Chronic rhinosinusitis with nasal polyps (CRSwNP) is often characterized by tissue eosinophilia that is associated with poor prognosis. Recent findings that proton pump inhibitors (PPIs) directly modulate the expression of eotaxin-3, an eosinophil chemoattractant, in patients with eosinophilic diseases suggest therapeutic potential for PPIs in those with CRSwNP.

We assessed the effect of type 2 mediators, particularly IL-13 and eotaxin-3, on tissue eosinophilia and disease severity in patients with chronic rhinosinusitis (CRS). Further investigation focused on PPI suppression of eotaxin-3 expression in vivo and in vitro, with exploration of underlying mechanisms.

Type 2 mediator levels in nasal tissues and secretions were measured by using a multiplex immunoassay. Eotaxin-3 and other chemokines expressed in IL-13-stimulated human sinonasal epithelial cells (HNECs) and BEAS-2B cells with or without PPIs were assessed by using ELISA, Western blotting, real-time PCR, and intracellular pH imaging.

Nasal tissues and secretions from patients with CRSwNP had increased IL-13, eotaxin-2, and eotaxin-3 levels, and these were positively correlated with tissue eosinophil cationic protein levels and radiographic scores in patients with CRS (P < .05). IL-13 stimulation of HNECs and BEAS-2B cells dominantly induced eotaxin-3 expression, which was significantly inhibited by PPIs (P < .05). Patients with CRS taking PPIs also showed lower in vivo eotaxin-3 levels compared with those without PPIs (P < .05). Using intracellular pH imaging and altering extracellular K+, we found that IL-13 enhanced H+,K+-exchange, which was blocked by PPIs and the mechanistically unrelated H,K-ATPase inhibitor, SCH-28080. Furthermore, knockdown of ATP12A (gene for the nongastric H,K-ATPase) significantly attenuated IL-13-induced eotaxin-3 expression in HNECs. PPIs also had effects on accelerating IL-13-induced eotaxin-3 mRNA decay.

Our results demonstrated that PPIs reduce IL-13-induced eotaxin-3 expression by airway epithelial cells. Furthermore, mechanistic studies suggest that the nongastric H,K-ATPase is necessary for IL-13-mediated epithelial responses, and its inhibitors, including PPIs, might be of therapeutic value in patients with CRSwNP by reducing epithelial production of eotaxin-3.

Myeloid-derived suppressor cells (MDSCs) expand in tumor-bearing host. They suppress anti-tumor immune response and promote tumor growth. Chemokines play a vital role in recruiting MDSCs into tumor tissue. They can also induce the generation of MDSCs in the bone marrow, maintain their suppressive activity, and promote their proliferation and differentiation. Here, we review CCL2/CCL12-CCR2, CCL3/4/5-CCR5, CCL15-CCR1, CX3CL1/CCL26-CX3CR1, CXCL5/2/1-CXCR2, CXCL8-CXCR1/2, CCL21-CCR7, CXCL13-CXCR5 signaling pathways, their role in MDSCs recruitment to tumor tissue, and their correlation with tumor development, metastasis and prognosis. Targeting chemokines and their receptors may serve as a promising strategy in immunotherapy, especially combined with other strategies such as chemotherapy, cyclin-dependent kinase or immune checkpoints inhibitors.

To investigate pathogenic mechanisms of primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and autoimmune hepatitis (AIH), serum levels of 26 chemokines and cytokines were determined and compared with patients with chronic hepatitis C or in healthy controls. The chemokine eotaxin-3 (E3; CCL26), which recruits eosinophils to sites of inflammation, was found to be highly elevated in all PSC, PBC, and AIH patients compared with HCV patients and healthy controls. Eotaxin-1 (E1; CCL11), another eosinophil-specific chemokine, was elevated in PSC but reduced in PBC and AIH, while the macrophage-derived chemokine (MDC; CCL22) was lower in all PSC, PBC, and AIH patients compared with HCV patients and controls. By incorporating levels of the interleukin (IL)-15 into a diagnostic algorithm, PSC, PBC, and AIH patients could each be differentiated with good sensitivity and specificity. These findings represent the first study to compare the level of serum cytokine/chemokine levels among these related autoimmune-like liver diseases. Furthermore, our data indicate that the measurement of serum E3, E1, CCL22, and IL-15 levels can aid in the diagnosis of these clinically challenging diseases and shed light on the potential pathogenic mechanisms underlying these diseases. By suggesting a potential role for an allergic phenomenon involving eosinophils, which may define them as liver-specific allergic diseases, this may open up potential new therapeutic avenues by abrogating the action of these disease-associated immune modulators.

BACKGROUND

Allergic rhinitis is an inflammatory disease characterized by local overproduction of type 2 cytokines and tissue eosinophilia. Recent research suggests the involvement of additional cytokines such as IL-17, chemokine (C-C motif) ligand (CCL) 26/eotaxin-3, and CCL13/monocyte chemoattractant protein-4 (MCP-4) in its pathophysiology. Furthermore, bronchial epithelial cells treated with IL-17 and type 2 cytokines distinctively up-regulated eotaxin-3 gene expression. In this study we investigated the kinetics of IL-4, IL-10, IL-17, eotaxin-3, and MCP-4 in seasonal allergic rhinitis volunteers after nasal allergen challenge (NAC) and their release during natural pollen exposure.

METHODS

The nasal lavages of 15 symptomatic allergic and 14 nonallergic subjects were collected during the pollination season. Additionally, six allergic subjects underwent a single unilateral nasal allergen and control challenge out of season, and nasal secretions were collected. Levels of IL-4, IL-10, IL-17, eotaxin-3, and MCP-4 in nasal lavages and secretions were measured using an electrochemiluminescent assay.

RESULTS

After NAC, allergic subjects had a significant immediate response of nasal symptoms as well as a significant increase at 5 hours of IL-4, IL-10, and IL-17 and at 2, 5, and 24 hours significantly raising levels of eotaxin-3. IL-17 and eotaxin-3 concentrations at 5 hours were correlated (r = 0.94; p = 0.005). During natural pollen exposure, barely detectable levels of IL-17 in allergic subjects were also correlated with eotaxin-3 (r = 0.62; p = 0.01). Eotaxin-3 and MCP-4 levels were significantly elevated 9- or 3.7-fold, respectively, and IL-10 and, unexpectedly, IL-4 were significantly lower in allergic subjects compared with nonallergic subjects.

CONCLUSIONS

Nasal IL-17, MCP-4, and, possibly, eotaxin-3 may aggravate and IL-10 may alleviate nasal mucosal allergy.

CC-chemokine receptor 3 (CCR3) is a chemokine receptor for which major ligands, CC-chemokine ligand (CCL) 11, CCL24, and CCL26, are known to be involved in chemotaxis for eosinophils. In the present study, we evaluated the effect of a low molecular weight CCR3-receptor antagonist, Ki19003 (4-[[5-(2,4-dichlorobenzylureido)pentyl][1-(4-chlorophenyl)ethyl]amino]butanoic acid), on airway remodeling in a mouse model of allergic asthma. BALB/c mice were sensitized twice by intraperitoneal injection of ovalbumin (OA) and exposed daily to 1% OA for 3 weeks. Twenty-four hours after the final antigen challenge, bronchoalveolar lavage and histological examinations were carried out. Ki19003 clearly inhibited antigen-induced increase in the number of eosinophils in bronchoalveolar lavage fluid (BALF), but did not affect the number of other cell types examined in this study. Ki19003 also inhibited the increased production of transforming growth factor-beta1 in BALF and the amount of hydroxyproline in the lungs in a dose-dependent manner. Furthermore, Ki19003 significantly attenuated allergen-induced subepithelial and peribronchial fibrosis. These findings indicate that CCR3 antagonism prevents not only the infiltration of eosinophils into the airways but also the development of allergen-induced subepithelial and peribronchial fibrosis. Therefore, a CCR3 antagonist may be useful in the treatment of airway remodeling, especially subepithelial and peribronchial fibrosis, in allergic asthma.

Moderate-to-severe atopic dermatitis (AD) has been associated with significant disease burden and systemic abnormalities and often requires systemic treatments. Currently, safe and effective oral systemic treatments for moderate-to-severe AD are not yet available. ASN002 is an oral inhibitor of the Janus kinase/spleen tyrosine kinase signaling pathways, targeting several cytokine axes (T_HHHHWe sought to evaluate the effect of ASN002 on the cellular and molecular biomarker profile of patients with moderate-to-severe AD and to correlate changes in biomarkers to improvements in clinical severity measures and pruritus.Thirty-six patients with moderate-to-severe AD were randomized to groups with dose escalation of ASN002 (20, 40, and 80 mg) and a placebo group. Skin biopsy specimens were performed at baseline, day 15, and day 29. Gene expression studies were conducted by using microarray and quantitative RT-PCR, and cellular infiltrates and protein expression were studied by using immunohistochemistry.

RESULTS

ASN002 reversed the lesional skin transcriptome toward a nonlesional phenotype. It also rapidly and significantly suppressed key inflammatory pathways implicated in AD pathogenesis, including T_HCCL26/eotaxin-3), T_HHHThe Janus kinase/spleen tyrosine kinase inhibitor ASN002 significantly suppressed key AD inflammatory pathways, corresponding to clinical response. ASN002 might be an effective novel therapeutic agent for moderate-to-severe AD.

BACKGROUND

The function of eosinophils has been attributed to host defense, immunomodulation, and fibrosis. Although eosinophils are found among infiltrating cells in a broad spectrum of skin diseases, their pathogenic role remains uncertain. This study aimed to analyze the cytokine expression by eosinophils in different skin diseases.

METHODS

Skin specimens from different skin diseases [allergic/reactive, infectious, autoimmune, and tumors/lymphomas (LY)] were stained by antibodies directed to eosinophil cationic protein, cytokines [tumor necrosis factor (TNF)-α, interleukin (IL)-5, IL-6, IL-10, IL-11, IL-13, IL-17, IL-25, IL-33, interferon-γ, transforming growth factor (TGF)-β, and thymic stromal lymphopoietin], eotaxins (CCL11, CCL24, and CCL26), metalloproteinase (MMP)-9 as well as extracellular matrix proteins (tenascin-C and procollagen-3) and then analyzed by laser scanning microscopy.

RESULTS

The number of eosinophils varied considerably in and between disease groups and did not correlate with the numbers of accompanying inflammatory cells. The expression of IL-5, IL-6, IL-11, TGF-β, CCL24, and MMP-9 by eosinophils significantly differed between disease groups. Eosinophils in tumors/LY predominantly expressed IL-6, TGF-β, and CCL24, but not IL-11. On the other hand, in autoimmune diseases, eosinophils largely contributed to MMP-9 production. IL-5-generating eosinophils were particularly obvious in allergic and infectious diseases.

CONCLUSIONS

In skin diseases, eosinophil expresses a broad spectrum of cytokines. The different cytokine expression patterns suggest distinct functional roles of eosinophils in these diseases that might be related to host defense, immunomodulation, fibrosis, and/or tumor development.

BACKGROUND

Rhinovirus infection is a major cause of asthma exacerbations.

OBJECTIVE

We studied nasal and bronchial mucosal inflammatory responses during experimental rhinovirus-induced asthma exacerbations.

METHODS

We used nasosorption on days 0, 2-5 and 7 and bronchosorption at baseline and day 4 to sample mucosal lining fluid to investigate airway mucosal responses to rhinovirus infection in patients with allergic asthma (n=28) and healthy non-atopic controls (n=11), by using a synthetic absorptive matrix and measuring levels of 34 cytokines and chemokines using a sensitive multiplex assay.

RESULTS

Following rhinovirus infection asthmatics developed more upper and lower respiratory symptoms and lower peak expiratory flows compared to controls (all P<0.05). Asthmatics also developed higher nasal lining fluid levels of an anti-viral pathway (including IFN-γ, IFN-λ/IL-29, CXCL11/ITAC, CXCL10/IP10 and IL-15) and a type 2 inflammatory pathway (IL-4, IL-5, IL-13, CCL17/TARC, CCL11/eotaxin, CCL26/eotaxin-3) (area under curve day 0-7, all P<0.05). Nasal IL-5 and IL-13 were higher in asthmatics at day 0 (P<0.01) and levels increased by days 3 and 4 (P<0.01). A hierarchical correlation matrix of 24 nasal lining fluid cytokine and chemokine levels over 7days demonstrated expression of distinct interferon-related and type 2 pathways in asthmatics. In asthmatics IFN-γ, CXCL10/IP10, CXCL11/ITAC, IL-15 and IL-5 increased in bronchial lining fluid following viral infection (all P<0.05).

CONCLUSIONS

Precision sampling of mucosal lining fluid identifies robust interferon and type 2 responses in the upper and lower airways of asthmatics during an asthma exacerbation. Nasosorption and bronchosorption have potential to define asthma endotypes in stable disease and at exacerbation.

BACKGROUND

Although eosinophilic esophagitis (EoE) is associated with certain gene variants, the rapidly increasing incidence of EoE suggests that environmental factors contribute to disease development.

OBJECTIVE

We tested for gene-environment interaction between EoE-predisposing polymorphisms (within TSLP, LOC283710/KLF13, CAPN14, CCL26, and TGFB) and implicated early-life factors (antibiotic use in infancy, cesarean delivery, breast-feeding, neonatal intensive care unit [NICU] admission, and absence of pets in the home).

METHODS

We conducted a case-control study using hospital-based cases (n = 127) and control subjects representative of the hospital catchment area (n = 121). We computed case-only interaction tests and in secondary analyses evaluated the combined and independent effects of genotype and environmental factors on the risk of EoE.

RESULTS

Case-only analyses identified interactions between rs6736278 (CAPN14) and breast-feeding (P = .02) and rs17815905 (LOC283710/KLF13) and NICU admission (P = .02) but not with any of the factors examined. Case-control analyses suggested that disease risk might be modifiable in subjects with certain gene variants. In particular, breast-feeding in those with the susceptibility gene variant at rs6736278 (CAPN14) reduced the risk of EoE (adjusted odds ratio, 0.08; 95% CI, 0.01-0.59). Admission to the NICU in those without the susceptibility gene variant at rs17815905 (LOC283710/KLF13) significantly increased the risk of having disease (adjusted odds ratio, 4.83; 95% CI, 1.49-15.66).

CONCLUSIONS

The interplay of gene (CAPN14 and LOC283710/KLF13) and early-life environment factors (breast-feeding and NICU admission) might contribute to EoE susceptibility.

Airway eosinophilia plays a major role in the pathogenesis of asthma with the inhibition of apoptosis by GM-CSF and IL-5 proposed as a mechanism underlying prolonged eosinophil survival. In vivo and ex vivo studies have indicated the capacity of interventions that drive human eosinophil apoptosis to promote the resolution of inflammation. Far less is known about the impact of transendothelial migration on eosinophil survival, in particular, the capacity of endothelial cell-derived factors to contribute toward the apoptosis-resistant phenotype characteristic of airway-resident eosinophils. We examined the effects of conditioned medium from human pulmonary artery endothelial cells (HPAEC-CM) on eosinophil apoptosis in vitro. HPAEC-CM inhibited eosinophil, but not neutrophil apoptosis. This effect was specific to HPAECs and comparable in efficacy to the survival effects of GM-CSF and IL-5. The HPAEC survival factor was shown, on the basis of GM-CSF, IL-5, and IL-3 detection assays, Ab neutralization, and sensitivity to PI3K inhibition, to be clearly discrete from these factors. Gel filtration of HPAEC-CM revealed a peak of eosinophil survival activity at 8-12 kDa, and PCR confirmed the presence of mRNA for CCL5, CCL11, CCL24, CCL26, and CCL27 in the HPAECs. The CCR3 antagonist GW782415 caused a major inhibition of the HPAEC-CM-induced survival effect, and Ab neutralization of individual CCR3 chemokines revealed CCL11 as the major survival factor present in the HPAEC-CM. Furthermore, chemokine Ab arrays demonstrated up-regulation of CCL11 in HPAEC-CM. These data demonstrate the capacity of HPAECs to generate CCR3 agonists and the ability of CCL11 to inhibit human eosinophil apoptosis.

Atopic dermatitis is a common pruritic and chronically relapsing inflammatory skin disease. Atopic dermatitis patients show disturbed skin barrier functions, frequent allergic responses against allergens, defects in the antimicrobial immune defense, and a genetic predisposition. Clinical and experimental evidence points to a role for staphylococcal superantigens during the initiation and amplification of atopic skin inflammation. In the past decade, numerous studies identified chemokines including CCL1, CCL2, CCL3, CCL4, CCL5, CCL11, CCL13, CCL17, CCL18, CCL20, CCL22, CCL26, CCL27 and CX3CL1 to be associated with atopic dermatitis. Here we summarize recent findings suggesting a role for staphylococcal superantigens in the production of chemokines during the development of atopic skin inflammation.

Eotaxins are the chemokines which are highly selective chemotactic agents for eosinophils. The aim of our study was the evaluation of the gene expression level for eotaxin 1/CCL11, eotaxin 2/CCL24, and eotaxin 3/CCL26, both in skin changes and in uninvolved skin of atopic dermatitis (AD) patients. The study comprised 19 patients with AD and 10 healthy controls. The gene expression level for eotaxins in the skin biopsies was evaluated by the real-time quantitative PCR. The change of the gene expression level, calculated as log10 skin lesions/non-lesional skin, was 0.635 for CCL11, 0.172 for CCL24 and 0.291 for CCL26. The change of the gene expression level, calculated as log10 non-lesional skin of AD patients/healthy control, was 0.394 for CCL11, -0.216 for CCL24, and 0.229 for CCL26, while skin lesions of AD patients/healthy control, was: 0.788, -0.046, and 0.483, respectively.

CONCLUSIONS

The mean gene expression level for CCL11, CCL24, CCL26 was higher in skin changes of AD patients than in uninvolved skin. The higher level of CCL26 in skin changes, indicates its role in their aetiology in AD. The gene expression level for CCL24 in AD patients was lower, both in involved and uninvolved skin vs. the healthy control.

Asthma is a chronic disorder characterized by airway inflammation, reversible bronchial obstruction, hyper-responsiveness and remodelling. Data from human in vitro studies and experimental in vivo models of asthma has implicated interleukin (IL)-13 in the asthma phenotype suggesting that a therapeutic agent against it could be effective in treating asthma. The role of biomarkers is becoming increasingly important in the clinical development of therapeutics. Here we describe the use of the GeneChip((R)) DNA microarray technology platform to explore and identify potential response to therapy biomarkers that are associated with the biology of IL-13. Peripheral blood mononuclear cells (PBMCs) from eight healthy donors were cultured in the presence of IL-13, IL-4, an anti-IL-13 monoclonal antibody (mAb) or an isotype control mAb, and RNA from the treated cells was subjected to microarray analysis. The results revealed a number of genes, such as CCL17 (TARC), CCL22 (MDC), CCL23 (MPIF-1), CCL26 (eotaxin 3) and WNT5A (human wingless-type MMTV integration site family member 5A), that showed increased expression in the IL-13 and IL-4 treatment groups. Real-time polymerase chain reaction (PCR) subsequently confirmed these results. A follow-up study in PBMCs from five additional healthy donors showed that the neutralization of IL-13 completely blocked IL-13-induced TARC, MDC and eotaxin 3 production at the protein level. These data suggest that TARC, MDC, eotaxin 3, CCL23 and WNT5A if validated could serve as potential biomarkers for anti-IL-13 therapeutics.

BACKGROUND

Recent data indicated that natural killer (NK) cells and chemokines could play a pivotal role in nasal inflammation. CX3CR1, the only receptor for fractalkine/CX3CL1, is abundantly expressed by NK cells, and was recently shown to also be a receptor for eotaxin-3/CCL26. However, no reports explored the NK cells-CX3CL1-CCL26 axis via CX3CR1 in allergy.

OBJECTIVE

Our goals were first to determine specifically NK cell recruitment pattern in nasal tissue of allergic chronic rhinosinusitis (ACRS) and non-allergic chronic rhinosinusitis (NACRS) patients in comparison with healthy controls, and secondly, to investigate the function of CX3CR1 in NK cell migration.

METHODS

Immunohistochemistry, microchemotaxis chambers, flow cytometry and confocal microscopy were used in this study.

RESULTS

Herein, we showed that NK cells infiltrated the epithelial layers of nasal tissue only in ACRS patients and not in NACRS patients or controls. NK cells were also more numerous in the stroma of the nasal tissue from ACRS patients compared with NACRS patients or controls. This migration could be mediated by both CX3CL1 and CCL26, as these two chemokines induced NK cell migration. Moreover, both molecules also stimulated cytoskeleton changes and F-actin reorganisation in NK cells. Chemotaxis and cytoskeleton changes were sensitive to genistein, a tyrosine kinase inhibitor. By flow cytometry, we demonstrated that a single antigen nasal provocation challenge increased the expression of CX3CR1 on NK cells in allergic rhinitis (AR) patients. The function of this receptor was associated with a significant augmentation of NK cell chemotaxis against the optimal doses of CX3CL1 and CCL26.

CONCLUSIONS

Our results highlight a novel role for CX3CR1 in NK cell migration that may contribute to the NK cell trafficking to the allergic upper airway. This could be mediated largely by CX3CL1 and CCL26 stimulation of the tyrosine kinase pathway.

BACKGROUND

Recent studies provided insights into the recruitment and activation pathways of leukocytes in atopic dermatitis, however, the underlying mechanisms of tissue remodeling in atopic skin inflammation remain elusive.

OBJECTIVE

To identify chemokine-mediated communication pathways regulating tissue remodeling during atopic skin inflammation.

METHODS

Analysis of the chemokine receptor repertoire of human dermal fibroblasts using flow cytometry and immunofluorescence. Quantitative real-time polymerase chain reaction and immunohistochemical analyses of chemokine expression in atopic vs. non-atopic skin inflammation. Investigation of the function of chemokine receptor CCR3 on human dermal fibroblasts through determining intracellular Ca(2+) mobilization, cell proliferation, migration, and repair capacity.

RESULTS

Analyses on human dermal fibroblasts showed abundant expression of the chemokine receptor CCR3 in vitro and in vivo. Among its corresponding ligands (CCL5, CCL8, CCL11, CCL24 and CCL26) CCL26 demonstrated a significant and specific up-regulation in atopic when compared to psoriatic skin inflammation. In vivo, epidermal keratinocytes showed most abundant CCL26 protein expression in lesional atopic skin. In structural cells of the skin, TH2-cytokines such as IL-4 and IL-13 were dominant inducers of CCL26 expression. In dermal fibroblasts, CCL26 induced CCR3 signaling resulting in intracellular Ca(2+) mobilization, as well as enhanced fibroblast migration and repair capacity, but no proliferation.

CONCLUSIONS

Taken together, findings of the present study suggest that chemokine-driven communication pathways from the epidermis to the dermis may modulate tissue remodeling in atopic skin inflammation.

The ontogeny of the C-C chemokines eotaxin-1, eotaxin-2, and eotaxin-3 has not been fully elucidated in human lung. We explored a possible role for eotaxin in developing lung by determining the ontogeny of eotaxin-1 (CCL11), eotaxin-2 (CCL24), eotaxin-3 (CCL26), and the eotaxin receptor, CCR3. We tested discarded surgical samples of developing human lung tissue using quantitative RT-PCR (QRT-PCR) and immunostaining for expression of CCL11, CCL24, CCL26, and CCR3. We assessed possible functionality of the eotaxin-CCR3 system by treating lung explant cultures with exogenous CCL11 and analyzing the cultures for evidence of changes in proliferation and activation of ERK1/2, a signaling pathway associated with CCR3. QRT-PCR analyses of 22 developing lung tissue samples with gestational ages 10-23 wk demonstrated that eotaxin-1 mRNA is most abundant in developing lung, whereas mRNAs for eotaxin-2 and eotaxin-3 are minimally detectable. CCL11 mRNA levels correlated with gestational age (P < 0.05), and immunoreactivity was localized predominantly to airway epithelial cells. QRT-PCR analysis detected CCR3 expression in 16 of 19 developing lung samples. Supporting functional capacity in the immature lung, CCL11 treatment of lung explant cultures resulted in significantly increased (P < 0.05) cell proliferation and activation of the ERK signaling pathway, which is downstream from CCR3, suggesting that proliferation was due to activation of CCR3 receptors by CCL11. We conclude that developing lung expresses the eotaxins and functional CCR3 receptor. CCL11 may promote airway epithelial proliferation in the developing lung.

Signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a central role in asthma pathogenesis, with its activation driving the development of airway hyper-reactivity and local inflammation. Therefore, inhibition of local STAT6 expression provides a rationale for therapeutic intervention in bronchial asthma. Given the absence of specific inhibitory drugs, we tested the ability of small interfering RNAs (siRNAs) to target STAT6 gene expression through the molecular process of RNA interference (RNAi). At pico-molar concentrations, STAT6-specific siRNAs potently inhibited STAT6 mRNA expression in lung epithelial cells (50% inhibitory concentration range = 134-861 pm) without inducing cellular interferon responses. Detectable STAT6 protein expression was rapidly abolished within 48 hr of treatment (t(1/2) range = or < 12-37 hr) and this was unaffected by pretreatment with STAT6-activating cytokines. Furthermore, STAT6 suppression by RNAi produced downstream functional inhibitory effects in that interleukin (IL)-13- or IL-4-driven eotaxin chemokine family [chemokine (C-C motif) ligand 11 (CCL11), CCL24 and CCL26] mRNA expression was markedly inhibited. Induction of detectable CCL26 protein synthesis was completely ablated by pretreating cells with STAT6-specific siRNA. The therapeutic potential of this approach is further demonstrated by novel findings that cells pre-exposed to IL-13 or IL-4 and subsequently treated with STAT6-targeting siRNA exhibited a rapid and significant attenuation of ongoing CCL26 protein expression, suggesting that chronic asthma-associated lung inflammation will be responsive to this approach.

Eotaxins induce the trafficking of eosinophils to the sites of inflammation via CC chemokine receptor 3 (CCR3). In this study, we investigated eotaxin-3/CC chemokine ligand 26 (CCL26) expression in the inflamed mucosa of patients with inflammatory bowel disease (IBD), and characterized the molecular mechanisms responsible for eotaxin-3 expression in human colonic myofibroblasts. Eotaxin-3 mRNA and protein expression was evaluated by real time-polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Eotaxin-3 mRNA expression was elevated significantly in the active lesions of ulcerative colitis (UC) patients. Significant elevations were also observed in the active lesions of Crohn's disease (CD) patients, but this was significantly lower than that detected in the active UC lesions. There were no significant increases in the inactive lesions of UC or CD patients. Colonic myofibroblasts were identified as a major source of eotaxin-3 in the colonic mucosa, and interleukin (IL)-4 and IL-13 enhanced eotaxin-3 mRNA and protein expression significantly in these cells. There was a significant positive correlation between mucosal eotaxin-3 and IL-4 mRNA expression in the active lesions of IBD patients. The IL-4- and IL-13-induced eotaxin-3 mRNA expression was regulated by the signal transducer and activator of transcription-6 (STAT-6) and suppressor of cytokine signalling (SOCS)1-mediated pathways. Interferon (IFN)-γ acts as a negative regulator on the IL-4- and IL-13-induced eotaxin-3 expression via STAT-1 activation. Eotaxin-3 expression was elevated specifically in the active lesions of IBD, in particular UC. Eotaxin-3 derived from colonic myofibroblasts may play an important role in the pathophysiology of UC.

Cancer-associated fibroblast (CAF) is one of the most crucial components of the tumor microenvironment to promote the invasiveness of cancer cells. The interactions between cancer cells and CAFs are bidirectional. Our recent study showed that up-regulations of chemokine (C-C motif) ligand 2 (CCL2), chemokine (C-C motif) ligand 26 (CCL26), interleukin 6 (IL6), and lysyl oxidase-like 2 (LOXL2) genes in cancer cells were parts of the common effects of CAFs on hepatocellular carcinoma (HCC) cells to promote proliferation, migration and invasion of cancer cells. However, the subject of how HCC cells to influence the gene expressions of CAFs still needs to be clarified. The purpose of this study was to investigate this issue. Two human HCC (HCC24/KMUH, HCC38/KMUH) and two human CAF cell lines (F26/KMUH, F28/KMUH) were studied. Influence of HCC38/KMUH cancer cells on differential expressions of genes in F28/KMUH CAFs was detected by microarray to select target genes for further analysis. Both HCC cell lines increased proliferation (all p < 0.005) and migration (all p < 0.0001) of two CAF cell lines. HCC24/KMUH cancer cells had stronger ability to promote migration of F26/KMUH CAFs than HCC38/KMUH cancer cells did (p < 0.0001). Eleven up-regulated cancer-promoting genes, including apelin (APLN), CCL2, CCL26, fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), IL6, mucin 1 (MUC1), LOXL2, platelet-derived growth factor alpha polypeptide (PDGFA), phosphoglycerate kinase 1 (PGK1), and vascular endothelial growth factor A (VEGFA) detected by microarray showed good correlation with results of quantitative reverse transcriptase-polymerase chain reaction study. Among these genes, HCC24/KMUH cancer cells had same tendency of effects on differential expressions of genes in F28/KMUH CAFs as HCC38/KMUH cancer cells did. However, the responses of F26/KMUH CAFs to different HCC cell lines were variable. Only PGK1 gene was consistently up-regulated and PDGFA gene was consistently down-regulated caused by both HCC cell lines in F26/KMUH CAFs. Besides PGK1 gene, HCC38/KMUH cancer cells only up-regulated APLN, LOXL2, and VEGFA genes and HCC24/KMUH cancer cells only up-regulated FGF2 gene in F26/KMUH CAFs. In conclusion, HCC cells can promote proliferation and migration of CAFs. However, the impact of HCC cells on differential expressions of cancer-promoting genes in CAFs is influenced by the characteristics of CAFs. This implies that blocking single or several particular cancer-promoting genes in CAFs is unable to become a common stratagem for the treatment of HCC.

Eotaxin-3/CCL26 is an agonist for chemokine receptor 3 (CCR3) and a natural antagonist for CCR1, CCR2 and CCR5. CCL26 expression by non-haematopoietic cells has been well documented; however, no studies to date have demonstrated CCL26 expression by leucocytes. In this study, we investigated the ability of human monocytic cells to produce CCL26 in response to cytokines. We found that interleukin-4 (IL-4) increased the expression of CCL26 messenger RNA (mRNA) and protein in U937 cells, in human monocytes and in human monocyte-derived macrophages. Tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) alone did not induce CCL26 expression, yet these pro-inflammatory cytokines synergized with IL-4 to increase CCL26 protein expression. Signal transducer and activator of transcription 6 (STAT6) was not affected by costimulation with TNF-alpha, suggesting that the synergy between IL-4 and TNF-alpha occurs at a step downstream of STAT6 activation. Co-incubation of interferon-gamma (IFN-gamma) with IL-4 had no effect on CCL26 protein release. By contrast, pretreatment with IFN-gamma decreased total STAT6 protein, blocked IL-4-mediated STAT6 phosphorylation and decreased IL-4-mediated CCL26 mRNA expression and protein release. These data show that IL-4 and pro-inflammatory cytokines such as TNF-alpha, IL-1beta and IFN-gamma regulate CCL26 synthesis in human monocytic cells, which may be important in regulating monocyte inflammatory responses.

OBJECTIVE

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?

CONCLUSIONS

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.

BACKGROUND

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.

METHODS

This study used the HTR8/SVneo cell line as a model for invasive EVT. All experiments were repeated on at least three separate occasions.

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.

RESULTS

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).

CONCLUSIONS

This work has been conducted in vitro with a commonly used cell line model of EVT, HTR8/SVneo.

CONCLUSIONS

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.

BACKGROUND

Chemokines ligands of CCR3 including eotaxin/CC chemokine ligand 11 (CCL11) may contribute to the pathogenesis of asthma. These chemokines and a growth factor (TGF-beta) may be involved in the process of airway remodelling.

OBJECTIVE

We analysed the effects of TGF-beta on the expression of CCR3 ligands in human airway smooth muscle (HASM) cells and investigated the mechanisms.

METHODS

HASM cells were cultured and treated with TGF-beta and Th2 cytokines IL-4 or IL-13. Expression of mRNA was analysed by real-time PCR. Secretion of CCL11 into the culture medium was analysed by ELISA. Transcriptional regulation of CCL11 was analysed by luciferase assay using CCL11 promoter-luciferase reporter plasmids.

RESULTS

IL-4 or IL-13 significantly up-regulated the expression of mRNAs for CCL11 and CCL26. TGF-beta alone did not increase the expression of chemokine mRNAs, but enhanced the induction of only CCL11 by IL-4 or IL-13 among CCR3 ligands. Activity of the CCL11 promoter was stimulated by IL-4, and this activity was enhanced by TGF-beta. Activation by IL-4 or IL-4 plus TGF-beta was lost by mutation of the binding site for signal transducers and activators of transcription-6 (STAT6) in the promoter. Cooperative activation by IL-4 and TGF-beta was inhibited by mutation of the binding site for nuclear factor-kappaB (NF-kappaB) in the promoter. Pretreatment with an inhibitor of NF-kappaB and glucocorticoid fluticasone propionate significantly inhibited the expression of CCL11 mRNA induced by IL-4 plus TGF-beta, indicating the importance of NF-kappaB in the cooperative activation of CCL11 transcription by TGF-beta and IL-4.

CONCLUSIONS

These results indicate that Th2 cytokines and TGF-beta may contribute to the pathogenesis of asthma by stimulating expression of CCL11. The transcription factors STAT6 and NF-kappaB may play pivotal roles in this process.

Knowledge of the molecules used by the maternal reproductive tract to regulate development of the preimplantation embryo is largely incomplete. The goal of the present experiment was to identify candidates for this function. The approach was to assess expression patterns in the endometrium and oviduct of 93 genes encoding for hormones, growth factors, chemokines, cytokines, and WNT-related molecules. Results show that all of the genes were expressed in the reproductive tract. Expression in oviduct was affected by day of the estrous cycle for 21 genes with 11 genes having highest expression at estrus (CCL21, CTGF, CXCL10, CXCL16, DKK3, FGF10, IL18, IL33, IL34, PGF, and SFRP2), 1 gene at d 3 (WNT4), 8 at d 5 (BMP7, HGF, IL6, SFRP1, TGFB1, WIF1, WNT2, and WNT5A), and 1 at d 7 (IK). For endometrium, expression of 34 genes was affected by day of the estrous cycle with 11 having highest expression at d 0 (BMP7, CCL14, CCL21, CCL26, CTGF, CXCL12, IGF2, IL16, IL33, SFRP2, and WIF1), 2 at d 3 (HDGF, IL15), 14 at d 5 (CSF2, CX3CL1, CXCL3, FGF1, FGF2, GRO1, HGF, IGF1, IL1B, IL8, SFRP1, SFRP4, WNT5A, and WNT16), and 7 at d 7 (CXCL16, FGF13, HDGFRP2, TDGF1, VEGFB, WNT7A, and WNT11). Results are consistent with a set of genes regulated by estradiol early in the estrous cycle and another set regulated by progesterone later in the cycle. The cell-signaling genes identified here as being expressed in the oviduct and endometrium could serve to regulate early embryonic development in a stage-of-pregnancy-specific manner.