Eotaxin-3 Secretion Research Articles

In Human Esophageal Epithelial and Muscle Cells Treated With Th2 Cytokines, Upadacitinib Decreases Eotaxin-3 Secretion and Muscle Tension

In Human Esophageal Epithelial and Muscle Cells Treated With Th2 Cytokines, Upadacitinib Decreases Eotaxin-3 Secretion and Muscle Tension

Th2 cytokine signaling through IL-4Rα increases eotaxin-3 secretion and tension in human esophageal smooth muscle.

In esophageal epithelial cells in eosinophilic esophagitis (EoE), Th2 cytokines (IL-4, IL-13) signal through IL-4Rα, activating JAK to increase eotaxin-3 secretion, which draws eosinophils into the mucosa. We explored whether Th2 cytokines also might stimulate eotaxin-3 secretion and increase tension in esophageal smooth muscle (ESM), which might impair esophageal distensibility, and whether those events could be blocked by proton pump inhibitors (PPIs) or agents that disrupt IL-4Rα signaling. We established human ESM cell cultures from organ donors, characterizing Th2 cytokine receptor and P-type ATPase expression by qPCR. We measured Th2 cytokine-stimulated eotaxin-3 secretion by enzyme-linked immunosorbent assay (ELISA) and ESM cell tension by gel contraction assay, before and after treatment with omeprazole, ruxolitinib (JAK inhibitor), or IL-4Rα blocking antibody. CPI-17 (inhibitor of a muscle-relaxing enzyme) effects were studied with CPI-17 knockdown by siRNA or CPI-17 phospho(T38A)-mutant overexpression. ESM cells expressed IL-4Rα and IL-13Rα1 but only minimal H+-K+-ATPase mRNA. Th2 cytokines increased ESM eotaxin-3 secretion and tension, effects blocked by ruxolitinib and IL-4Rα blocking antibody but not consistently blocked by omeprazole. IL-13 increased ESM tension by increasing CPI-17 expression and phosphorylation, effects blocked by CPI-17 knockdown. Blocking IL-4Rα decreased IL-13-stimulated eotaxin-3 secretion, CPI-17 expression, and tension in ESM. Th2 cytokines increase ESM eotaxin-3 secretion and tension via IL-4Rα signaling that activates CPI-17. Omeprazole does not reliably inhibit this process, but IL-4Rα blocking antibody does. This suggests that ESM eosinophilia and impaired esophageal distensibility might persist despite elimination of mucosal eosinophils by PPIs, and IL-4Rα blocking agents might be especially useful in this circumstance.NEW & NOTEWORTHY We have found that Th2 cytokines increase eotaxin-3 secretion and tension in esophageal smooth muscle (ESM) cells via IL-4Rα signaling. Unlike esophageal epithelial cells, ESM cells do not express H+-K+-ATPase, and omeprazole does not inhibit their cytokine-stimulated eotaxin-3 secretion or tension. An IL-4Rα blocking antibody reduces both eotaxin-3 secretion and tension induced by Th2 cytokines in ESM cells, suggesting that an agent such as dupilumab might be preferred for patients with EoE with esophageal muscle involvement.

Immune Profiling of Secreted Factors from Human Vestibular Schwannoma Cells and Tumor-associated Macrophages.

This study compared the immune-related secretory capacity of human vestibular schwannoma (VS) and tumor-assisted macrophages (TAMs) with their normal counterparts (Schwann cells [SC] and peripheral blood monocyte-derived macrophages [Mo-MFs], respectively), and examined relationships with presurgical hearing and tumor size. VS tumors (n = 16), auditory nerve (n = 1), blood (n = 9), and great auricular nerves (n = 3) were used. SCs (S100B+ ) and TAMs (CD68+ ) were isolated from VS tissue for culture. The secreted levels of 65 immune-related factors were measured and compared using unpaired t-tests with Welch correction (schwannoma vs. SCs) or Mann-Whitney tests (TAMs and Mo-MFs). Associations between factor concentration and word recognition (WR), pure-tone average (PTA), and tumor size were evaluated with Spearman correlation. Secreted factors with significantly higher concentrations in schwannoma versus SC supernatants included IL-2 and BAFF, whereas MMP-1, IL-6, FGF-2, VEGF-A, MIP-3α, and GRO-α concentrations were significantly higher in TAMs versus Mo-MFs (all p < 0.05). Worse WR was significantly associated with higher secretion of fractalkine, eotaxin-3, CD30, and IL-16 by VS cells; IP-10, eotaxin-3, multiple interleukins, GM-CSF, SCF, and CD30 by TAMs; and TNF-α and MIP-1α by Mo-MFs (all p < 0.05). Worse PTA was significantly correlated with higher secretion of IL-16 by VS cells (p < 0.05). Larger tumor size was significantly correlated with higher secretion of eotaxin by VS cells, and of IL-7, IL-21, and LIF by TAMs (all p = 0.017). Differential secretion of immune-related factors was observed in schwannoma versus normal SCs and in TAMs versus Mo-MFs, some of which were correlated with worse hearing and larger VS tumors. N/A Laryngoscope, 134:S1-S14, 2024.

Type-2 Cytokines Promote the Secretion of the Eosinophil-Attractant CCL26 by Intestinal Epithelial Cells in Food-Sensitized Patients.

Several inflammatory processes of the bowel are characterized by an accumulation of eosinophils at inflammation sites. The mechanisms that govern mucosal infiltration with eosinophils are not fully understood. In this work, we studied the colorectal polyp-confined tissue containing eosinophils and we hypothesized that intestinal epithelial cells are the cell source of eotaxin-3 or CCL26, a potent chemoattractant for eosinophils. We analyzed colorectal polyps (n=50) from pediatric patients with rectal bleeding by H&E staining and eosin staining, and different pro-inflammatory cytokines were assessed by RT-qPCR and ELISA. IgE and CCL26 were investigated by RT-qPCR, ELISA and confocal microscopy. Finally, the intracellular signaling pathway that mediates the CCL26 production was analyzed using a kinase array and immunoblotting in human intestinal Caco-2 cell line. We found a dense cell agglomeration within the polyps, with a significantly higher frequency of eosinophils than in control adjacent tissue. IL-4 and IL-13 were significantly up-regulated in polyps and CCL26 was elevated in the epithelial compartment. Experiments with Caco-2 cells showed that the type-2 cytokine IL-13 increased STAT3 and STAT6 phosphorylation and eotaxin-3 secretion. The addition of the blocking antibody Dupilumab or the inhibitor Ruxolitinib to the cytokine-stimulated Caco-2 cells diminished the CCL26 secretion to basal levels in a dose-dependent manner. In conclusion, our findings demonstrate a high frequency of eosinophils, and elevated levels of type-2 cytokines and eotaxin-3 in the inflammatory stroma of colorectal polyps from pediatric patients. Polyp epithelial cells showed to be the main cell source of CCL26, and IL-13 was the main trigger of this chemokine through the activation of the STAT3/STAT6/JAK1-2 pathway. We suggest that the epithelial compartment actively participates in the recruitment of eosinophils to the colonic polyp-confined inflammatory environment.

Angelica gigas extract ameliorates allergic rhinitis in an ovalbumin-induced mouse model by inhibiting Th2 cell activation

BackgroundAllergic rhinitis (AR) is a well-documented type 2 helper T (Th2) cell-mediated allergic disease that is accompanied by symptoms such as nasal rubbing, sneezing, itching, and rhinorrhea. Angelica gigas (AG) is traditional oriental medicine, and its dried root is widely used for the treatment of anemia, as a sedative, and as a blood tonic. PurposeThe effects of AG on allergic diseases including AR are currently unclear; therefore, we aimed to investigate the effects of AG extract (AG-Ex) in ameliorating AR. Study design/methodsThe cytotoxicity of AG-Ex was analyzed by EZ-Cytox or MTS assay in splenocytes, differentiated Th2 cells, and human nasal epithelial cells (HNEpC). The changes of Th2 cells activation were determined by the secretion levels of cytokines and chemokines using cytometric bead array in splenocytes and differentiated Th2 cells. The expression levels of eotaxin-3 and periostin were analyzed using an ELISA. AR was induced by ovalbumin in BALB/c mice and the ameliorating effects of AG-Ex were assessed by their clinical symptoms. ResultsThe secretion of Th2 cytokines such as IL-4, IL-5, and IL-13 was inhibited by the AG-Ex treatment in the splenocytes and differentiated Th2 cells. The treatment also suppressed allergic responses including the secretion of eotaxin-3 and periostin in human nasal epithelial cells (HNEpC). Moreover, the administration of AG-Ex to the OVA-induced AR mice improved their clinical symptoms, including behavioral tests, immune cell counts, histopathological analysis, and changes in serum parameters. ConclusionThe results of this study suggest that AG-Ex ameliorates AR by inhibiting Th2 cell activation and could thus be utilized as a treatment for Th2-mediated allergic diseases in the future.

In Esophageal Squamous Cells From Eosinophilic Esophagitis Patients, Th2 Cytokines Increase Eotaxin-3 Secretion Through Effects on Intracellular Calcium and a Non-Gastric Proton Pump

In upper airway cells, T helper 2 cytokines that signal through interleukin-4 (IL-4) receptor-α have been shown to stimulate eotaxin-3 secretion via a nongastric proton pump (ngH+,K+ATPase). To seek novel targets for eosinophilic esophagitis (EoE) treatments, we evaluated ngH+,K+ATPase expression in EoE squamous cells, and explored molecular pathways involved in eotaxin-3 secretion by IL-4 receptor-α signaling. ngH+,K+ATPase expression in EoE cells was evaluated by quantitative real-time polymerase chain reaction and Western blotting. IL-4-stimulated eotaxin-3 secretion was measured by enzyme-linked immunosorbent assay after treatment with omeprazole, SCH 28080 (potassium-competitive acid blocker), ethylene glycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester (calcium chelator), 2-aminoethoxydiphenyl borate (inhibitor of endoplasmic reticulum calcium release), verapamil, and diltiazem (L-type calcium channel inhibitors). Intracellular calcium transients were measured by Fluo-4 fluorescence. Key experiments were confirmed in EoE primary cells and in RNA sequencing datasets from mucosal biopsies of patients with EoE and controls. EoE cells expressed ngH+,K+ATPase messenger RNA and protein. Omeprazole and SCH 28080 decreased IL-4-stimulated eotaxin-3 secretion. IL-4 increased intracellular calcium transients, and IL-4-stimulated eotaxin-3 secretion was blocked by ethylene glycol-bis(β-aminoethyl)-N,N,N',N'-tetraacetoxymethyl ester, 2-aminoethoxydiphenyl borate, verapamil, and diltiazem. The combination of omeprazole and verapamil suppressed IL-4-stimulated eotaxin-3 secretion more than either agent alone. EoE biopsies expressed higher ngH+,K+ATPase and exhibited more calcium signaling than controls. EoE cells express a nongastric proton pump that mediates T helper 2 cytokine-stimulated eotaxin-3 secretion. IL-4 induces calcium release from the endoplasmic reticulum and calcium entry via L-type calcium channels, increasing intracellular calcium that contributes to eotaxin-3 secretion by EoE cells. L-type calcium channel inhibitors block T helper 2 cytokine-stimulated eotaxin-3 secretion, suggesting a potential role for these agents in EoE treatment.

Eotaxin-3 as a Plasma Biomarker for Mucosal Eosinophil Infiltration in Chronic Rhinosinusitis.

Objective: Chronic rhinosinusitis with nasal polyps exhibits marked eosinophilic infiltration and its mucosal eosinophilia is associated with more severe symptoms. The Japanese epidemiological survey of refractory eosinophilic chronic rhinosinusitis found that patients with nasal polyps required multiple surgeries when there were higher infiltrating eosinophils in the mucosa. In order to identify plasma biomarkers for local eosinophil infiltration in rhinosinusitis for surgery, we examined the levels of molecules in the plasma of patients and compared the number of infiltrating eosinophils in the nasal mucosa.Materials and Methods: Mucosal tissues from 97 patients with chronic rhinosinusitis (CRS) were obtained from the nasal polyps during surgery. Tissues were immediately fixed and sections were stained with hematoxylin-eosin. The number of eosinophils in the mucosa was counted at HPF (x 400). Blood samples were obtained and the plasma was stored at −80°C. We measured the plasma cytokine and chemokine levels using multiple assay systems according to the manufacturers' protocols. The tissues were divided into high- and low-eosinophil mucosal infiltration group for recurrence after endoscopic sinus surgery (ESS). We also observed chemokine secretion from nasal fibroblasts.Results: The plasma level of eotaxin-3/ CC chemokine ligand 26 (CCL26) was significantly higher in the high-eosinophil mucosal infiltration group (p < 0.005). The number of infiltrating eosinophils in the mucosa was significantly higher in the group with the higher eotaxin-3 level (p < 0.001), but there was no significant difference in the blood eosinophil numbers among two groups. A significant positive correlation was found between the mucosal eosinophil count and the plasma levels of eotaxin-3 (p < 0.005). The levels of interleukin 33 (IL-33) (p < 0.001) and thymic stromal-derived lymphopoietin (TSLP) (p < 0.005) were significantly higher in the high-level eotaxin-3 group. IL-13 strongly induced the secretion of eotaxin-3 from human nasal fibroblasts (p < 0.05).Conclusion: This is the first report suggesting eotaxin-3 as a plasma biomarker for mucosal eosinophil infiltration. Furthermore, the level of eotaxin-3 was found to be closely related to IL-33 and TSLP levels which indicate respiratory diseases.

Farnesyltransferase Inhibition Exacerbates Eosinophilic Inflammation and Airway Hyperreactivity in Mice with Experimental Asthma: The Complex Roles of Ras GTPase and Farnesylpyrophosphate in Type 2 Allergic Inflammation.

Ras, a small GTPase protein, is thought to mediate Th2-dependent eosinophilic inflammation in asthma. Ras requires cell membrane association for its biological activity, and this requires the posttranslational modification of Ras with an isoprenyl group by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). We hypothesized that inhibition of FTase using FTase inhibitor (FTI)-277 would attenuate allergic asthma by depleting membrane-associated Ras. We used the OVA mouse model of allergic inflammation and human airway epithelial (HBE1) cells to determine the role of FTase in inflammatory cell recruitment. BALB/c mice were first sensitized then exposed to 1% OVA aerosol or filtered air, and half were injected daily with FTI-277 (20 mg/kg per day). Treatment of mice with FTI-277 had no significant effect on lung membrane-anchored Ras, Ras protein levels, or Ras GTPase activity. In OVA-exposed mice, FTI-277 treatment increased eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity. Human bronchial epithelial (HBE1) cells were pretreated with 5, 10, or 20 μM FTI-277 prior to and during 12 h IL-13 (20 ng/ml) stimulation. In HBE1 cells, FTase inhibition with FTI-277 had no significant effect on IL-13-induced STAT6 phosphorylation, eotaxin-3 peptide secretion, or Ras translocation. However, addition of exogenous FPP unexpectedly augmented IL-13-induced STAT6 phosphorylation and eotaxin-3 secretion from HBE1 cells without affecting Ras translocation. Pharmacological inhibition of FTase exacerbates allergic asthma, suggesting a protective role for FTase or possibly Ras farnesylation. FPP synergistically augments epithelial eotaxin-3 secretion, indicating a novel Ras-independent farnesylation mechanism or direct FPP effect that promotes epithelial eotaxin-3 production in allergic asthma.

Sa1153 - Esophageal Squamous Cells from Eosinophilic Esophagitis Patients Express a Non-Gastric Proton Pump, and their Th2 Cytokine-Stimulated Secretion of Eotaxin-3 can be Blocked by a Potassium-Competitive Acid Blocker and a Histamine H2-Receptor Antagonist

Sa1153 - Esophageal Squamous Cells from Eosinophilic Esophagitis Patients Express a Non-Gastric Proton Pump, and their Th2 Cytokine-Stimulated Secretion of Eotaxin-3 can be Blocked by a Potassium-Competitive Acid Blocker and a Histamine H2-Receptor Antagonist

JAK-STAT6 Pathway Inhibitors Block Eotaxin-3 Secretion by Epithelial Cells and Fibroblasts from Esophageal Eosinophilia Patients: Promising Agents to Improve Inflammation and Prevent Fibrosis in EoE

BackgroundAlthough most studies on treatments for eosinophilic esophagitis (EoE) have focused on effects in the epithelium, EoE is a transmural disease. Eosinophils that infiltrate the subepithelial layers of the esophagus lead to fibrosis and the serious complications of EoE, and current therapies have shown minimal effects on this fibrosis. We aimed to elucidate T helper (Th)2 cytokine effects on esophageal fibroblasts and to explore potential fibroblast-targeted therapies for EoE.MethodsWe established telomerase-immortalized fibroblasts from human esophageal biopsies. We stimulated these esophageal fibroblasts with Th2 cytokines, and examined effects of omeprazole and inhibitors of the Janus kinase (JAK)—signal transducer and activator of transcription (STAT6) pathway (AS1517499, leflunomide, and ruxolitinib) on STAT6 phosphorylation, STAT6 nuclear translocation, and eotaxin-3 expression. We also measured the effects of these inhibitors in esophageal epithelial cells stimulated with Th2 cytokines.ResultsAs in esophageal epithelial cells, Th2 cytokines increased STAT6 phosphorylation, STAT6 nuclear translocation, eotaxin-3 transcription and protein secretion in esophageal fibroblasts. Unlike in epithelial cells, however, omeprazole did not inhibit cytokine-stimulated eotaxin-3 expression in fibroblasts. In contrast, JAK-STAT6 pathway inhibitors decreased cytokine-stimulated eotaxin-3 expression in both fibroblasts and epithelial cells.ConclusionsOmeprazole does not inhibit Th2 cytokine-stimulated eotaxin-3 expression by esophageal fibroblasts, suggesting that PPIs will have limited impact on subepithelial EoE processes such as fibrosis. JAK-STAT6 pathway inhibitors block Th2 cytokine-stimulated eotaxin-3 expression both in fibroblasts and in epithelial cells, suggesting a potential role for JAK-STAT inhibitors in treating both epithelial inflammation and subepithelial fibrosis in EoE.

Altered esophageal histamine receptor expression in Eosinophilic Esophagitis (EoE): implications on disease pathogenesis.

Eosinophilic Esophagitis (EoE) is a chronic allergic disorder, whose pathobiology is incompletely understood. Histamine-producing cells including mast cells and basophils have been implicated in EoE. However, very little is currently known about the role of histamine and histamine receptor (HR) expression and signaling in the esophageal epithelium. Herein, we characterized HR (H1R, H2R, H3R, and H4R) expression in human esophageal biopsies and investigate the role of histamine signaling in inducible cytokine expression in human esophageal epithelial cells in vitro. HR expression was quantified in esophageal biopsies from non-EoE control (N = 23), inactive EoE (<15 eos/hpf, N = 26) and active EoE (>15 eos/hpf, N = 22) subjects using qRT-PCR and immunofluorescent localization. HR expression and histamine-mediated cytokine secretion were evaluated in human primary and telomerase-immortalized esophageal epithelial cells. H1R, H2R, and H4R expression were increased in active EoE biopsies compared to inactive EoE and controls. H2R was the most abundantly expressed receptor, and H3R expression was negligible in all 3 cohorts. Infiltrating eosinophils expressed H1R, H2R, and H4R, which contributed to the observed increase in HR in active subjects. H1R and H2R, but not H3R or H4R, were constitutively expressed by primary and immortalized cells, and epithelial histamine stimulation induced GM-CSF, TNFα, and IL-8, but not TSLP or eotaxin-3 secretion. Epithelial priming with the TLR3 ligand poly (I:C) induced H1R and H2R expression, and enhanced histamine-induced GM-CSF, TNFα, and IL-8 secretion. These effects were primarily suppressed by H1R antagonists, but unaffected by H2R antagonism. Histamine directly activates esophageal epithelial cytokine secretion in vitro in an H1R dependent fashion. However, H1R, H2R and H4R are induced in active inflammation in EoE in vivo. While systemic antihistamine (anti-H1R) therapy may not induce clinical remission in EoE, our study suggests that further study of histamine receptor signaling in EoE is warranted and that targeting of additional histamine receptors may lead to novel treatment strategies for this important disease.

Proton pump inhibitors decrease eotaxin-3 expression in the proximal esophagus of children with esophageal eosinophilia.

ObjectiveBesides reducing gastric acid secretion, proton pump inhibitors (PPIs) suppress Th2-cytokine-stimulated expression of an eosinophil chemoattractant (eotaxin-3) by esophageal epithelial cells through acid-independent, anti-inflammatory mechanisms. To explore acid-inhibitory and acid-independent, anti-inflammatory PPI effects in reducing esophageal eosinophilia, we studied eotaxin-3 expression by the proximal and distal esophagus of children with esophageal eosinophilia before and after PPI therapy. In vitro, we studied acid and bile salt effects on IL-13-stimulated eotaxin-3 expression by esophageal epithelial cells.DesignAmong 264 children with esophageal eosinophilia seen at a tertiary pediatric hospital from 2008 through 2012, we identified 10 with esophageal biopsies before and after PPI treatment alone. We correlated epithelial cell eotaxin-3 immunostaining with eosinophil numbers in those biopsies. In vitro, we measured eotaxin-3 protein secretion by esophageal squamous cells stimulated with IL-13 and exposed to acid and/or bile salt media, with or without omeprazole.ResultsThere was strong correlation between peak eosinophil numbers and peak eotaxin-3-positive epithelial cell numbers in esophageal biopsies. Eotaxin-3 expression decreased significantly with PPIs only in the proximal esophagus. In esophageal cells, exposure to acid-bile salt medium significantly suppressed IL-13-induced eotaxin-3 secretion; omeprazole added to the acid-bile salt medium further suppressed that eotaxin-3 secretion, but not as profoundly as at pH-neutral conditions.ConclusionIn children with esophageal eosinophilia, PPIs significantly decrease eotaxin-3 expression in the proximal but not the distal esophagus. In esophageal squamous cells, acid and bile salts decrease Th2 cytokine-stimulated eotaxin-3 secretion profoundly, possibly explaining the disparate PPI effects on the proximal and distal esophagus. In the distal esophagus, where acid reflux is greatest, a PPI-induced reduction in acid reflux (an effect that could increase eotaxin-3 secretion induced by Th2 cytokines) might mask the acid-independent, anti-inflammatory PPI effect of decreasing cytokine-stimulated eotaxin-3 secretion.

430 Different Effects of Proton Pump Inhibitors (PPIs) on Eotaxin-3 Protein Expression by the Proximal and Distal Esophagus of Children With Esophageal Eosinophilia

Introduction: PPIs can reduce esophageal eosinophilia, an effect usually attributed to decreased acid reflux. However, PPIs can suppress Th2-cytokine-stimulated expression of an eosinophil chemoattractant (eotaxin-3) by esophageal cells through mechanisms independent of effects on gastric acid. To explore the relative contributions of these acid-suppressive and acid-independent PPI actions, we studied eotaxin-3 expression by the proximal and distal esophagus of children with esophageal eosinophilia before and after PPI therapy. In vitro, we studied acid effects on Th2-cytokine-stimulated eotaxin-3 expression by esophageal epithelial cells.Methods:We identified 10 children with initial esophageal biopsies showing ≥15 eos/hpf who had follow-up endoscopy after PPI treatment alone for ≥8 weeks. Eosinophils were quantified on HE eotaxin-3 secretion was determined by ELISA. Results: For the group as a whole, mean maximum eos/hpf decreased with PPI treatment (50 to 40 eos/hpf); in 5 of the 10 patients, counts fell to <15 eos/hpf. There was strong correlation between maximum eos/hpf and maximum eotaxin-3-positive epithelial cells/hpf (R=0.8332, P<0.0001). Although PPI effects in reducing acid reflux are strongest in the distal esophagus, eotaxin-3 expression decreased significantly with PPIs only in the proximal esophagus (28 to 6 cells/hpf proximal, P=0.049; 14 to 24 cells/hpf distal esophagus, P=0.41). In vitro, EoE1-T and EoE2-T cells stimulated with IL-13 showed strong suppression of eotaxin-3 secretion at pH4; weakly acidic pH levels had no consistent effects on eotaxin3 in the same cell lines (Table). Treatment with omeprazole at weakly acidic pH did not suppress IL-13-stimulated eotaxin-3 secretion as profoundly as exposure to pH4 alone (Table). Conclusions: In children with esophageal eosinophilia, PPIs significantly decrease expression of eotaxin-3 protein in the proximal but not distal esophagus. In esophageal squamous cells in vitro, acid at pH4 decreases eotaxin-3 secretion significantly. This in vitro finding might explain the disparate PPI effects on the proximal and distal esophagus. In the distal esophagus, where acid exposure that inhibits eotaxin-3 secretion is greatest, suppression of acid reflux by PPIs might increase eotaxin-3 secretion and mask acid-independent PPI suppressive effects on the protein. In the proximal esophagus, where acid exposure is least, acid-independent PPI effects in suppressing eotaxin-3 secretion are manifest most greatly. Acid pH Effects on IL-13-Driven Eotaxin-3 Protein Secretion by Esophageal Squamous Cell Lines (EoE1-T and EoE2-T)

408 TH2 Cytokines Stimulate Eotaxin-3 Secretion by Esophageal Fibroblasts: An Effect That Is Not Blocked by Omeprazole.

408 TH2 Cytokines Stimulate Eotaxin-3 Secretion by Esophageal Fibroblasts: An Effect That Is Not Blocked by Omeprazole.

Su1126 Omeprazole Blocksth2-Cytokine-Stimulated Secretion of Eotaxin-3 in Esophageal Squamous Cells From Patients With EoE and From Patients With GERD

Eosinophilic esophagitis (EoE) has been recognized as a chronic disease of the esophagus in children and adults. Chronic inflammation of the esophagus may lead to malignant transformation of cells. Malignant or pre-malignant (dysplasia) conditions of the esophagus in EoE has not been reported. p53, a mutated intracellular oncogene marker, and Ki-67, a nuclear antigen associated with cell proliferation, were found to be increased in patients with Barrett's esophagus and esophageal cancer. Aim: To evaluate the presence of p53 and Ki-67 in children with EoE. Materials: Esophageal biopsies of children with EoE (Test Group), children with GERD (Control 1), adults with EoE (Control 2), adults with esophageal cancer (Control 3), and children with normal esophagus (Control 4), comprised our patient population. The immunreactivity of monoclonal oncogene p53 (DAKO, DO-7) and monoclonal Ki-67 cell marker (DAKO, Clone MIB-1) were utilized. Immunoreactivity of p53 was considered positive when at least 10% of the cells stained positive (Wang 1993). Ki-67 immunoreactivity was calculated as a percentage of positive cells per at least 500 cells (Feith 2004). Results: A total of 50 esophageal biopsies were available, 25 children with EoE, 15 children with GERD, 15 children with normal esophagus, 3 adults with EoE, and 2 adults with esophageal cancer. p53 was negative in normal mucosa but was Ki-67 positive in the basal layer (proliferative zone). Adult biopsies with esophageal cancer were positive for both markers. Immunoreactivity of p53 and Ki-67 was significantly higher in EoE vs. normal esophagus (Table). Conclusion: Oncogene p53 and Ki-67 immunoreactivity were higher in the EoE compared to the GERD and the normal group. Our data suggested an active cell proliferation in the esophagus of children with EoE and GERD. Whether the positive immunoreactivity of p53 in EoE children represents a real genetic mutation or false positive results is yet to be resolved (Rice 1994). Oncogene immunoreactivity in EoE

599 in Esophageal Squamous Epithelial Cells From Patients With EoE, Omeprazole Blocks Cytokine-Stimulated Eotaxin-3 Secretion by Reducing Binding of STAT6 to the Eotaxin-3 Promoter.

599 in Esophageal Squamous Epithelial Cells From Patients With EoE, Omeprazole Blocks Cytokine-Stimulated Eotaxin-3 Secretion by Reducing Binding of STAT6 to the Eotaxin-3 Promoter.

Vitamin E forms inhibit IL-13/STAT6-induced eotaxin-3 secretion by up-regulation of PAR4, an endogenous inhibitor of atypical PKC in human lung epithelial cells

Vitamin E forms inhibit IL-13/STAT6-induced eotaxin-3 secretion by up-regulation of PAR4, an endogenous inhibitor of atypical PKC in human lung epithelial cells

STAT6 phosphorylation inhibitors block eotaxin-3 secretion in bronchial epithelial cells

The STAT6 (signal transducer and activator of transcription 6) protein facilitates T-helper cell 2 (Th2) mediated responses that control IgE-mediated atopic diseases such as asthma. We have identified compounds that bind to STAT6 and inhibit STAT6 tyrosine phosphorylation induced by IL-4. In the bronchial epithelial cell line BEAS-2B, compound (R)-84 inhibits the secretion of eotaxin-3, a chemokine eliciting eosinophil infiltration. (R)-84 appears to prevent STAT6 from assuming the active dimer configuration by directly binding the protein and inhibiting tyrosine phosphorylation.

Vitamin E forms inhibit IL-13/STAT6-induced eotaxin-3 secretion by up-regulation of PAR4, an endogenous inhibitor of atypical PKC in human lung epithelial cells

Eotaxin-3 (CCL-26), a potent chemokine for eosinophil recruitment and contributing significantly to the pathogenesis of asthma, is secreted by lung epithelial cells in response to T helper 2 cytokines including interleukin 13 (IL-13). Here we showed that vitamin E forms, but not their metabolites, differentially inhibited IL-13-stimulated generation of eotaxin-3 in human lung epithelial A549 cells. The relative inhibitory potency was γ-tocotrienol (γ-TE) (IC50 ∼15 μM)>γ-tocopherol, δ-tocopherol (IC50 ∼25–50 μM)>α-tocopherol. Consistent with suppression of eotaxin, γ-TE treatment impaired IL-13-induced phosphorylation of STAT6, the key transcription factor for activation of eotaxin expression, and consequently blocked IL-13-stimulated DNA-binding activity of STAT6. In search of the upstream target of γTE by using inhibitor and siRNA approaches, we discovered that the atypical protein kinase C (aPKC) signaling, instead of classical PKC, p38 MAPK, JNK or ERK, played a critical role in IL-13-stimulated eotaxin generation and STAT6 activation. While showing no obvious effect on aPKC expression or phosphorylation, γ-TE treatment resulted in increased expression of prostate-apoptosis-response 4 (PAR4), an endogenous negative regulator of aPKCs. Importantly, γ-TE treatment led to enhanced formation of aPKC/PAR4 complex that is known to reduce aPKC activity via protein–protein crosstalk. Our study demonstrated that γ-TE inhibited IL-13/STAT6-activated eotaxin secretion via up-regulation of PAR4 expression and enhancement of aPKC–PAR4 complex formation. These results support the notion that specific vitamin E forms may be useful anti-asthmatic agents.

Blackcurrant proanthocyanidins augment IFN‐γ‐induced suppression of IL‐4 stimulated CCL26 secretion in alveolar epithelial cells

Epidemiological studies reveal that fruit consumption reduces the prevalence of airway inflammation and childhood asthma. In particular, blackcurrant polyphenolic extracts have been shown to alleviate lung inflammation. Since IL-4-stimulated eotaxin-3 (CCL26) secretion is a major factor in the continuous eosinophil recruitment observed in atopic asthma, our focus was to evaluate the effectiveness of blackcurrant polyphenolic compounds on CCL26 secretion in human alveolar epithelial cells. Our results indicate that a proanthocyanin-enriched blackcurrant extract (BC-P), but not anthocyanin-enriched blackcurrant extract suppressed both IL-4- and IL-13-stimulated CCL26 secretion in a dose-dependent manner. Furthermore pre-incubation of cells with BC-P caused a time-dependent suppression of IL-4-stimulated CCL26 secretion. Moreover, epigallocatechin (EGC), and to a lesser extent epicatechin, metabolites identified in the proanthocyanidin extract, suppressed IL-4-stimulated CCL26 secretion. EGC was also effective at reducing the cellular phosphorylated STAT-6/STAT-6 ratio. Furthermore, both BC-P and purified EGC potentiated the ability of IFN-gamma to suppress IL-4-stimulated CCL26 secretion. The progression of an allergic immune response is complex, identifying plant compounds that target specific cellular events and complement the body's own immune actions is important for the development of functional foods. Our findings support the potential for blackcurrant polyphenolic compounds to reduce eosinophil recruitment and alleviate eosinophilic-driven airway inflammation.