Discovery of Potent Cyclic Sulfopeptide Chemokine Inhibitors via Reprogrammed Genetic Code mRNA Display.

Targeting chemokine signaling is an attractive avenue for the treatment of inflammatory disorders. Tyrosine sulfation is an important post-translational modification (PTM) that enhances chemokine-receptor binding and is also utilized by a number of pathogenic organisms to improve the binding affinity of immune-suppressive chemokine binding proteins (CKBPs). Here we report the display selection of tyrosine-sulfated cyclic peptides using a reprogrammed genetic code to discover high-affinity ligands for the chemokine CCL11 (eotaxin-1). The selected cyclic sulfopeptides possess high affinity for the target chemokine (as well as one or more of the related family members CCL2, CCL7 and CCL24) and inhibit CCL11 activation of CC chemokine receptor 3 (CCR3). This work demonstrates the utility of exploiting native PTMs as binding motifs for the generation of new leads for medicinal chemistry.

Stem Cell-Induced Pulp Regeneration Can Be Enhanced by Administration of CCL11-Neutralizing Antibody in the Ectopic Tooth Transplantation Model in the Aged Mice.

OBJECTIVE: Pulp regeneration by stem cell transplantation declines due to age-related reduction. We hypothesized that administration of a cytokine together with the cell transplantation may improve the stem cell niche microenvironment and promote regeneration. CCL11 is implicated as a factor in aging. This investigation was performed to investigate the changes in the quality of the regenerated pulp by administration of CCL11 antibody in the aged mice and elucidate the underlying mechanisms. MATERIALS AND METHODS: Mobilized dental pulp stem cell (MDPSC) transplants were characterized in an ectopic tooth root transplantation model in both the aged and young mice. The amount of regenerated pulp tissue was analyzed in the transplants with continuous administration of CCL11 antibody compared with those without the antibody administration. Blood CCL11 levels were assessed at the onset of the experiment. Furthermore, immunostaining of CD68 together with CD11c or CD206 for M1 and M2 macrophage, respectively, were performed. Each double-positive cell count of M1 and M2 macrophages and M1/M2 ratio in the transplants with administration were compared with those without administration both in the aged and young mice. RESULTS: The administration of CCL11 antibody enhanced pulp regeneration and significantly reduced the blood CCL11 level in the aged mice. As the number of M1 macrophages decreased, the M1/M2 ratio in the treated aged mouse was less than that in the untreated aged mouse. There was, however, significant difference between the treated aged mouse and the untreated young mouse. CONCLUSION: CCL11 antibody has the potential to enhance and stimulate pulp regeneration in the aged mice.

IL-37 inhibits IL-4/IL-13-induced CCL11 production and lung eosinophilia in murine allergic asthma.

BACKGROUND: IL-37 is emerging as an anti-inflammatory cytokine, particularly in innate inflammation. However, the role of IL-37 in Th2-mediated allergic lung inflammation remains uncertain. We sought to determine the role and the underlying mechanisms of IL-37 in the development of house dust mites (HDM)-induced murine asthma model. METHODS: We examined the effect of IL-37 administration during the sensitization or challenge phase on Th2-mediated allergic asthma induced by inhaled HDM. Cellular source of CCL11 and distribution of IL-37 receptors, IL-18Rα and IL-1R8, were determined in HDM-exposed lungs. Finally, we examined the effect of IL-37 on CCL11 production and STAT6 activation in different primary lung structural cell types upon IL-4/IL-13 stimulation. RESULTS: IL-37 had no effect on HDM sensitization, but when administrated during the challenge phase, significantly attenuated pulmonary eosinophilia, CCL11 production, and airway hyper-reactivity (AHR). Interestingly, IL-37 treatment had no significant effects on lung infiltrating T cells and Th2 cytokine production. Intranasal co-administration of CCL11 reversed the inhibiting effect of IL-37 on HDM-induced pulmonary eosinophilia and AHR. Furthermore, we demonstrated that CCL11 was primarily expressed by fibroblasts and airway smooth muscle cells (AMSC), while IL-37 receptors by tracheobronchial epithelial cells (TEC). In vitro study showed that IL-37 inhibited IL-4/IL-13-induced STAT6 activation and CCL11 production by fibroblasts and AMSC, which was dependent on its direct action on TEC. Moreover, cell contact was required for the inhibitory effect of IL-37-treated TEC. CONCLUSIONS: IL-37 attenuates HDM-induced asthma, possibly by inhibiting IL-4/IL-13-induced CCL11 production by fibroblasts and AMSC via its direct act on TEC.

1,6-O,O-Diacetylbritannilactone Inhibits Eotaxin-1 and ALOX15 Expression Through Inactivation of STAT6 in A549 Cells.

1,6-O,O-Diacetylbritannilactone (OODBL), a plant sesquiterpene lactone, was previously reported to show multiple pharmacological effects such as anti-cancer and anti-inflammatory activities. In this study, we investigated the anti-inflammatory effect of OODBL on interleukin (IL)-4-induced signal transducer and activator of transcription 6 (STAT6) signaling pathway in human lung A549 cells. We found that OODBL dramatically inhibited IL-4-induced messenger RNA (mRNA) expression of eotaxin-1 and arachidonate 15-lipoxygenase-1 (ALOX15) in a dose-dependent manner. To clarify the action mechanism of OODBL, we examined the effect of OODBL on activation of STAT6. OODBL decreased both STAT6 phosphorylation and reporter gene activity. Furthermore, OODBL suppressed phosphorylation of Janus Kinase 3 (JAK3) without affecting JAK1. Taken together, OODBL abolished IL-4-induced eotaxin-1 and ALOX15 mRNA expressions by repressing the activation of STAT6 and JAK3. These results suggest that OODBL has a potential therapeutic efficacy on inflammatory diseases especially allergic airway disease as a lead compound.

Blackcurrant anthocyanins modulate CCL11 secretion and suppress allergic airway inflammation.

CCL11, a chemokine, is linked to the early development of airways eosinophilia in allergic asthma. Therefore, CCL11 production is a target for abrogating eosinophilic-driven airway inflammation. Blackcurrants are high in compounds that regulate inflammation, particularly anthocyanins. In this study, we investigated the effect of oral blackcurrant supplementation on allergen-induced eosinophilia and CCL11 production; we also profiled key compounds in blackcurrants that were linked to this effect. Ten milligram per kilogram (total anthocyanins) of a commercially available, anthocyanin-rich New Zealand "Ben Ard" blackcurrant extract ("Currantex 30") attenuated ovalbumin-induced inflammation, eosinophilia (by 52.45 ± 38.50%), and CCL11 production (by 48.55 ± 28.56%) in a mouse model of acute allergic lung inflammation. Ten blackcurrant polyphenolic extracts were also found to suppress CCL11 secretion by stimulated human lung epithelial cells in vitro. Correlation analysis identified potential blackcurrant polyphenolic anthocyanin constituents specifically delphinidins and cyanidins, involved in CCL11 suppression. Our findings show oral supplementation with New Zealand blackcurrant is effective in reducing lung inflammation, and highlight the potential benefit of developing cultivars with specific polyphenolic profiles for the creation of functional foods with desirable biological activity.

Neutralization of RANTES and Eotaxin Prevents the Loss of Dopaminergic Neurons in a Mouse Model of Parkinson Disease.

Parkinson disease (PD) is second only to Alzheimer disease as the most common human neurodegenerative disorder. Despite intense investigation, no interdictive therapy is available for PD. Recent studies indicate that both innate and adaptive immune processes are active in PD. Accordingly, we found a rapid increase in RANTES (regulated on activation normal T cell expressed and secreted) and eotaxin, chemokines that are involved in T cell trafficking, in vivo in the substantia nigra pars compacta and the serum of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. RANTES and eotaxin were also up-regulated in the substantia nigra pars compacta of post-mortem PD brains as compared with age-matched controls. Therefore, we investigated whether neutralization of RANTES and eotaxin could protect against nigrostriatal degeneration in MPTP-intoxicated mice. Interestingly, after peripheral administration, functional blocking antibodies against RANTES and eotaxin reduced the infiltration of CD4(+) and CD8(+) T cells into the nigra, attenuated nigral expression of proinflammatory molecules, and suppressed nigral activation of glial cells. These findings paralleled dopaminergic neuronal protection, normalized striatal neurotransmitters, and improved motor functions in MPTP-intoxicated mice. Therefore, we conclude that attenuation of the chemokine-dependent adaptive immune response may be of therapeutic benefit for PD patients.

Zingiber mioga (Thunb.) Roscoe attenuates allergic asthma induced by ovalbumin challenge.

Zingiber mioga (Thunb.) Roscoe (ZM) is a traditional medicine, used to treat inflammatory diseases. The present study aimed to evaluate the inhibitory effects of ZM on the inflammatory response in lipopolysaccharide (LPS)­stimulated RAW264.7 murine macrophage cells and in a mouse model of ovalbumin (OVA)­induced allergic asthma. Mice received OVA sensitization on day 0 and 14, and were challenged with OVA between days 21 and 23. ZM was administered to the mice at a dose of 30 mg/kg, 1 h prior to OVA challenge. In LPS­stimulated RAW264.7 cells, ZM significantly decreased nitric oxide (NO) and tumor necrosis factor (TNF)­α production in a concentration­dependent manner, and mRNA expression of inducible NO synthase (iNOS), TNF­α and matrix metalloproteinase (MMP)­9 was reduced. In addition, treatment with ZM decreased the inflammatory cell count in bronchoalveolar lavage fluid from the mice, and reduced the expression of interleukin (IL)­4, IL­5, IL­13, eotaxin and immunoglobulin E. ZM also reduced airway hyperresponsiveness in OVA­challenged mice, and attenuated the infiltration of inflammatory cells and mucus production in the airways, with a decrease in the expression of iNOS and MMP­9 in lung tissue. In conclusion, the results of the present study indicate that ZM effectively inhibits inflammatory responses. Therefore, it may be that ZM has potential as a therapeutic agent for use in inflammatory diseases.

Astragalin inhibits airway eotaxin-1 induction and epithelial apoptosis through modulating oxidative stress-responsive MAPK signaling.

BACKGROUND: Eotaxin proteins are a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Since inflammation is often associated with an increased generation of reactive oxygen species (ROS), oxidative stress is a mechanistically imperative factor in asthma. Astragalin (kaempferol-3-O-glucoside) is a flavonoid with anti-inflammatory activity and newly found in persimmon leaves and green tea seeds. This study elucidated that astragalin inhibited endotoxin-induced oxidative stress leading to eosinophilia and epithelial apoptosis in airways. METHODS: Airway epithelial BEAS-2B cells were exposed to lipopolysaccharide (LPS) in the absence and presence of 1-20 µM astragalin. Western blot and immunocytochemical analyses were conducted to determine induction of target proteins. Cell and nuclear staining was also performed for ROS production and epithelial apoptosis. RESULTS: When airway epithelial cells were exposed to 2 µg/ml LPS, astragalin nontoxic at ≤ 20 µM suppressed cellular induction of Toll-like receptor 4 (TLR4) and ROS production enhanced by LPS. Both LPS and H2O2 induced epithelial eotaxin-1 expression, which was blocked by astragalin. LPS activated and induced PLCγ1, PKCß2, and NADPH oxidase subunits of p22phox and p47phox in epithelial cells and such activation and induction were demoted by astragalin or TLR4 inhibition antagonizing eotaxin-1 induction. H2O2-upregulated phosphorylation of JNK and p38 MAPK was dampened by adding astragalin to epithelial cells, while this compound enhanced epithelial activation of Akt and ERK. H2O2 and LPS promoted epithelial apoptosis concomitant with nuclear condensation or caspase-3 activation, which was blunted by astragalin. CONCLUSIONS: Astragalin ameliorated oxidative stress-associated epithelial eosinophilia and apoptosis through disturbing TLR4-PKCß2-NADPH oxidase-responsive signaling. Therefore, astragalin may be a potent agent antagonizing endotoxin-induced oxidative stress leading to airway dysfunction and inflammation.

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.

S-nitrosoglutathione reductase inhibition regulates allergen-induced lung inflammation and airway hyperreactivity.

Allergic asthma is characterized by Th2 type inflammation, leading to airway hyperresponsivenes, mucus hypersecretion and tissue remodeling. S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of intracellular levels of S-nitrosothiols. GSNOR activity has been shown to be elevated in human asthmatic lungs, resulting in diminished S-nitrosothiols and thus contributing to increased airway hyperreactivity. Using a mouse model of allergic airway inflammation, we report that intranasal administration of a new selective inhibitor of GSNOR, SPL-334, caused a marked reduction in airway hyperreactivity, allergen-specific T cells and eosinophil accumulation, and mucus production in the lungs in response to allergen inhalation. Moreover, SPL-334 treatment resulted in a significant decrease in the production of the Th2 cytokines IL-5 and IL-13 and the level of the chemokine CCL11 (eotaxin-1) in the airways. Collectively, these observations reveal that GSNOR inhibitors are effective not only in reducing airway hyperresponsiveness but also in limiting lung inflammatory responses mediated by CD4(+) Th2 cells. These findings suggest that the inhibition of GSNOR may provide a novel therapeutic approach for the treatment of allergic airway inflammation.

ß2-Agonists inhibit TNF-α-induced ICAM-1 expression in human airway parasympathetic neurons.

BACKGROUND: Major basic protein released from eosinophils to airway parasympathetic nerves blocks inhibitory M(2) muscarinic receptors on the parasympathetic nerves, increasing acetylcholine release and potentiating reflex bronchoconstriction. Recruitment of eosinophils to airway parasympathetic neurons requires neural expression of both intercellular adhesion molecular-1 (ICAM-1) and eotaxin. We have shown that inflammatory cytokines induce eotaxin and ICAM-1 expression in parasympathetic neurons. OBJECTIVE: To test whether the ß(2) agonist albuterol, which is used to treat asthma, changes TNF-alpha-induced eotaxin and ICAM-1 expression in human parasympathetic neurons. METHODS: Parasympathetic neurons were isolated from human tracheas and grown in serum-free medium for one week. Cells were incubated with either (R)-albuterol (the active isomer), (S)-albuterol (the inactive isomer) or (R,S)-albuterol for 90 minutes before adding 2 ng/ml TNF-alpha for another 4 hours (for mRNA) or 24 hours (for protein). RESULTS AND CONCLUSIONS: Baseline expression of eotaxin and ICAM-1 were not changed by any isomer of albuterol as measured by real time RT-PCR. TNF-alpha induced ICAM-1 expression was significantly inhibited by (R)-albuterol in a dose dependent manner, but not by (S) or (R,S)-albuterol. Eotaxin expression was not changed by TNF-alpha or by any isomer of albuterol. The ß-receptor antagonist propranolol blocked the inhibitory effect of (R)-albuterol on TNF-alpha-induced ICAM-1 expression. CLINICAL IMPLICATION: The suppressive effect of (R)-albuterol on neural ICAM-1 expression may be an additional mechanism for decreasing bronchoconstriction, since it would decrease eosinophil recruitment to the airway nerves.

Lactococcus lactis NCC 2287 alleviates food allergic manifestations in sensitized mice by reducing IL-13 expression specifically in the ileum.

OBJECTIVE: Utilizing a food allergy murine model, we have investigated the intrinsic antiallergic potential of the Lactococcus lactis NCC 2287 strain. METHODS: BALB/c mice were sensitized at weekly intervals with ovalbumin (OVA) plus cholera toxin (CT) by the oral route for 7 weeks. In this model, an oral challenge with a high dose of OVA at the end of the sensitization period leads to clinical symptoms. Lactococcus lactis NCC 2287 was given to mice via the drinking water during sensitization (prevention phase) or after sensitization (management phase). RESULTS: Lactococcus lactis NCC 2287 administration to sensitized mice strikingly reduced allergic manifestations in the management phase upon challenge, when compared to control mice. No preventive effect was observed with the strain. Lactococcus lactis NCC 2287 significantly decreased relative expression levels of the Th-2 cytokine, IL-13, and associated chemokines CCL11 (eotaxin-1) and CCL17 (TARC) in the ileum. No effect was observed in the jejunum. CONCLUSION/SIGNIFICANCE: These results taken together designate Lactococcus lactis NCC 2287 as a candidate probiotic strain appropriate in the management of allergic symptoms.

The patents on glucocorticosteroids and selected new therapies for the management of asthma in children.

Despite the continuous increase in the prevalence of asthma in the most underdeveloped parts of the world, nowadays, we can generally speak of a better understanding and management of this disease. The remarkable role played by the inflammatory process in asthmatic patients is well known. The aim of most asthma guidelines is to suppress inflammatory process with a combination of anti-inflammatory drugs and immunotherapy. The management of asthma in children is a challenge because of their inability to express warning signs and seek medical attention in a timely manner. Unlike adults, asthmatic children must rely on their parents or caregivers for the administration of asthma medications. This inability to carry and self-administer asthma drugs may increase the risk of non-compliance. Glucocorticosteroids, the most important drugs for patients with asthma, are associated with an increased level of side effects and compliance issues mostly in children. In an attempt to solve that dilemma, emphasis is being placed on the modification of current management tactics and the introduction of other drugs. This review presents more recent patent therapies for the management of asthma in children.

Increase effect of transforming growth factor on eotaxin production by normal cultured dermal fibroblasts stimulated with interleukin-4: inhibitory effect of suplatast tosilate on eotaxin production.

Eotaxin plays a central role in the development of allergic disease, including atopic dermatitis, asthma, and nasal allergy. Interleukin (IL)-4 induces eotaxin production in normal human dermal fibroblasts. On the other hands, Transforming growth factor-beta (TGF-beta), a multifunctional regulatory cytokine, affects many biological functions, including fibroblast growth and differentiation and Th2 cytokine regulation. In this study, we investigated the effect of TGF-beta on IL-4-induced eotaxin production by normal human fibroblasts, as well as the effect of suplatast tosilate, an antiallergic drug that selectively inhibits Th2 cytokine production. Dermal fibroblast treatment with IL-4 and TGF-beta for 24 h increased eotaxin production and expression of eotaxin mRNA, as measured by enzyme-linked immunosorbent assay (ELISA) and reverse-transcriptase polymerase chain reaction (RT-PCR), respectively. TGF-beta synergistically up-regulated eotaxin production and eotaxin mRNA expression when stimulated with IL-4. Suplatast tosilate dose-dependently inhibited eotaxin production induced by IL-4 or IL-4 plus TGF-beta. These results suggest that TGF-beta may regulate skin allergic inflammation by up-regulating eotaxin production in dermal fibroblasts. Suplatast tosilate might suppress this inflammation by inhibiting eotaxin production.

Targeting CCL11 in the treatment of ovarian cancer.

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

CCR3 is a target for age-related macular degeneration diagnosis and therapy.

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

Licorice flavonoids inhibit eotaxin-1 secretion by human fetal lung fibroblasts in vitro.

Glycyrrhiza uralensis (Gan-Cao), commonly called "licorice", is one of the most commonly used herbs in traditional Chinese medicine (TCM). In the United States, licorice products are most often consumed as flavoring and sweetening agents in food products. The licorice triterpenoid glycyrrhizin has several biological activities, including anti-inflammatory activity. Other potential anti-inflammatory constituents in G. uralensis have not been fully investigated. Airway eosinophilic inflammation is a major feature of allergic asthma. Eotaxin-1 (eotaxin) is involved in the recruitment of eosinophils to sites of antigen-induced inflammation in asthmatic airways. Because human lung fibroblasts are the major source of eotaxin, inhibition of eosinophil recruitment by suppression of fibroblast eotaxin production is a potentially valuable approach for the pharmacological intervention in asthma. A systematic bioassay-guided purification of G. uralensis yielded five flavonoids: liquiritin, liquiritigenin, isoliquiritigenin, 7,4'-dihydroxyflavone, and isoononin. The structures of the compounds were established by (1)H and (13)C nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) studies. The potential ability of these isolated pure compounds and glycyrrhizin to inhibit secretion of eotaxin-1 by human fetal lung fibroblasts (HFL-1) was tested. Liquiritigenin, isoliquiritigenin, and 7,4'-dihydroxyflavone were more effective than liquiritin, isoononin, and glycyrrhizin in suppressing eotaxin secretion. A concentration-response study showed the IC(50) concentrations of liquiritigenin, isoliquiritigenin, and 7,4'-dihydroxyflavone were 4.2, 0.92, and 0.21 microg/mL, respectively, demonstrating that Glycyrrhiza flavonoids inhibit eotaxin-1 secretion in vitro.

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.