflammation. Combined treatment with D8 and MTX caused additional protection. Significant reduction of inflammation in D8-treated animals was also demonstrated in pathological and X-ray examinations. Inhibition of eotaxin-2 by monoclonal antibodies has a significant protective effect in adjuvant arthritis. These results may introduce a novel therapeutic target in rheumatoid arthritis and additional inflammatory joint disorders.

Eotaxin‐1, ‐2, and ‐3 immunoreactivity and protein concentration in the nasal polyps of eosinophilic chronic rhinosinusitis patients

Eosinophilic chronic rhinosinusitis (CRS) is characterized by the accumulation of numerous eosinophils in the sinus mucosa and nasal polyps, which are frequently difficult to control, even with surgery. The present study was designed to evaluate the expression and localization of eotaxins, which are well known to be potent and selective chemoattractants for eosinophils in CRS. Randomized study. The patients were classified into eosinophilic and noneosinophilic groups. Histopathological profiles of the nasal polyp were observed with hematoxylin-eosin staining. Eotaxin-1, -2, and -3 were immunohistochemically stained in the nasal polyps. Furthermore, the protein content of eotaxin subtypes inside the nasal polyp and sinus effusion was measured using enzyme-linked immunosorbent assay (ELISA). In the nasal polyps, immunoreactivities of the eotaxin subfamily, eotaxin-1, -2, and -3, were noted in most of the infiltrating eosinophils, as well as in other inflammatory cells, epithelial cells, and endothelial cells. Compared with noneosinophilic CRS groups, eosinophilic CRS groups had a significant expression of eotaxins in their eosinophils. The eotaxin concentrations of nasal polyp and sinus effusion as measured by ELISA were significantly increased in the eosinophilic CRS group compared to the noneosinophilic CRS group. The present findings suggest that enhanced eotaxin family production by eosinophils results in the recruitment of eosinophils into the tissue by a self-amplifying process. Laryngoscope, 2009.

TP receptor-mediated release of eosinophil chemotactic activity from human bronchial smooth muscle cells

There are reports indicating that thromboxane A 2 receptors (TP receptors) may stimulate the eosinophil accumulation in the lower airways of asthmatics, however, the mechanisms behind such an effect remain unknown. We quantified the synthesis of eosinophil chemotactic activity and eosinophilic CC chemokines, including CCL5, CCL7, CCL8, CCL11, CCL13, CCL24, and CCL26 in primary cultures of human bronchial smooth muscle cells (BSMC) stimulated with a prostanoid TP receptor agonist, IBOP (10 − 9 –10 − 7 M). The activation of prostanoid TP receptors in BSMC induced the release of potent eosinophil chemoattractant(s) in the presence of interleukin (IL)-4. CCL11/eotaxin-1 was the only synthesis significantly increased by IBOP co-stimulated with IL-4, and pretreatment with an anti-CCL11 antibody abrogated the eosinophil chemotactic activity released from IBOP/IL-4-stimulated BSMC. The effect of IBOP was also completely blocked by pretreatment with a prostanoid TP receptor-specific antagonist, AA-2414. IBOP had no effect on the expression of IL-4 receptor-α, or on the IL-4-induced phosphorylation of STAT6 in BSMC. In conclusion, activation of prostanoid TP receptors in a Th2-dominant microenvironment might exacerbate the eosinophilic inflammation of the airways by synthesis and release of CCL11 from BSMC.

Structural Requirements for Activation of the 5-Oxo-6E,8Z, 11Z,14Z-eicosatetraenoic Acid (5-Oxo-ETE) Receptor: Identification of a Mead Acid Metabolite with Potent Agonist Activity

The 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) is a potent chemoattractant for neutrophils and eosinophils, and its actions are mediated by the oxoeicosanoid (OXE) receptor, a member of the G protein-coupled receptor family. To define the requirements for activation of the OXE receptor, we have synthesized a series of 5-oxo-6E,8Z-dienoic acids with chain lengths between 12 and 20 carbons, as well as a series of 20-carbon 5-oxo fatty acids, either fully saturated or containing between one and five double bonds. The effects of these compounds on neutrophils (calcium mobilization, CD11b expression, and cell migration) and eosinophils (actin polymerization) were compared with those of 5-oxo-ETE. The C12 and C14 analogs were without appreciable activity, whereas the C16 5-oxo-dienoic acid was a weak partial agonist. In contrast, the corresponding C18 analog (5-oxo-18:2) was nearly as potent as 5-oxo-ETE. Among the C20 analogs, the fully saturated compound had virtually no activity, whereas 5-oxo-6E-eicosenoic acid had only weak agonist activity. In contrast, 5-oxo-6E,8Z,11Z-eicosatrienoic acid (5-oxo-20:3) and its 8-trans isomer were approximately equipotent with 5-oxo-ETE in activating granulocytes. Because of the potent effects of 5-oxo-20:3, we investigated its formation from Mead acid (5Z,8Z,11Z-eicosatrienoic acid), which accumulates in dietary essential fatty acid deficiency, by neutrophils. The main Mead acid metabolite identified was 5-hydroxy-6,8,11-eicosatrienoic acid, followed by 5-oxo-20:3 and two 6-trans isomers of leukotriene B(3). We conclude that optimal activation of the OXE receptor is achieved with 5-oxo-ETE, 5-oxo-18:2, and 5-oxo-20:3, and that the latter compound could potentially be formed under conditions of essential fatty acid deficiency.

Lentiviral-mediated GATA-3 RNAi Decreases Allergic Airway Inflammation and Hyperresponsiveness

GATA-3 is the key transcriptional factor for Th2 commitment in T cells and is strongly associated with asthma and allergic disease. We studied the silencing of the GATA-3 gene expression using RNA interference (RNAi) delivered by a lentiviral vector, to evaluate the therapeutic role of GATA-3 short hairpin RNAs (shRNAs) in a murine model of asthma. Mice were sensitized with OVA and instilled intratracheally (IT) with GATA-3 shRNAs lentiviral vector (Lenti-si-GATA-3) once, 48 hours before challenge. After three challenges with the OVA antigen, the mice were assessed for airway hyperresponsiveness (AHR) and inflammation. With infection of Lenti-si-GATA-3 in EL-4 cells, GATA-3 gene expression was abrogated and downstream Th2 cytokines, such as interleukin-4 (IL-4) and IL-5, were also significantly inhibited. IT delivery of Lenti-si-GATA-3 in OVA-immunized mice resulted in a strong inhibition of local GATA-3 gene expression. Treatment with Lenti-si-GATA-3 successfully alleviated OVA-induced airway eosinophilia and Th2 cytokine release. While evaluating AHR by means of enhanced pause (Penh) and pulmonary resistance (R(L)) using body plethysmography, it was found that the administration of Lenti-si-GATA-3 had significantly decreased AHR in OVA-immunized mice. These results suggest that inhibition of GATA-3 gene expression by shRNAs lentiviral vectors strongly attenuates antigen-induced airway inflammation and hyper-responsiveness in mice.

Airway epithelial cells synthesize the lipid mediator 5-oxo-ETE in response to oxidative stress

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent eosinophil chemoattractant that is synthesized from the 5-lipoxygenase product 5 S-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by the NADP +-dependent enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH), previously reported only in inflammatory cells. Because of their critical location at the interface of the lung with the external environment, we sought to determine whether epithelial cells could also synthesize this substance. We found that HEp-2, T84, A549, and BEAS-2B cells all synthesize 5-oxo-ETE from 5-HETE in amounts comparable to leukocytes. The epithelial dehydrogenase is localized in the microsomal fraction, requires NADP +, and is selective for the S-isomer of 5-HETE, suggesting that it is identical to leukocyte 5-HEDH. Normal human bronchial epithelial cells have an even greater capacity to synthesize 5-oxo-ETE. H 2O 2 dramatically stimulates its synthesis in association with increased levels of intracellular GSSG and NADP +. These responses were all blocked by removal of GSH/GSSG with N-ethylmaleimide, suggesting that H 2O 2 stimulates 5-oxo-ETE synthesis by raising NADP + levels through activation of the GSH redox cycle. Airway smooth muscle cells can also synthesize 5-oxo-ETE, but to a lesser extent. These results suggest that epithelial cells may be a major source of 5-oxo-ETE under conditions of oxidative stress, which may contribute to eosinophil infiltration in allergic diseases.

Association between Eotaxin (CCL11), C-Reactive Protein, and Antimicrobial Antibodies in Patients Undergoing Coronary Angioplasty

Eotaxin (CCL11) is a potent chemoattractant for eosinophils and lymphocytes. Apart from its functions in the eosinophilic system, eotaxin has been shown to be overexpressed in atherosclerosis. We therefore sought...

Gastroesophageal Reflux Disease–Associated Esophagitis Induces Endogenous Cytokine Production Leading to Motor Abnormalities

Gastroesophageal reflux disease is a condition frequently associated with esophagitis and motor abnormalities. Recent evidence suggests that proinflammatory cytokines, such as interleukin (IL)-1beta and IL-6, may be implicated because they reduce esophageal muscle contractility, but these results derive from in vitro or animal models of esophagitis. This study used human esophageal cells and tissues to identify the cellular source of cytokines in human esophagitis investigate whether cytokines can be induced by gastric refluxate, and examine whether esophageal tissue- or cell-derived mediators affect muscle contractility. Endoscopic mucosal biopsy specimens were obtained from patients with and without esophagitis, organ-cultured, and undernatants were assessed for cytokine content. The cytokine profile of esophageal epithelial, fibroblast, and muscle cells was analyzed, and esophageal mucosa and cell products were tested in an esophageal circular muscle contraction assay. The mucosa of esophagitis patients produced significantly greater amounts of IL-1beta and IL-6 compared with those of control patients. Cultured esophageal epithelial cells produced IL-6, as did fibroblasts and muscle cells. Epithelial cells exposed to buffered, but not denatured, gastric juice produced IL-6. Undernatants of mucosal biopsy cultures from esophagitis patients reduced esophageal muscle contraction, as did supernatants from esophageal epithelial cell cultures. The human esophagus produces cytokines capable of reducing contractility of esophageal muscle cells. Exposure to gastric juice is sufficient to stimulate esophageal epithelial cells to produce IL-6, a cytokine able to alter esophageal contractility. These results indicate that classic cytokines are important mediators of the motor disturbances associated with human esophageal inflammation.

Rosmarinic acid as a downstream inhibitor of IKK-beta in TNF-alpha-induced upregulation of CCL11 and CCR3.

1. Tumor necrosis factor (TNF)-alpha is known to induce the expression of CCL11 and CCR3 via the activation of NF-kappaB. CCL11 (eotaxin), the C-C chemokine, is a potent chemoattractant for eosinophils and Th2 lymphocytes, and CCR3 is the receptor for CCL11. 2. In order to determine the effects of rosmarinic acid on the TNF-alpha-induced upregulation of CCL11 and CCR3 in human dermal fibroblasts, we performed an enzyme-linked immunosorbent assay for CCL11 and a Western blot assay for CCR3. The TNF-alpha-induced expression of CCL11 and CCR3 genes was attenuated by rosmarinic acid. 3. In our NF-kappaB luciferase reporter system, TNF-alpha-induced NF-kappaB activation was observed to be reduced by rosmarinic acid. In accordance with this result, rosmarinic acid also inhibited TNF-alpha-induced phosphorylation and degradation of IkappaB-alpha, as well as nuclear translocation of NF-kappaB heterodimer induced by TNF-alpha. This suggests that rosmarinic acid downregulates the expression of CCL11 and CCR3 via the inhibition of NF-kappaB activation signaling. 4. Using the NF-kappaB luciferase reporter system, Western blot analysis, and IKK-beta activity assay, we determined that rosmarinic acid inhibits IKK-beta activity in NF-kappaB signaling, which upregulates the expression of CCL11 and CCR3. Additionally, TNF-alpha-induced secretion of soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1 molecules was found to be attenuated by rosmarinic acid. 5. Our results show that rosmarinic acid inhibits the expression of CCL11 and CCR3 by suppressing the IKK-beta activity in NF-kappaB activation signaling. Further, these results suggest that rosmarinic acid might inhibit the expression of NF-kappaB promoter-related genes.

Eotaxin polymorphisms and serum total IgE levels in children with asthma.

Eotaxin (chemokine, CC motif, ligand; CCL11) is a potent eosinophil chemoattractant strongly implicated in the pathobiology of asthma. Genetic variation at the CCL11 locus has been correlated with serum total IgE, blood eosinophil counts, and circulating eotaxin protein levels in several case-control asthma studies. Family-based association studies of CCL11 genetic variants have not been reported to date. To evaluate 9 common CCL11 single nucleotide polymorphisms (SNPs) in nuclear families ascertained through patients with asthma participating in the Childhood Asthma Management Program study. Single nucleotide polymorphism genotyping was performed by using minisequencing and probe hybridization platforms. Family-based association analysis for asthma and 4 asthma-related intermediate quantitative phenotypes was performed by using FBAT. One SNP, -384A>G, was associated with asthma among African American families (P = .01). CCL11 SNPs and haplotypes were not associated with asthma among white or Hispanic families. Two low-frequency alleles in strong pairwise linkage disequilibrium, -426C and IVS2+199A, were associated with lower serum total IgE levels (P = .0006 and P = .009, respectively) in white families, whereas 2 more common variants, -576C and g.4438C, were associated with higher IgE levels in African American families (P = .01-.04). Haplotype analysis in the white cohort provided additional evidence of association with serum total IgE, implicating 2 haplotypes. No single SNP or haplotype associations were observed with blood eosinophil levels, FEV(1), or airway responsiveness. These findings provide further evidence that genetic variation at the CCL11 locus is an important determinant of serum total IgE levels among patients with asthma.

Lack of association between the -403G/A promoter polymorphism in the human CCL5/RANTES chemokine gene in liver transplant outcome

Chemokines play a major role in the inflammatory and immune responses that mediate allograft outcome. CCL5/RANTES expansion chemokine is potent eosinophil, monocyte, basophils and lymphocyte chemoattractant and has recently been studied in transplantation with discrepant results, but systemic concentrations have been correlated to liver graft survival and incidence of rejection. Recent studies revealed that a functional mutation at -403 in the promoter may have a significance for inflammatory and infectious immune responses. Our objective was to investigate CCL5/RANTES promoter polymorphism in rejection and graft survival in liver transplant. We examined the CCL5/RANTES polymorphism in a series of 218 liver transplants and 101 healthy Caucasian subjects. CCL5/RANTES genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). After comparing recipients (with acute rejection episodes versus without rejection) with the control population, we found no significant deviation in the distribution of the alleles or genotypes of CCL5/RANTES dimorphism in any comparison (P > 0.05). Indeed, 5 years allograft survival was 61.3% in recipients with the GG genotype against 58.8% in recipients with the GA and AA genotypes. These differences were also not statistically significant. In conclusion, human CCL5/RANTES gene promoter polymorphism does not seem to influence acute rejection development and allograft survival in liver recipients.

Differential Regulation of Eotaxin-1/CCL11 and Eotaxin-3/CCL26 Production by the TNF-.ALPHA. and IL-4 Stimulated Human Lung Fibroblast

Allergic asthma and allergic dermatitis are chronic inflammatory diseases and are characterized by an accumulation of eosinophils at sites of inflammation. Eotaxin-1/CCL11 and eotaxin-3/CCL26 are members of the CC chemokine family, which are known to be potent chemoattractants for eosinophils. We observed that a human lung fibroblast, HFL-1 produces eotaxin-1 and -3 in response to TNF-alpha plus IL-4 stimulation, accompanied with NF-kappaB and STAT6 activation. We explored which signaling pathways are operative in the production of eotaxin-1 and -3 using several inhibitors. Eotaxin-1/CCL11 production was inhibited by a p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, but not by the MEK (MAPK/ERK kinase) inhibitors, PD98059 and U0126. In contrast, eotaxin-3/CCL26 production was inhibited similarly by PD98059 as well as U0126 and SB203580. In addition, two proteasome inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and bortezomib with significant inhibitory activity on NF-kappaB activation, inhibited eotaxin-1/CCL11 production with IC50 8 microM for ALLN and IC50 16 nM for bortezomib. In contrast, eotaxin-3/CCL26 production was not inhibited significantly up to 10 microM of ALLN (IC50 16 microM) and up to 10 nM of bortezomib (IC50 11 nM), giving inhibition of eotaxin-3/CCL26 less sensitive than eotaxin-1/CCL11 production by the proteasome inhibitors. Synergistic inhibition was observed among lower doses of SB203580 and proteasome inhibitors, particularly in the eotaxin-1/CCL11 production. No such prominent synergism was found on the eotaxin-3/CCL26 production. The suppression of eotaxin family production by these inhibitors may be efficacious against allergic diseases.