Tumour-associated fibroblasts (TAFs) are key elements of the tumour microenvironment, but their role in pituitary neuroendocrine tumours (PitNETs) has been little explored. We hypothesised that TAF-derived cytokines may play a role in tumour aggressiveness, and that their release can be inhibited by somatostatin analogues. TAFs were isolated and cultured from 16 PitNETs (11 clinically non-functioning tumours and 5 somatotropinomas). The fibroblast secretome was assessed with a 42-plex cytokine array before and after multiligand somatostatin receptor agonist pasireotide treatment. Angiogenesis and epithelial-to-mesenchymal transition pathway assessment included CD31, E-cadherin and ZEB1 expression. GH3 cells treated with TAF- or skin fibroblast-conditioned medium were assessed for migration, invasion and cell morphology changes. PitNET TAFs secreted significant amounts of cytokines including CCL2, CCL11, VEGF-A, CCL22, IL-6, FGF-2 and IL-8. TAFs from PitNETs with cavernous sinus invasion secreted more IL-6 levels compared to fibroblasts from non-invasive tumours (p=0.027). Higher CCL2 release from TAFs correlated with more capillaries (r=0.672, p=0.004), and TAFs from PitNETs with a higher Ki-67 tended to secrete more CCL2 (p=0.058). SST1 is the predominant somatostatin receptor in TAFs, and pasireotide decreased TAF-derived IL-6 by 80% (p<0.001) and CCL2 by 35% (p=0.038). GH3 cells treated with TAF-conditioned medium showed increased migration and invasion compared to cells treated with skin fibroblast-conditioned medium, with morphological and E-cadherin and ZEB1 expression changes suggesting epithelial-to-mesenchymal transition. TAF-derived cytokines may increase PitNETs aggressiveness, alter angiogenesis and induce epithelial-to-mesenchymal transition changes. Pasireotide's inhibitory effect on TAF-derived cytokines suggest that this effect may play a role in its anti-tumour effects.

BACKGROUND

Silencing of genes using small interfering RNA (siRNA) is a recently developed strategy to regulate the synthesis of target molecules. Signal transducer and activator of transcription 6 (STAT6) is a nuclear transcription factor that mediates Th2-type immunity.

METHODS

To elucidate the therapeutic potential of using siRNA to inhibit STAT6 in allergic reactions, we determined the nucleotide sequences of siRNA specific for STAT6.

RESULTS

The selected sequences of STAT6 siRNA specifically inhibited the generation of STAT6 synthesis in dermal fibroblasts and eotaxin (CCL11) production in response to IL-4/TNF-α in vitro. Local administration of STAT6 siRNA in vivo alleviated contact hypersensitivity responses to chemical haptens. This was accompanied by reduced local production of IL-4, IL-13, eotaxin (CCL11), TARC (CCL17) and MDC (CCL22). Similarly, consecutive intranasal instillation of STAT6 siRNA markedly inhibited inflammatory cellular infiltration of mucosal tissues in allergic rhinitis responses in association with reduced IL-4 and IL-5 production from regional lymph node cells. Immediate responses, such as sneezing and nasal rubbing behaviors, were also improved by STAT6 siRNA.

CONCLUSIONS

Local administration of STAT6 siRNA is thus a promising therapeutic strategy for both Th2-mediated cutaneous diseases and allergic rhinitis.

Macrophage-derived chemokine (MDC/CCL22) plays a role in Experimental Autoimmune Encephalomyelitis (EAE), the animal model of Multiple Sclerosis (MS). MDC/CCL22 gene is part of a chemokine cluster, which includes also thymus and Activation-Regulated Chemokine (TARC/CCL17). The frequency of the C/T and C/A Single Nucleotide Polymorphisms (SNPs) in the promoter and coding sequence of CCL22 as well as the C/T SNP in the promoter of CCL17 were determined in 370 patients with Multiple Sclerosis (MS) compared with 380 controls. A trend towards a decreased allelic frequency of the A allele of the CCL22 C/A SNP as well as of the T allele of the CCL17 C/T SNP was found in patients compared with controls. The frequency of the AT haplotype was significantly decreased in MS patients (P=0.017, OR: 0.49, CI: 0.28-0.87). Stratifying patients according to gender, the observed association was even more pronounced in male patients compared with male controls (P=0.004, OR=0.18, 95% CI: 0.06-0.50), whereas no significant differences were observed in females. Therefore, the presence of the AT haplotype in chromosome 16 chemokine cluster is likely to confer a decreased risk of developing MS, particularly in males.

Vibrio cholerae induces acute inflammatory response at intestinal epithelial surface; the underlying cellular immune mechanisms for such effects are largely unexplored. Mucosal immune response is controlled by crosstalk between the intestinal epithelial cells (ECs) and dendritic cells (DCs). An EC-derived cytokine thymic stromal lymphopoietin (TSLP) has been found a critical regulator of several human inflammatory conditions. TSLP is highly elevated in ECs stimulated with V. cholerae and its recombinant flagellin (rFlaA). V. cholerae treated human ECs produce DC-attracting chemokine MIP-3α (CCL20). Flagellin, a potent V. cholerae factor was responsible for maximum stimulation of epithelial CCL20 production and subsequent DC activation. Activated DCs express high levels of costimulatory molecules and secrete inflammatory cytokines TNF-α, IL-6 and IL-1β. Bacteria stimulated ECs conditioned DCs to produce Th2 cell-attracting chemokines CCL17 and CCL22. TSLP and other mediators present in the V. cholerae stimulated EC-culture filtrate potently activated DCs, which subsequently primed CD4(+)T cells to differentiate into T helper type 2 (Th2) cells that produce high amounts of IL-4, IL-13 and TNF-α and low IFN-γ. TSLP-induced proinflammatory response in DCs involved the transcriptional mechanisms, MAPKs (ERK1/2, p38 and JNK) and STAT3 activation. This study suggests TSLP and other mediators released from ECs in response to V. cholerae colonization actively influence DCs in initiating inflammatory response.

T cell infiltration at the tumor site has been identified as a major predictor for the efficacy of adoptive T cell therapy. The chemokine C-C motif ligand 22 (CCL22) is highly expressed by immune cells in murine and human pancreatic cancer. Expression of its corresponding receptor, C-C chemokine receptor type 4 (CCR4), is restricted to regulatory T cells (Treg). We show that transduction of cytotoxic T cells (CTL) with CCR4 enhances their immigration into a pancreatic cancer model. Further, we show that binding of CCR4 with CCL22 strengthens the binding of T cell LFA-1 to dendritic cell (DC) ICAM-1 and increases CTL activation. In vivo, in a model of subcutaneous pancreatic cancer, treatment of tumor-bearing mice with CCR4-transduced CTL led to the eradication of established tumors in 40% of the mice. In conclusion, CCR4 overexpression in CTL is a promising therapeutic strategy to enhance the efficacy of adoptive T cell transfer (ACT).

BACKGROUND

CC chemokine ligand 17 (CCL17) and CCL22 are the functional ligands for CCR4. We previously reported that inhibitors of nuclear factor-kappa B and p38 mitogen-activated protein kinase (p38 MAPK), but not of extracellular signal-related kinase (ERK), inhibited tumor necrosis factor (TNF)-α- and interferon (IFN)-γ-induced production of CCL17 by the human keratinocyte cell line, HaCaT. Further, an inhibitor of epidermal growth factor receptor (EGFR) enhanced the CCL17 production by these keratinocytes.

OBJECTIVE

To identify the mechanism underlying CCL22 production by HaCaT cells.

METHODS

We investigated the signal transduction pathways by which TNF-α and IFN-γ stimulate HaCaT cells to produce CCL22 by adding various inhibitors.

RESULTS

TNF-α- and IFN-γ-induced CCL22 production was inhibited by PD98059, PD153035, Bay 11-7085, SB202190, c-Jun N-terminal kinase (JNK) inhibitor II, and Janus kinase (JAK) inhibitor 1.

CONCLUSIONS

Our results indicate that CCL22 production in HaCaT cells is dependent on ERK, EGFR, p38 MAPK, JNK, and JAK and is mediated by different signal pathways from those regulating CCL17 production. Altogether, our previous and present results suggest that EGFR activation represses CCL17 but enhances CCL22 production by these cells.

OBJECTIVE

The aim of this study was to explore the tape strip sampling technique in the assessment of stratum corneum levels of inflammatory mediators in a clinical trial setting.

METHODS

Thirty-eight inflammatory mediators were analyzed by a multiplex-assay in the stratum corneum, collected by adhesive tapes before and after 6 weeks of therapy, in mild and moderate atopic dermatitis (AD) patients (n = 90). Treatment was a ceramide- and magnesium-containing emollient.

RESULTS

Twenty-four mediators could quantitatively be determined. The Th2 mediators interleukin (IL)-4, IL-13, CCL2 (monocyte chemotactic protein-1), CCL22 (macrophage-derived chemokine), and CCL17 [thymus and activation-regulated chemokine (TARC)] were significantly decreased after therapy as well as IL-1β, IL-2, IL-8 (CXCL8), IL-10, acute-phase protein serum amyloid A, C-reactive protein, and vascular adhesion molecule-1. The decrease of CCL17 and IL-8 was correlated with the decrease in disease severity in a subgroup of moderate AD individuals.

CONCLUSIONS

Stratum corneum tape stripping offers a minimally invasive approach for studying local levels of immunomodulatory molecules in the skin. CCL17 (TARC) and IL-8 were found to be the most promising biomarkers of AD and might be useful for investigating the course of skin diseases and the effect of local therapy.

BACKGROUND

Atopic dermatitis (AD) affects children of all skin types. Most research has focused on light skin types. Studies investigating biomarkers in people with AD with dark skin types are lacking.

OBJECTIVE

To explore skin barrier and immune response biomarkers in stratum corneum (SC) tape strips from children with AD with different skin types.

METHODS

Tape strips were collected from lesional and nonlesional forearm skin of 53 children with AD and 50 controls. We analysed 28 immunomodulatory mediators, and natural moisturizing factors (NMF) and corneocyte morphology.

RESULTS

Interleukin (IL)-1β, IL-18, C-X-C motif chemokine (CXCL) 8 (CXCL8), C-C motif chemokine ligand (CCL) 22 (CCL22), CCL17, CXCL10 and CCL2 were significantly higher (P < 0·05) in lesional AD skin compared with nonlesional AD skin; the opposite trend was seen for IL-1α. CXCL8, CCL2 and CCL17 showed an association with objective SCORing Atopic Dermatitis score. NMF levels showed a gradual decrease from healthy skin to nonlesional and lesional AD skin. This gradual decreasing pattern was observed in skin type II but not in skin type VI. Skin type VI showed higher NMF levels in both nonlesional and lesional AD skin than skin type II. Corneocyte morphology was significantly different in lesional AD skin compared with nonlesional AD and healthy skin.

CONCLUSIONS

Minimally invasive tape-stripping is suitable for the determination of many inflammatory mediators and skin barrier biomarkers in children with AD. This study shows differences between children with AD with skin type II and skin type VI in NMF levels, suggesting that some aspects of pathophysiological mechanisms may differ in AD children with light versus dark skin types.

We examined the inhibitory effect of a histamine 4 receptor (H4R) antagonist (JNJ7777120) on CCL17 and CCL22 chemokine production by human monocyte-derived Langerhans cells (MoLC) in patients with atopic dermatitis (AD) and healthy controls (HC). We confirmed the significantly higher production of both CCL17 and CCL22 in the MoLC of AD patients compared with HC. The H4R antagonist significantly inhibited the production of both CCL17 and CCL22 in the MoLC of AD patients. With regard to TLR2-signaled enhancement, peptidoglycan (PGN)-enhanced production of CCL17 and CCL22 by MoLC was inhibited by the H4R. Immunoblotting analysis demonstrated that phosphorylated p38 mitogen-activated protein kinase was induced by PGN and that this enhancement was attenuated by the application of the H4R antagonist. These data indicate that H4 signaling modulates the production of T-helper 2 chemokine in MoLC and contributes to chronic inflammation in AD patients. Our data suggest a possible novel therapeutic approach using a H4R antagonist in the treatment of patients with AD.

Usual type VIN is a premalignant disorder caused by persistent HPV infection. High prevalence of VIN in immuno-suppressed women suggests that a good innate and adaptive immune response is important for defense against HPV. Here, we explored expression levels of chemokines and related these to the presence or absence of immuno-competent cells (dendritic and T-cells) in affected (HPV-positive VIN) and non-affected (HPV-negative) vulvar tissues from the same patients. Combining microarray data with quantitative real-time RT-PCR, it was observed that several important chemokines were differentially expressed between VIN and control samples (up-regulation of IL8, CXCL10, CCL20 and CCL22 and down-regulation of CXCL12, CCL21 and CCL14). Furthermore, an increased number of mature dendritic cells (CD208+) seemed to be bottled up in the dermis, and although a T-cell response (increased CD4+ and CD8+ cells) was observed in VIN, a much larger response is required to clear the infection. In summary, it seems that most mature dendritic cells do not receive the proper chemokine signal for migration and will stay in the dermis, not able to present viral antigen to naive T-cells in the lymph node. Consequently the adaptive immune response diminishes, resulting in a persistent HPV infection with increased risk for neoplasia.

The aggressiveness of melanoma is believed to be correlated with tumor-stroma-associated immune cells. Cytokines and chemokines act to recruit and then modulate the activities of these cells, ultimately affecting disease progression. Because melanoma frequently metastasizes to the brain, we asked whether global differences in immunokine profiles could be detected in the cerebrospinal fluid (CSF) of melanoma patients and reveal aspects of tumor biology that correlate with patient outcomes. We therefore measured the levels of 12 cytokines and 12 chemokines in melanoma patient CSF and the resulting data were analyzed to develop unsupervised hierarchical clustergrams and heat maps. Unexpectedly, the overall profiles of immunokines found in these samples showed a generalized reconfiguration of their expression in melanoma patient CSF, resulting in the segregation of individuals with melanoma brain metastasis from nondisease controls. Chemokine CCL22 and cytokines IL1α, IL4, and IL5 were reduced in most samples, whereas a subset including CXCL10, CCL4, CCL17, and IL8 showed increased expression. Further, analysis of clusters identified within the melanoma patient set comparing patient outcome suggests that suppression of IL1α, IL4, IL5, and CCL22, with concomitant elevation of CXCL10, CCL4, and CCL17, may correlate with more aggressive development of brain metastasis. These results suggest that global immunokine suppression in the host, together with a selective increase in specific chemokines, constitute a predominant immunomodulatory feature of melanoma brain metastasis. These alterations likely drive the course of this disease in the brain and variations in the immune profiles of individual patients may predict outcomes.

OBJECTIVE

Chemokines are small soluble molecules which mediate leukocyte migration and may be involved in the pathophysiology of preterm labor. We aimed to determine if serum concentrations of selected chemokines are changed in preterm labor and delivery.

METHODS

A novel array-based enzyme-linked immunosorbent assay was used to quantitate serum levels of nine chemokines from a single sample: MDC/CCL22, TARC/CCL17, ITAC/CXCL11, I-309/CCL1, IP-10/CXCL10, MIP-1alpha/CCL3, -1beta/CCL4, -3alpha/CCL20 and -3beta/CCL19. Women in preterm labor who delivered (n = 17), women at preterm pregnancy not in labor (n = 13) and women in labor at term (n = 8) participated.

RESULTS

In the preterm delivery group of patients, the MIP-3beta/CCL19 concentration was in mean (+/-S.D.) 70.4+/-31.7 pg/mL, which was significantly lower than that in preterm gravidas not in labor of 123+/-34 pg/mL (p < 0.001) and those in labor at term of 118+/-25.6 pg/mL (p < 0.01). The other measured chemokines did not differ significantly.

CONCLUSIONS

Of a small number of examined chemokines, we were able to show that one of them, MIP-3beta/CCL19 was significantly lower in women with preterm labor and delivery. Whether or not this chemokine has a potential as biochemical marker of preterm delivery remains to be determined.

Intestinal failure is common in patients with septic shock, with dysfunction of the gut often manifesting as both a cause and consequence of their critical illness. Most studies investigating the pathogenesis of intestinal failure focus on the systemic aspect, although few data examine the inflammatory signaling in the intestinal lumen. Having previously demonstrated apical/luminal chemokine secretion in an in vitro model of intestinal inflammation, we hypothesized that endotoxemia would induce secretion of proinflammatory chemokines into the intestinal lumen. In addition, we examined the contribution of these mediators to intestinal dysmotility. C57/BL6 male mice were injected intraperitoneally with LPS. Serum, intestinal tissue, and intestinal luminal contents were harvested for cytokine analysis. For intestinal motility studies, a transit assay was performed after oral gavage of chemokines. Caco-2 cells grown on Transwell culture inserts were used to examine the role of the intestinal epithelium in chemokine secretion. Monocyte chemoattractant protein 1 (MCP-1/CCL2) and macrophage-derived chemokine (MDC/CCL22) were secreted into the lumen of multiple segments of the gut during endotoxemia in mice. In vitro work showed that the intestinal epithelium participates in monocyte chemoattractant protein 1 and MDC secretion and expresses the CCR2 and CCR4 receptors for these chemokines. Intestinal transit studies show that oral gavage of MDC results in impaired gut motility. This study demonstrates that the intestinal lumen is an active compartment in the gut's inflammatory response. Proinflammatory chemokines are secreted into the intestinal lumen during endotoxemia. These intraluminal chemokines contribute to intestinal dysmotility, complicating intestinal failure.

Kawasaki disease (KD) is a childhood-onset vascular disease. In order to determine whether KD is associated with altered chemokine production, we measured CCL2, CCL22, and CXCL10 levels in the serum of KD patients and healthy control subjects. The mean serum concentration of CCL2 in KD subjects was 829.0 +/- 388.2 pg/ml, significantly higher than that seen in healthy controls (223.4 +/- 92.6 pg/ml; p < 0.001). In addition, the mean serum CXCL10 level in KD subjects was 2,469.4 +/- 998.8 pg/ml, again significantly higher than that in healthy controls (127.7 +/- 64.2 pg/ml; p < 0.001). No difference was observed in serum concentrations of CCL22 between KD and healthy controls (1,685 +/- 1,985 microg/ml and 1,539 +/- 380 microg/ml, respectively). Thus, we observed the selective induction of a TH1-associated (CXCL10) and a TH2-associated chemokine (CCL2) in the serum of individuals with KD, suggesting a mixed TH1/TH2 response at the level of chemokine production and subsequent cell recruitment and thus pointing at a potential role for these chemokines in the pathology of KD.

Macrophages can be polarized into classically (CAM) or alternatively (AAM) activated macrophages with IFN-γ or IL-4, respectively. CAM are associated with type 1 immune responses and are implicated in autoimmunity; AAM are associated with type 2 responses and are implicated in allergic diseases. An impediment in investigating macrophage biology using primary human monocyte derived macrophages is the wide inter-donor heterogeneity and the limited quantity of cells that survive in vitro polarization. To overcome this impediment, we established a protocol to generate CAM and AAM cultures derived from the THP-1 human promonocytic cell line. In this report, we demonstrate that THP-CAM and -AAM express gene and protein markers that define their primary human monocyte derived counterparts, such as IL-1β, CXCL10, and CXCL11 for CAM, and MRC1, IL-4 and CCL22 for AAM. In addition, we demonstrate that STAT6 is selectively activated in THP-AAM which, upon LPS stimulation, have an attenuated or delayed expression of IFN-β, IFN-λ1, and IFN α/β pathway genes compared to their CAM counterparts. Taken together, these findings may help further investigate human diseases associated with the alternatively activated macrophage phenotype using this reproducible in vitro macrophage model.

Allergic bronchopulmonary aspergillosis (ABPA) is a frequent syndrome in patients with cystic fibrosis (CF) or asthma. Animal models revealed distinct roles for the chemokines CCL2, CCL3, CCL5, CCL6, CCL17 and CCL22 and their receptors in the pathogenesis of allergic aspergillosis. In humans, serum levels of the CCR4 ligand CCL17 identified ABPA in patients with CF or asthma, suggesting CCL17 as novel diagnostic marker and future therapeutical target in ABPA. This review illustrates the manifold role of chemokines in animal models of allergic aspergillosis and translates these findings to human lung diseases.

Inorganic arsenic is an environmental contaminant toxic for key immune cells. We recently reported that low micromolar concentrations of arsenic trioxide (As(2)O(3)) alter functions and differentiation gene program of human macrophages. Particularly, prolonged treatment with As(2)O(3) concomitantly reverses expression of a macrophage-specific gene subset and triggers reactive oxygen species (ROS) production, suggesting a possible role of cell stress in As(2)O(3) gene effects. This study was thus designed to determine whether redox-sensitive signaling pathways could mediate gene expression in metalloid-exposed macrophages. Our results show that As(2)O(3)-dependent alterations of stress (HMOX1 and GCLM) and macrophage-specific (MMP9, CCL22, and CXCL2) gene expression are not mediated by ROS or related signaling pathways. Notably, As(2)O(3) alters neither activity of the redox-sensitive transcription factor Sp1 nor that of AP-1 or NF-kappaB. In contrast, N-acetylcysteine, a potent cysteine reductive compound, significantly prevents up-regulation of HMOX1, GCLM, and CXCL2 genes, and repression of MMP9 and CCL22 genes induced by As(2)O(3). In addition, we demonstrate that As(2)O(3) markedly alters nuclear levels of Nrf2 and Bach1, two redox-sensitive regulators of stress genes, and represses expression of the transcription factor EGR2 which is involved in mouse macrophage differentiation; such effects are reduced by N-acetylcysteine. Finally, we report that genetic invalidation of EGR2 gene partially mimics metalloid effects; it significantly represses CCL22 gene expression and weakly induces that of CXCL2. In conclusion, our results demonstrate that As(2)O(3) alters macrophage gene expression through redox-sensitive signaling pathways unrelated to ROS production and reveal the transcription factor EGR2 as a new molecular target of arsenic.

Monocyte-derived dendritic cell functions have been explored for identification of contact allergens in vitro. Current methods, including measurement of changes in cell surface marker expression (e.g. CD83, CD86) do not provide a sensitive method for detecting the sensitising potential of a chemical. In this study, we investigated whether chemokine production by monocyte-derived dendritic cells is increased upon maturation and whether chemokine production can provide methodology for the detection of allergens. Monocyte-derived dendritic cells were exposed to allergens (nickel sulphate, cobalt chloride, palladium chloride, copper sulphate, chrome-(III)-chloride, potassium dichromate, p-phenylenediamine and dinitrochlorobenzene) and irritants (sodium dodecyl sulphate, dimethylsulphoxide, benzalkoniumchloride and propane-1-ol). CD83 and CD86 expression was analysed by flow cytometry and chemokine production (CXCL8, CCL5, CCL17, CCL18, CCL19, CCL20, CCL22) was determined by ELISA. Significant up regulation of CD83 and CD86 expression could only be induced by three out of seven and five out of seven allergens, respectively. In contrast, CXCL8 production was significantly increased after stimulation with all allergens tested, whereas irritant exposure led to decreased CXCL8 production. All other chemokines tested, failed in identifying contact allergens. In conclusion, CXCL8 production, next to CD83 and CD86 up regulation, by monocyte-derived dendritic cells provides a promising in vitro tool for discrimination between allergens and irritants.

BACKGROUND

Chemerin has been implicated to play opposing roles, either pro-inflammatory or anti-inflammatory, in various tissue inflammation processes primarily through the regulation of tissue recruitment of immune cells. However, the effect of chemerin in allergic asthma has not yet been explored. We sought to investigate the role of chemerin in the murine model of allergic asthma and explore the underlying mechanism.

METHODS

We examined the effect of intranasal (i.n.) administration of chemerin during antigen challenge in murine models of asthma. Moreover, we examined whether administration of CCL2 or bone marrow-derived dendritic cells (BMDCs) transfer reversed the effects of chemerin on ovalbumin-induced asthma. We finally examined the effect of chemerin on CCL2 expression in activated lung epithelial cells in vitro.

RESULTS

The administration of chemerin attenuated allergic airway inflammation and airway hyperreactivity during antigen challenge. Chemerin treatment caused significant decreases in BALF CD4(+) T-cell accumulation and mRNA expression of Th2-attracting chemokines, CCL17 and CCL22, which was accompanied by significantly decreased BALF CD11c(+) CD11b(+) inflammatory DC accumulation and CCL2 production. Furthermore, airway administration of exogenous CCL2 or adoptive transfer of CD11c(+) CD11b(+) BMDCs abrogated the suppressive effects of chemerin on allergic asthma. Finally, in vitro study showed that chemerin inhibited CCL2 secretion by low-dose LPS-stimulated lung epithelial cells, which led to decreased chemotaxis of BMDCs.

CONCLUSIONS

Our study demonstrates that chemerin plays a protective role in allergic asthma by suppressing airway recruitment of inflammatory CD11c(+) CD11b(+) DCs through the inhibition of CCL2 secretion by active lung epithelial cells.

OBJECTIVE

The aim was to investigate the mucosal activation of a broad range of genes associated with the T-helper 17 cell (Th17) signaling pathway in children at different stages of celiac disease (CD), including children with increased risk for CD and children with untreated and gluten-free diet (GFD)-treated CD.

METHODS

Small intestinal biopsies were taken from children with untreated and GFD-treated CD, transglutaminase antibody (TGA)-positive children with potential CD, and reference children. Real-time polymerase chain reaction (PCR) arrays were used to study the gene expression pattern of Th17-related genes, and quantitative PCR was used to study the interleukin (IL)-17A expression.

RESULTS

The mucosal expression of CD8A was elevated at all stages of CD. Children with untreated CD had diminished levels of IL-17RE, IL-23R, RORc, STAT6, CCL22, NFATC2, IL-18, CD4, CD247, and matrix metalloproteinase (MMP)9 but had elevated levels of MMP3, IL-17, interferon-γ (IFN-γ) and CD8A, compared to references. The majority of the aforementioned genes, being differentially expressed in untreated CD, displayed similar expression in GFD-treated children and references. Children with untreated and GFD-treated CD had elevated expression of IFN-γ but had reduced expression of CD247. Interestingly, children with potential CD displayed reduced FOXP3, IL-21, and IL-17A levels.

CONCLUSIONS

Mucosal upregulation of Th17 immunity occurs at the late stage of disease and is downregulated with dietary treatment, thus indicating that IL-17 immunity is not a fundamental feature of CD as Th1 immunity, which is not fully downregulated by GFD.

To define cytokine concentrations and detectability in children with noninflammatory neurological disorders (NIND). The multiplex bead assay technology was used for simultaneous measurement of 34 soluble cytokines/chemokines in cerebrospinal fluid (CSF) from 73 NIND. Sera from 36 healthy children and 37 NIND also were analyzed. In CSF, CXCL10 had the highest concentration; CCL2, CXCL10, and interleukin (IL)-6 were detectable in all samples, and CXCL8, CCL22, CXCL1, IL-16, and IL-1 receptor antagonist were found in ≥50% of the samples. In serum, CXCL1 had the highest concentration; sIL-2Ra, CXCL1, CXCL10, and CCL22 were detectable in all samples, and CCL2, IL-12, CCL5, and granulocyte monocyte colony-stimulating factor (GM-CSF) were found in ≥50% of the samples. The mean CSF:serum ratio for CCL2 was several-fold higher than the rest, with the CXCL10 and CXCL8 ratios also >1. Intercorrelations between CSF cytokines included CCL2 versus CXCL8 and IL-6, and CXCL1 versus CCL22, reflecting both T-helper-1 (Th1)/Th1 and Th1/Th2 relations. Serum correlations included CCL11 versus CCL2, GM-CSF, and IL-4. For serum cytokines, the agreement between healthy children and NIND was good, with the exception of higher CCL4 in NIND. Cytokines in children varied greatly in concentration and detectability, with chemokines predominating in the CSF. These data allow investigators to select their own kit cytokines, instead of manufacturer-selected cytokines, for greater cost-effectiveness and interpretability.

Chemokines are believed to play a crucial role in local immunoresponse by regulating leukocyte movement in various tissues, including the intestinal mucosa. It has been suggested that they are key players in cancer biology, and several studies have identified leukocyte infiltration as a hallmark of most cancers. The chemokines CCL17 and CCL22 attract CCR4-bearing cells, which are especially polarised to Th2-type cells and regulatory T cells (Treg). Recent studies have revealed the participation of the CCL17 and CCL22 proteins in diseases such as atopic dermatitis and lymphoma. The purpose of this study was to assess the role of CCL17 and CCL22 protein expression in colorectal cancer (CRC) and to ascertain whether an association exists between promoter -431C>T CCL17 and -961G>A CCL22 gene polymorphisms in CRC versus non-CRC subjects. Using the ELISA assay, we noted a significantly higher expression of CCL22 in tumour tissue with a 2.3-fold up-regulation (tumour vs. paired normal tissue, n=78) but no significant difference in CCL17 protein expression. Immunohistochemistry revealed protein expression of CCL22 and CCL17 in the epithelial compartment of cancer tissue, in epithelial cells at the resection border that reflects normal tissue, and in some stromal cells such as lymphocytes, macrophages, and fibroblasts. Using a TaqMan system we screened for -431C>T CCL17 and -961G>A CCL22 gene variants in 245 CRC patients and 256 controls, but could not find any significant difference in genotype distribution or in allelic frequencies between the two groups. The genotype and allelic distributions of CRC patients were not related to tissue levels of CCL17 and CCL22 protein, and none of the variables were associated with plasma levels or clinical characteristics. To ascertain whether the tissue expression of CCL17 and CCL22 exerts an influence on the pathogenesis of CRC, a forthcoming study on the 5-year survival rate of CRC patients will be conducted.

Chemokines may contribute to the systemic inflammation that is linked to the increased risk of co-morbidities in patients with psoriasis. The aim of this study was to investigate circulating chemokines in patients with psoriasis and their relationship to disease severity. Analysis of plasma levels of chemokines in patients with psoriasis before narrowband ultraviolet B (UVB) therapy revealed increased expression of Th1-associated CXCL9 and -10, Th2-associated CCL17 and CCL22, and Th17-associated CCL20. CCL20 correlated with disease severity. UVB therapy reduced skin symptoms, but did not affect the chemokine levels in plasma. Anti-CD3 and anti-CD28-mediated activation of peripheral blood mononuclear cells (PBMCs) caused a higher secretion of Th2 cytokine interleukin (IL)-13 by PBMCs from patients with psoriasis than from healthy controls. The sustained high expression of inflammatory chemokines is a potential link to systemic inflammation in psoriasis. UVB therapy may be a more effective treatment of local rather than systemic inflammation.

Atopic dermatitis (AD) is characterized by skin inflammation, barrier dysfunction and chronic pruritus. In this review, recent advances in the pathogenesis of AD are summarized. Clinical efficacy of the anti-IL-4 receptor antibody dupilumab implies that type 2 cytokines IL-4 and IL-13 have pivotal roles in atopic inflammation. The expression of IL-4 and IL-13 as well as type 2 chemokines such as CCL17, CCL22 and CCL26 is increased in the lesional skin of AD. In addition, IL-4 and IL-13 down-regulate the expression of filaggrin in keratinocytes and exacerbate epidermal barrier dysfunction. Keratinocytes in barrier-disrupted epidermis produce large amounts of thymic stromal lymphopoietin, IL-25 and IL-33, conducing to type 2 immune deviation via OX40L/OX40 signaling. IL-31, produced by type 2 T cells, is a cardinal pruritogenic cytokine. IL-4 and IL-13 also amplify the IL-31-mediated sensory nerve signal. These molecules are particularly important targets for future drug development for AD.