<AbstractText>Various profibrotic and proinflammatory cytokines have been found upregulated in uncomplicated primary retinal detachment (pRD), but without providing a uniform picture. Here, we compare the cyto- and chemokine profiles in pRD with and without proliferative vitreoretinopathy (PVR) in an attempt to unravel relevant differences not in single cytokines, but in the cytokine profiles at diagnosis.</AbstractText><AbstractText>Undiluted vitreous fluid (VF) was obtained at the beginning of surgery from 174 eyes with pRD without relevant PVR (maximally grade B; group 1; n = 81) and with moderate or advanced PVR requiring a gas tamponade (group 2; n = 49) or silicon oil filling (group 3; n = 44). VF of eyes undergoing macular hole (MH) surgery served as controls (group 4; n = 26). Forty-three cytokines were quantified in parallel using a multiplex cytokine analysis system (Bioplex). For all comparisons we applied Holm's correction to control for multiple comparisons.</AbstractText><AbstractText>44.9% of group 2 eyes presented grade C1 and 55.1% C2-C3, whereas 86.4% of group 3 eyes exhibited a PVR grade of C2-D. CCL19 was the only cytokine that displayed higher concentrations in the vitreous of eyes with PVR C1 compared to lower PVR grades. Eyes with PVR C2-D showed higher levels of CCL27, CXCL6, IL4, IL16, CXCL10, CCL8, <em>CCL22</em>, MIG/CXCL9, CCL15, CCL19, CCL 23 and CXCL12 compared to controls. Interestingly, no difference of cytokine levels was detected between C1 and C2-D PVR.</AbstractText><AbstractText>CCL19 may represent a potential biomarker for early PVR progression that holds promise for future diagnostic and therapeutic applications.</AbstractText>

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are chronic neuroinflammatory autoimmune diseases characterized by axonal loss, demyelination and neurodegeneration of the central nervous system (CNS). Overactivation of CD4(+)T cells, especially the Th1 and Th17 subsets, is thought to play a causal role in this disease. In this study, we investigated the immunomodulatory effects of IFN-β treatment in EAE. IFN-β significantly inhibits disease severity, and decreases levels of CCR2, CCR4, CCR5, CCR6 and CXCR3 in the CNS. This was associated with fewer Th1/Th17 cells expressing these chemokine receptors. Furthermore, levels of their corresponding ligands CCL2, CCL3, CCL4, CCL5, CCL20, CCL22 and CXCL10 were also reduced, coinciding with reduced CNS inflammation and demyelination. Chemokine expression significantly correlated with disease severity. Furthermore, we demonstrate that IFN-β reduces CCL2/CCL5 induced-T cell migration by inhibiting p38-MAPK and ERK1/2 activation. Our results reveal that IFN-β reduces the expression of chemokines and chemokine receptors expressed by encephalitogenic Th1/Th17 cells, thereby decreasing their migration into the CNS.

BACKGROUND

Obesity negatively impacts asthma control, but the inflammatory mechanisms are poorly understood.

OBJECTIVE

To explore which systemic inflammatory mediators mediate the effects of obesity on asthma control.

METHODS

The subjects with stable asthma (n = 108) underwent assessment of clinical characteristics, which included using The Asthma Control Questionnaire (ACQ)-6. Obesity was defined as a body mass index (BMI) of ≥30 kg/m2, overweight was defined as BMI between 25 to 29.9 kg/m2, and lean weight was defined as BMI < 25 kg/m2. Body composition, including fat mass (FM), visceral fat area (VFA), and percentage body fat (PBF) was analyzed by bioimpedance. Serum interleukin (IL) 4, IL-5, IL-8, IL-13, IL-17, chemokine (C-C motif) ligand (CCL) 17, CCL22, leptin, adiponectin, C-reactive protein (CRP), and interferon (IFN) gamma were measured by using ELISA. Linear regression models were fitted according to the Baron and Kenny procedures for mediation analysis.

RESULTS

FM (12.73 ± 3.95 versus 18.59 ± 2.95 versus 27.82 ± 5.17 kg; p < 0.0001), VFA (65.99 ± 23.17 versus 93.96 ± 10.28 versus 123.10 ± 18.34 cm2; p < 0.0001), PBF (23.86 ± 7.46 versus 30.74 ± 5.08 versus 36.21 ± 6.28 %; p = 0.0003) and ACQ-6 values (0.83 [0, 1.17]) versus 1.15 [0.50, 1.75] versus 1.33 [0.83, 1.83] score; p = 0.002) were different among lean (n = 52), overweight (n = 37), and obese (n = 19) subjects. Serum levels of leptin, IL-5, IL-13, IL-17, CCL17, CRP, and IFN-gamma in the obese group were significantly elevated compared with the subjects who were lean or overweight (all p < 0.05). The mediation analyses found that the effect of obesity, assessed by BMI, on ACQ-6 was significantly mediated through IL-13 and CCL17. Furthermore, IL-13 and CCL17 mediated the effects of body composition (FM, VFA and PBF) on ACQ-6. The effects of obesity assessed by body composition, but not by using BMI, on ACQ-6 were mediated by leptin.

CONCLUSIONS

Our mediation analysis confirmed that systemic inflammation biomarkers, such as leptin, CCL17, IL-4, and IL-13, mediated the effects of obesity on asthma control. This warrants prospective exploration in this distinct asthma phenotype in the future.

Corticosteroids are extensively used in pediatrics, yet the burden of side effects is significant. Availability of a simple, fast, and reliable biochemical read out of steroidal drug pharmacodynamics could enable a rapid and objective assessment of safety and efficacy of corticosteroids and aid development of corticosteroid replacement drugs. To identify potential corticosteroid responsive biomarkers we performed proteome profiling of serum samples from DMD and IBD patients with and without corticosteroid treatment using SOMAscan aptamer panel testing 1,129 proteins in <0.1 cc of sera. Ten pro-inflammatory proteins were elevated in untreated patients and suppressed by corticosteroids (MMP12, IL22RA2, CCL22, IGFBP2, FCER2, LY9, ITGa1/b1, LTa1/b2, ANGPT2 and FGG). These are candidate biomarkers for anti-inflammatory efficacy of corticosteroids. Known safety concerns were validated, including elevated non-fasting insulin (insulin resistance), and elevated angiotensinogen (salt retention). These were extended by new candidates for metabolism disturbances (leptin, afamin), stunting of growth (growth hormone binding protein), and connective tissue remodeling (MMP3). Significant suppression of multiple adrenal steroid hormones was also seen in treated children (reductions of 17-hydroxyprogesterone, corticosterone, 11-deoxycortisol and testosterone). A panel of new pharmacodynamic biomarkers for corticosteroids in children was defined. Future studies will need to bridge specific biomarkers to mechanism of drug action, and specific clinical outcomes.

The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptor-ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 and HGF and reducing the secretion of TIMP-1.

Asthma is a complex disease linked to various pathophysiological events including the activity of proteinases. The multifunctional A disintegrin and metalloproteinases (ADAMs) displaying the ability to cleave membrane-bound mediators or cytokines appear to be key mediators in various inflammatory processes. In the present study, we investigated ADAM-8 expression and production in a mouse model of allergen-induced airway inflammation. In allergen-exposed animals, increased expression of ADAM-8 was found in the lung parenchyma and in DC purified from the lungs. The potential role of ADAM-8 in the development of allergen-induced airway inflammation was further investigated by the use of an anti-ADAM-8 antibody and ADAM-8 knockout animals. We observed a decrease in allergen-induced acute inflammation both in BALF and the peribronchial area in anti-ADAM-8 antibody-treated mice and in ADAM-8-deficient mice (ADAM-8(-/-) ) after allergen exposure. ADAM-8 depletion led to a significant decrease of the CD11c(+) lung DC. We also report lower levels of CCL11 and CCL22 production in antibody-treated mice and ADAM-8- deficient mice that might be explained by decreased eosinophilic inflammation and lower numbers of DC, respectively. In conclusion, ADAM-8 appears to favour allergen-induced acute airway inflammation by promoting DC recruitment and CCL11 and CCL22 production.

Atopic dermatitis (AD) is a prevalent chronic inflammatory skin disease. The use of immunomodulatory corticosteroids in AD treatment causes adverse side effects. Therefore, novel natural anti-inflammatory therapeutics are needed. The aim of the present study was to investigate the anti-allergic and anti-inflammatory activities of kuwanon G and morusin. To investigate the effect of kuwanon G and morusin on skin inflammation, enzyme-linked immunosorbent assays (ELISA) to quantitate secreted (RANTES/CCL5), thymus- and activation-regulated chemokine (TARC/CCL17), and macrophage-derived chemokine (MDC/CCL22) were performed, followed by Western blotting to measure the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and nuclear transcription factor-κB (NF-κB) p65 in tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-stimulated HaCaT keratinocytes. In order to evaluate the anti-allergic effects, ELISA to quantify histamine and leukotriene C₄ (LTC₄) production and Western blotting to measure 5-lipoxygenase (5-LO) activation were performed using PMA and A23187-stimulated MC/9 mast cells. Kuwanon G reduced the release of RANTES/CCL5, TARC/CCL17, and MDC/CCL22 via down-regulation of STAT1 and NF-κB p65 signaling in TNF-α and IFN-γ-stimulated HaCaT keratinocytes. Kuwanon G also inhibited histamine production and 5-LO activation in PMA and A23187-stimulated MC/9 mast cells. Morusin inhibited RANTES/CCL5 and TARC/CCL17 secretion via the suppression of STAT1 and NF-κB p65 phosphorylation in TNF-α and IFN-γ-stimulated HaCaT keratinocytes, and the release of histamine and LTC₄ by suppressing 5-LO activation in PMA and A23187-stimulated MC/9 mast cells. Kuwanon G and morusin are potential anti-inflammatory mediators for the treatment of allergic and inflammatory skin diseases such as AD.

BACKGROUND

Previous studies suggest that dendritic cells and macrophages play an important role in inflammation of eosinophilic pneumonia. The mechanism of dendritic cell and macrophage accumulation into the lung, however, is unknown. Here, we hypothesized that CCR7 ligands, CCL19 and CCL21, contribute to the accumulation of dendritic cells and alveolar macrophages in the inflamed lung of patients with eosinophilic pneumonia.

METHODS

Concentrations of the CCR7 ligands as well as CCL16, CCL17 and CCL22 in the bronchoalveolar lavage fluid of 53 patients with eosinophilic pneumonia, 29 patients with sarcoidosis, 18 patients with idiopathic pulmonary fibrosis and 12 healthy volunteers were measured by enzyme-linked immunosorbent assay. Cell sources of CCR7 ligands and CCR7-expressing cells in the bronchoalveolar lavage fluid were evaluated by immunocytochemistry.

RESULTS

CCL19 and CCL21 levels in the bronchoalveolar lavage fluid were significantly higher in patients with eosinophilic pneumonia than in controls. Levels of CCL19, but not CCL21, were statistically correlated with the levels of CCL16, CCL17 and CCL22 in patients with eosinophilic pneumonia. Immunocytochemistry revealed CCL19 expression in dendritic cells, macrophages and T-lymphocytes harvested from patients with eosinophilic pneumonia, and CCR7 expression in dendritic cells and macrophages. Levels of CCL19, but not CCL21, were significantly decreased after remission in patients with eosinophilic pneumonia. After provocation tests, CCL19 levels were elevated in all patients with eosinophilic pneumonia.

CONCLUSIONS

These findings indicate that CCL19 rather than CCL21 may contribute to the accumulation of dendritic cells and macrophages in the inflamed lungs of patients with eosinophilic pneumonia.

Immune molecules, which have been found to be important in tumor microenvironment, seem prospective in tumor therapy, but they are still not effective enough to use in clinical practice. C-C motif chemokine 22 (CCL22) exists in various malignancies and correlates with migration of regulatory T cells, but its clinical significance in gastric cancer is still unclear. In this study, a combined data set of 466 patients with gastric cancer after surgical resection, comprised of a discovery (n = 319) and a validation data set (n = 147), was enrolled. CCL22 expression was assessed by immunohistochemical staining and we evaluated prognostic values of CCL22 staining and clinical outcomes with use of Kaplan-Meier curve and Multivariate Cox regression analysis. Positive CCL22 expression predicted adverse overall survival independent of traditional pathological grade. Multivariate analysis defined CCL22 and TNM stage as two independent prognostic factors for overall survival. Besides, in patients with TNM stage II/III disease, the rate of overall survival was higher among patients with CCL22-positive tumors who were treated with 5-fluorouracil based adjuvant chemotherapy than that among those who were not (P = 0.012, P < 0.001 and P < 0.001, in discovery, validation and combined data set). But for these with CCL22-negative tumors, whether to undergo adjuvant chemotherapy showed no statistical significance (P = 0.595, P = 0.085 and P = 0.252, respectively). To conclude, CCL22 was identified as an independent adverse prognostic immunobiomarker for patients with gastric cancer after surgery, which is associated with tumor-infiltrating immunocytes and could be incorporated into TNM staging system to redefine a high-risk subgroup who were more likely to benefit from 5-fluorouracil based adjuvant chemotherapy.

Ex vivo expansion of CD8+ T-cells has been a hindrance for the success of adoptive T cell transfer in clinic. Currently, preconditioning with chemotherapy is used to modulate the patient immunity before ACT, however, the tumor microenvironment beneficial for transferring T cells may also be damaged. Here preconditioning with single low dose of doxorubicin or paclitaxel combined with fewer CD8+ T-cells was investigated to verify whether the same therapeutic efficacy of ACT could be achieved. An E.G7/OT1 animal model that involved adoptive transfer of OVA-specific CD8+ T-cells transduced with a granzyme B promoter-driven firefly luciferase and tomato fluorescent fusion reporter gene was used to evaluate this strategy. The result showed that CD8+ T-cells were activated and sustained longer in mice pretreated with one low-dose Dox or Tax. Enhanced therapeutic efficacy was found in Dox or Tax combined with 2x106 CD8+ T-cells and achieved the same level of tumor growth inhibition as that of 5x106 CD8+ T-cells group. Notably, reduced numbers of Tregs and myeloid derived suppressor cells were shown in combination groups. By contrast, the number of tumor-infiltrating cytotoxic T lymphocytes and IL-12 were increased. The NF-κB activity and immunosuppressive factors such as TGF-β, IDO, CCL2, VEGF, CCL22, COX-2 and IL-10 were suppressed. This study demonstrates that preconditioning with single low dose Dox or Tax and combined with two fifth of the original CD8+ T-cells could improve the tumor microenvironment via suppression of NF-κB and its related immunosuppressors, and activate more CD8+ T-cells which also stay longer.

Chemokines are important mediators of cell migration, and thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well-known typical inflammatory chemokines involved in atopic dermatitis (AD). (+)-Nootkatone is the major component of Cyperus rotundus. (+)-Nootkatone has antiallergic, anti-inflammatory, and antiplatelet activities. The purpose of this study was to investigate the effect of (+)-nootkatone on tumor necrosis factor α (TNF-α)/interferon γ (IFN-γ)-induced expression of Th2 chemokines in HaCaT cells. We found that (+)-nootkatone inhibited the TNF-α/IFN-γ-induced expression of TARC/CCL17 and MDC/CCL22 mRNA in HaCaT cells. It also significantly inhibited TNF-α/IFN-γ-induced activation of nuclear factor kappa B (NF-κB), p38 mitogen-activated protein kinase (MAPK), and protein kinase Cζ (PKCζ). Furthermore, we showed that PKCζ and p38 MAPK contributed to the inhibition of TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression by blocking IκBα degradation in HaCaT cells. Taken together, these results suggest that (+)-nootkatone may suppress TNF-α/IFN-γ-induced TARC/CCL17 and MDC/CCL22 expression in HaCaT cells by inhibiting of PKCζ and p38 MAPK signaling pathways that lead to activation of NF-κB. We propose that (+)-nootkatone may be a useful therapeutic candidate for inflammatory skin diseases such as AD.

Although chemokines are critical elements for the selective attraction and activation of various leukocyte subsets in the inflammatory process, there are few findings concerning T helper (Th) 1 or Th2 chemokines in autoimmune blistering disease (ABD). To determine whether serum levels of chemokines that are preferentially chemotactic for Th1 (monokine induced by IFN-gamma (MIG/CXCL9)) and Th2 (thymus and activation regulated chemokine (TARC/CCL17) and macrophage derived chemokine (MDC/CCL22)) cells were elevated and whether they correlated with the clinical features in patients with ABD. Serum chemokine levels were examined using ELISA in patients with pemphigus vulgaris (PV, n=19), pemphigus foliaceous (PF, n=14), or bullous pemphigoid (BP, n=27) and normal controls (n=20). Serum MIG levels were significantly higher in patients with PV, PF, or BP than those in the control subjects. Serum levels of TARC and MDC were also significantly elevated in patients with PV, PF, or BP relative to the normal controls. Among the ABD subgroups, the levels of each chemokine tended to be higher in BP patients than in PV patients. Furthermore, serum TARC levels correlated positively with serum IgE levels in patients with ABD. Levels of TARC, MDC, and MIG were significantly decreased after treatment when the skin lesions disappeared in these patients. Furthermore, serum MIG levels correlated positively with serum levels of TARC and MDC in the ABD patients. These results suggest that both a Th1 chemoattractant MIG and Th2 chemoattractants, TARC and MDC, cooperatively play a role in the development of ABD.

Recent studies have demonstrated that regulatory T cells (Tregs) are recruited to tumor sites where they can suppress antitumor immunity. The chemokine receptor CCR4 is expressed at high levels on functional CD4+CD25+FoxP3+ Tregs and production of the CCR4 ligand CCL22 by tumor cells and tumor-associated macrophages is associated with Treg recruitment to the tumor site. Here, we tested IgG1 and IgG4 isotypes of human anti-CCR4 mAb2-3 for their in vitro activity and in vivo capacity in a NSG mouse model bearing CCL22-secreting ovarian cancer (OvCA) xenograft to modulate Tregs and restore antitumor activity. Both mAb2-3 isotypes blocked in vitro chemoattraction of Tregs to CCL22-secreting OvCA cells. However, they differed in their in vivo mode of action with IgG1 causing Treg depletion and IgG4 blocking migration to the tumors. Primary T cells that were primed with OvCA-pulsed dendritic cells (DCs) demonstrated INFγ secretion that could be enhanced through Treg depletion by mAb2-3. Humanized mice reconstructed with allogeneic tumor-primed T cells (TP-T) were used to evaluate the restoration of OvCA immunity by depletion or blockade of Tregs with mAb2-3. We observed that IgG1 was more potent than IgG4 in inhibiting tumor growth. Mechanism studies demonstrated that mAb2-3 treatment lead to inhibition of IL-2 binding to its receptor. Further studies showed that mAb2-3 induced CD25 shedding (sCD25) from Tregs which lead to a decrease in IL-2-dependent survival. Together, the results demonstrate that mAb2-3 is an agonist antibody that can restore anti-OvCA immunity through modulation of Treg activity.

Macrophages (MΦs) are critical regulators of immune response and serve as a link between innate and acquired immunity. The precise mechanism of involvement of tissue-resident MΦs in the pathogenesis of autoimmune diseases is not clear. Here, using a murine model for Sjögren's syndrome (SS), we investigated the role of tissue-resident MΦs in the onset and development of autoimmunity. Two unique populations of CD11bhigh and CD11blow resident MΦs were observed in the target tissue of the SS model. Comprehensive gene expression analysis of chemokines revealed effective production of CCL22 by the CD11bhigh MΦs. CCL22 upregulated the migratory activity of CD4+ T cells by increasing CCR4, a receptor of CCL22, on T cells in the SS model. In addition, CCL22 enhanced IFN-γ production of T cells of the SS model, thereby suggesting that CCL22 may impair the local immune tolerance in the target organ of the SS model. Moreover, administration of anti-CCL22 antibody suppressed autoimmune lesions in the SS model. Finally, histopathological analysis revealed numerous CCL22-producing MΦs in the minor salivary gland tissue specimens of the SS patients. CCL22-producing tissue-resident MΦs may control autoimmune lesions by enhancing T cell response in the SS model. These results suggest that specific chemokines and their receptors may serve as novel therapeutic or diagnostic targets for SS.

Within hours after stroke, potentially cytotoxic pro-inflammatory mediators are elevated within the brain; thus, one potential therapeutic strategy is to reduce them and skew the brain toward an anti-inflammatory state. Because interleukin-4 (IL-4) treatment induces an anti-inflammatory, "alternative-activation" state in microglia and macrophages in vitro, we tested the hypothesis that early supplementation of the brain with IL-4 can shift it toward an anti-inflammatory state and reduce damage after transient focal ischemia. Adult male rat striata were injected with endothelin-1, with or without co-injection of IL-4. Inflammation, glial responses and damage to neurons and white matter were quantified from 1 to 7 days later. At 1 day, IL-4 treatment increased striatal expression of several anti-inflammatory markers (ARG1, CCL22, CD163, PPARγ), increased phagocytic (Iba1-positive, CD68-positive) microglia/macrophages, and increased VEGF-A-positive infiltrating neutrophils in the infarcts. At 7 days, there was evidence of sustained, propagating responses. IL-4 increased CD206, CD200R1, IL-4Rα, STAT6, PPARγ, CD11b, and TLR2 expression and increased microglia/macrophages in the infarct and astrogliosis outside the infarct. Neurodegeneration and myelin damage were not reduced, however. The sustained immune and glial responses when resolution and repair processes have begun warrant further studies of IL-4 treatment regimens and long-term outcomes.

The potential use of lymphoid chemokines to generate a dendritic cell (DC) cancer vaccine is not yet clear. We investigated the effect of lymphoid chemokines on DC function and on the production of effective cytotoxic T lymphocytes (CTLs) for application of cancer vaccine using monocyte-derived mature DCs (mDCs) prestimulated with lymphoid chemokines. mDCs exposed to a secondary lymphoid organ chemokine (SLC/CCL21) dramatically induced CTL response by increasing cytolytic activity without any significant alterations on expression of cell surface markers (e.g. CD80, CD83, CD86 and CCR7) or on the production of cytokines (e.g. IL-12p70, IL-10 and IL-23). Interestingly, mDCs prestimulated with CCL21 showed higher levels of CXCL10 (IP-10) production, but not the production of CCL22, compared with untreated mDCs. IP-10 treatment during CTL generation with DCs dramatically enhanced tumour-specific CTL response compared with untreated CTLs, and these enhanced CTL-inducing functions of CCL21-treated DCs were inhibited by anti-IP-10 treatment. Taken together, our data suggested an important role of the lymphoid-endothelium-associated chemokine, CCL21, on DCs in the induction of CTL responses.

The increased generation of reactive oxygen species (ROS) induces inflammation in different cell types. However, it is unclear whether ROS play an essential role in the production of thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) in keratinocytes. Here, we investigated the function of ROS in the production of these two Th2 chemokines in interferon-gamma (IFN-γ)-treated HaCaT keratinocytes. We found that IFN-γ-induced production of both chemokines in parallel with the increased generation of intracellular ROS. A ROS scavenger, N-acetyl cysteine (NAC), significantly inhibited the IFN-γ-induced production of chemokines as well as the activation of I kappa-B (IκB)-nuclear factor-kappa B (NF-κB). Inhibitors of Janus family kinases (JAKs), p38 mitogen-activated kinase (MAPK), and NF-κB suppressed IFN-γ-induced production of TARC and MDC. NF-κB activation was inhibited by both inhibitors of JAKs and p38 MAPK. Importantly, IFN-γ-stimulated phosphorylation of p38 MAPK was significantly suppressed by JAKs inhibitors, but not significantly affected by NAC or L-buthionine sulfoximine (L-BSO). However, IFN-γ-stimulated activation of IκB and NF-κB was suppressed by NAC but enhanced by BSO. Furthermore, inhibition of p38 MAPK and JAKs did not affect ROS generation in IFN-γ-stimulated HaCaT cells. These results indicate that intracellular ROS and JAKs/p38 MAPK both contribute independently to IFN-γ-stimulated production of TARC and MDC in HaCaT keratinocytes, by increasing NF-κB activation.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a chronically progressive interstitial lung disease of unknown etiology. Previously, we have demonstrated the selective upregulation of the macrophage-derived chemokine CCL22 and the thymus activation-regulated chemokine CCL17 among chemokines, in a rat model of radiation pneumonitis/pulmonary fibrosis and preliminarily observed an increase in bronchoalveolar (BAL) fluid CCL22 levels of IPF patients.

METHODS

We examined the expression of CCR4, a specific receptor for CCL22 and CCL17, in bronchoalveolar lavage (BAL) fluid cells, as well as the levels of CCL22 and CCL17, to elucidate their pathophysiological roles in pulmonary fibrosis. We also studied their immunohistochemical localization.

RESULTS

BAL fluid CCL22 and CCL17 levels were significantly higher in patients with IPF than those with collagen vascular diseases and healthy volunteers, and there was a significant correlation between the levels of CCL22 and CCL17 in patients with IPF. CCL22 levels in the BAL fluid did not correlate with the total cell numbers, alveolar lymphocytes, or macrophages in BAL fluid. However, the CCL22 levels significantly correlated with the numbers of CCR4-expressing alveolar macrophages. By immunohistochemical and immunofluorescence analysis, localization of CCL22 and CCR4 to CD68-positive alveolar macrophages as well as that of CCL17 to hyperplastic epithelial cells were shown. Clinically, CCL22 BAL fluid levels inversely correlated with DLco/VA values in IPF patients.

CONCLUSIONS

We speculated that locally overexpressed CCL22 may induce lung dysfunction through recruitment and activation of CCR4-positive alveolar macrophages.

Macrophages (MΦ), well-known to play an important role in immune response, also respond to environmental toxic chemicals such as diesel exhaust particles (DEP). Potential effects of DEPs towards MΦ polarization, a key hall-mark of MΦ physiology, remain however poorly documented. This study was therefore designed to evaluate the effects of a reference DEP extract (DEPe) on human MΦ polarization. Human blood monocytes-derived MΦ were incubated with IFNγ+LPS or IL-4 to obtain M1 and M2 subtypes, respectively; a 24 h exposure of polarizing MΦ to 10 μg/ml DEPe was found to impair expression of some macrophagic M1 and M2 markers, without however overall inhibition of M1 and M2 polarization processes. Notably, DEPe treatment increased the secretion of the M1 marker IL-8 and the M2 marker IL-10 in both MΦ subtypes, whereas it reduced lipopolysaccharide-induced IL-6 and IL-12p40 secretion in M1 MΦ. In M2 MΦ, DEPe exposure led to a reduction of CD200R expression and of CCL17, CCL18 and CCL22 secretion, associated with a lower chemotaxis of CCR4-positive cells. DEPe activated the Nrf2 and AhR pathways and induced expression of their reference target genes such as Hmox-1 and cytochrome P-4501B1 in M1 and M2 MΦ. Nrf2 or AhR silencing through RNA interference prevented DEPe-related down-regulation of IL-6. AhR silencing also inhibited the down-secretion of IL-12p40 and CCL18 in M1- and M2-DEPe-exposed MΦ, respectively. DEPs are therefore likely to alter expression of some M1 and M2 markers in an AhR- and Nrf2-dependent manner; such regulations may contribute to deleterious immune effects of atmospheric DEP.

BACKGROUND

Chronic Obstructive Pulmonary Disease (COPD) is characterized by lung and systemic inflammation as well as airway goblet cell hyperplasia (GCH). Mucin production is activated in part by stimulation of the epidermal growth factor (EGF) receptor pathway through neutrophils and macrophages. How circulating cytokine levels relate to GCH is not clear.

METHODS

We performed phlebotomy and bronchoscopy on 25 subjects (six nonsmokers, 11 healthy smokers, and eight COPD subjects FEV1 30-60 %). Six endobronchial biopsies per subject were performed. GCH was measured by measuring mucin volume density (MVD) using stereological techniques on periodic acid fast-Schiff stained samples. We measured the levels of chemokines CXCL8/IL-8, CCL2/MCP-1, CCL7/MCP-3, CCL22/MCD, CCL3/MIP-1α, and CCL4/MIP-1β, and the cytokines IL-1, IL-4, IL-6, IL-9, IL-17, EGF, and vascular endothelial growth factor (VEGF). Differences between groups were assessed using one-way ANOVA, t test, or Chi squared test. Post hoc tests after ANOVA were performed using Bonferroni correction.

RESULTS

MVD was highest in healthy smokers (27.78 ± 10.24 μL/mm(2)) compared to COPD subjects (16.82 ± 16.29 μL/mm(2), p = 0.216) and nonsmokers (3.42 ± 3.07 μL/mm(2), p < 0.0001). Plasma CXCL8 was highest in healthy smokers (11.05 ± 8.92 pg/mL) compared to nonsmokers (1.20 ± 21.92 pg/mL, p = 0.047) and COPD subjects (6.01 ± 5.90 pg/mL, p = 0.366). CCL22 and CCL4 followed the same trends. There were no significant differences in the other cytokines measured. When the subjects were divided into current smokers (healthy smokers and COPD current smokers) and non/ex-smokers (nonsmokers and COPD ex-smokers), plasma CXCL8, CCL22, CCL4, and MVD were greater in current smokers. No differences in other cytokines were seen. Plasma CXCL8 moderately correlated with MVD (r = 0.552, p = 0.003).

CONCLUSIONS

In this small cohort, circulating levels of the chemokines CXCL8, CCL4, and CCL22, as well as MVD, attain the highest levels in healthy smokers compared to nonsmokers and COPD subjects. These findings seem to be driven by current smoking and are independent of airflow obstruction.

CONCLUSIONS

These data suggest that smoking upregulates a systemic pattern of neutrophil and macrophage chemoattractant expression, and this correlates significantly with the development of goblet cell hyperplasia.

BACKGROUND

Calcineurin inhibitors (CNIs) exhibit remarkable efficacy in atopic dermatitis (AD). Tacrolimus, one type of CNI, is prevalently used to treat AD. AD is a chronic inflammatory disease that exhibits predominant infiltration of T-helper type 2 (Th2) cell in the acute phase and a mixed Th1 and Th0 cell pattern in chronic lesions. Cytokines such as tumor necrosis factor-α (TNF-α), Th2-related chemokines [e.g., macrophage-derived chemokine (MDC)/CCL22 and I-309/CCL1], Th1-related chemokines [e.g., interferon γ-induced protein 10 (IP-10)/CXCL10], and neutrophil chemoattractant growth-related oncogene-α (GRO-α)/CXCL1 are involved in the pathogenesis of AD. However, whether tacrolimus modulates the expression of AD-associated cytokines and chemokines remains unknown. The intracellular mechanisms of tacrolimus are also unclear.

METHODS

Human monocytic cell line THP-1 cells were pretreated with tacrolimus and stimulated with lipopolysaccharide (LPS). The MDC, I-309, IP-10, GRO-α, and TNF-α concentrations of the cell supernatants were measured using enzyme-linked immunosorbent assay. Intracellular signaling was investigated using the Western blot analysis.

RESULTS

Tacrolimus suppressed the expression of MDC, IP-10, I-309, GRO-α, and TNF-α in LPS-stimulated THP-1 cells in a dose- and time-dependent manner. All three mitogen-activated protein kinase (MAPK) inhibitors and the nuclear factor-κB inhibitor suppressed LPS-induced MDC, I-309, and TNF-α expressions in THP-1 cells. Only MAPK inhibitors suppressed LPS-induced expression of IP-10 and GRO-α. Tacrolimus suppressed the LPS-induced phosphorylation of MAPK-extracellular signal-related kinase (ERK).

CONCLUSIONS

Tacrolimus suppressed LPS-induced MDC, I-309, IP-10, GRO-α, and TNF-α expressions in monocytes through the MAPK-ERK pathway; thus, tacrolimus may yield therapeutic efficacy by modulating AD-associated cytokines and chemokines.

BACKGROUND

The chemokine receptor CCR4 has been implicated in Th2 cell-mediated immune responses. However, other T cell subsets are also known to participate in allergic inflammation.

OBJECTIVE

The role of CCR4 in Th1, Th2, and Th17 cell-mediated allergic airway inflammation was investigated.

METHODS

We generated an allergic airway inflammation model by adoptive transfer of in vitro-polarized ovalbumin (OVA)-specific Th1, Th2, and Th17 cells. The effect of a low-molecular weight CCR4 antagonist, Compound 22, on this model was examined.

RESULTS

Upon in vitro polarization of DO11.10 naïve T cells, Th1- and Th2-polarized cells dominantly expressed CXCR3 and CCR4, respectively, while Th17-polarized cells expressed CCR6 and CCR4. Intranasal OVA-challenge of mice transferred with each T cell subset induced accumulation of T cells in the lungs. Eosinophils were also massively accumulated in Th2-transferred mice, whereas neutrophils were preferentially recruited in Th1- and Th17-transferred mice. Compound 22, as well as anti-CCL17 or anti-CCL22 antibody selectively suppressed accumulation of Th2 cells and eosinophils in the lungs of Th2-transferred and OVA-challenged mice. Compound 22 also inhibited bronchial hyperresponsiveness but had little effect on goblet cell hyperplasia in Th2-transferred and OVA-challenged mice.

CONCLUSIONS

There were notable differences in allergic lung inflammation mediated by different T cell subsets. CCR4 blockage was selectively effective for suppression of Th2-mediated allergic inflammation by blocking infiltration of Th2 cells.