Pancreatic ductal adenocarcinoma (PDA) has an aggressive natural history and is resistant to therapy. The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor for many damage-associated molecular pattern molecules. RAGE is overexpressed in both human and murine models of PDA as well as most advanced epithelial neoplasms. The immunosuppressive nature of the PDA microenvironment is facilitated, in part, by the accumulation of regulatory immune cell infiltrates such as myeloid-derived suppressor cells (MDSCs). To study the role of RAGE expression in the setting of mutant Ras-promoted pancreatic carcinogenesis (KC), a triple-transgenic model of spontaneous murine PDA in a RAGE-null background (KCR) was generated. KCR mice had markedly delayed pancreatic carcinogenesis and a significant diminution of MDSCs compared with KC mice at comparable time points postweaning. Although RAGE was not required for the development or suppressor activity of MDSCs, its absence was associated with temporally limited pancreatic neoplasia and altered phenotype and function of the myeloid cells. In lieu of MDSCs, KCR animals at comparable time points exhibited mature CD11b(+)Gr1(-)F4/80(+) cells that were not immunosuppressive in vitro. KCR mice also maintained a significantly less suppressive milieu evidenced by marked decreases in CCL22 in relation to CXCL10 and diminished serum levels of IL-6.

BACKGROUND

Patients with localized tuberculoid and generalized lepromatous leprosy show respectively Th1 and Th2 cytokine profile. Additionally, other patients in both types of leprosy also show a non discriminating Th0 cytokine profile with both interferon-γ and IL-4. The present study investigated the role of Th17 cells which appear to be a distinct subtype of Th subtypes in 19 tuberculoid and 18 lepromatous leprosy patients. Five healthy subjects with long term exposure to infection and 4 skin biopsies from healthy subjects undergoing cosmetic surgery were used as controls.

RESULTS

An array of Th17 related primers for cytokines, chemokines and transcription factors was used in real time reverse transcribed PCR to evaluate gene expression, ELISA for cytokine secretion in the supernatants of antigen stimulated PBMC cultures and flow cytometry for establishing the phenotype of the IL-17, IL-21 producing cells.

CONCLUSIONS

IL-17 isoforms showed significantly higher expression and release in supernatants of antigen stimulated PBMC cultures and dermal lesions of healthy contacts and tuberculoid leprosy as compared to lepromatous leprosy (p<0.003). This was further confirmed by Th17 associated transcription factor RORC, cytokines IL-21, IL-22, and IL-23, chemokines MMP13, CCL20, CCL22. Of interest was the association of IL-23R and not IL-6R with IL-17(+) cells. The Th17 cells were CD4(+) CCR6(+) confirming their effector cell lineage. Polarized Th1 cytokines were seen in 3/7 tuberculoid and Th2 cytokines in 5/10 lepromatous leprosy patients. Of importance was the higher association of Th17 pathway factors with the non-polarized Th0 types as compared to the polarized Th1 and Th2 (p<0.01). Our study draws attention to a third type of effector Th cell that may play a role in leprosy.

Various immune functions of different types of immune cells are strongly impaired in patients with head and neck squamous cell carcinoma (HNSCC). Regulatory T-lymphocyte cells (Tregs) have been suggested to be involved in the immunomodulation of immune responses and contribute to HNSCC progression and immune escape. 'Naturally' occurring CD4+ CD25+ Tregs represent a small fraction within the different subsets of regulatory T cells, which are known to inhibit numerous immune functions of different types of immune cells. In this study, the cellular ratio of CD4+ CD25(high) Tregs to the entire population of CD4+ T-lymphocytes was analyzed with respect to different stages of tumor progression and disease. Our data indicate a significantly high increased abundance of CD4+ CD25(high) CD127(low) Tregs in the peripheral blood of patients with HNSCC, which in addition show modulated expression levels of various functional proteins. Surprisingly, increased Treg levels were found even in patients with no active disease several years after tumor resection, with no significant correlation to the individual tumor stage. Additionally, increased levels of chemokine CCL22, which mediates migration of Tregs to the tumor, and upregulation of the corresponding receptor protein CCR4 were observed in HNSCC. Our data strongly suggest that HNSCC leads to a permanent shift of Treg levels with hardly recognizable recovery rates.

OBJECTIVE

To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response.

METHODS

The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an "antitumor" (M1) or a "protumor" (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 hours, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction.

RESULTS

Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpin RNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture.

CONCLUSIONS

These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

To investigate the pathogenesis of localized autoimmune damage in Sjögren's syndrome (SS) by examining the expression patterns of cytokines, chemokines and chemokine receptors at sites of autoimmune damage. mRNA expression of these molecules in the labial salivary glands (LSGs) and peripheral blood mononuclear cells (PBMCs) from 36 SS patients was examined using a real-time polymerase chain reaction-based method. Subsets of the infiltrating lymphocytes and chemokines/chemokine receptors expression in the LSG specimens were examined by immunohistochemistry. Cytokines/chemokine concentrations in the saliva were analysed using flow cytometry or enzyme-linked immunosorbent assay. mRNA expression of T helper type 1 (Th1) cytokines, chemokines and chemokine receptors was higher in LSGs than in PBMCs. In contrast, mRNA expression of Th2 cytokines, chemokines [thymus and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22)] and chemokine receptor (CCR4) was associated closely with strong lymphocytic accumulation in LSGs. Furthermore, TARC and MDC were detected immunohistochemically in/around the ductal epithelial cells in LSGs, whereas CCR4 was detected on infiltrating lymphocytes. The concentrations of these cytokines/chemokines were significantly higher in the saliva from SS patients than those from controls, and the concentrations of Th2 cytokines/chemokines were associated closely with strong lymphocytic accumulation in LSGs. These results suggest that SS might be initiated and/or maintained by Th1 and Th17 cells and progress in association with Th2 cells via the interaction between particular chemokines/chemokine receptors. Furthermore, the measurement of cytokines/chemokines in saliva is suggested to be useful for diagnosis and also to reveal disease status.

Atopic dermatitis (AD) is a chronic skin disease that affects millions of people worldwide. Keratinocytes and macrophages are two cells types that play a pivotal role in the development of AD. These cells produced different chemokines and cytokines, especially thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22), as well as nitric oxide (NO) through inducible nitric oxide synthase (iNOS) and COX2 in response to stimulation by TNF-α/IFN-γ and lipopolysaccharide (LPS) respectively. These mediators are thought to be crucial regulators of the pathogenesis of AD. Although several natural compounds to treat AD have been studied, the effect of Rg5:Rk1 from Panax ginseng (P. ginseng) on AD has not yet been investigated. In this study, we evaluated the inhibitory effect of Rg5:Rk1 on TNF-α/IFN-γ stimulated keratinocytes (HaCaT cells) and LPS-stimulated macrophages (RAW 264.7 cells). Enzyme-linked immunosorbent assay (ELISA) data showed that pretreatment of HaCaT cells with Rg5:Rk1 significantly reduced the TNF-α/IFN-γ-induced increase in TARC/CCL17 expression in a dose-dependent manner. In addition, Rg5:Rk1 decreased LPS-mediated nitric oxide (NO) and reactive oxygen species (ROS) production in RAW 264.7 cells. A considerable reduction in messenger RNA (mRNA) expression of the aforementioned AD mediators was also observed. Pretreatment with Rg5:Rk1 attenuated the TNF-α/IFN-γ-induced phosphorylation of p38 MAPK, STAT1, and NF-κB/IKKβ in HaCaT cells. Together, these findings suggest that ginsenoside Rg5:Rk1 may have a potential anti-AD effect by suppressing NF-κB/p38 MAPK/STAT1 signaling.

BACKGROUND

Atopic dermatitis (AD) is a recurrent inflammatory skin disease characterized by high serum levels of IgE and Th2-type cytokines such as IL-4, IL-5 or IL-13. Chemokines attract leukocytes in inflamed tissues. We have previously found that thymus and activation regulated chemokine (TARC)/CCL17 and macrophage-derived chemokine (MDC)/CCL22 are highly secreted in the plasma levels of AD patients. Dendritic cells (DCs) are antigen-presenting cells that are divided into two subgroups including monocyte derived DCs (MoDCs) and plasmacytoid DCs (pDCs).

OBJECTIVE

The aim of the study was to elucidate CCL17 and CCL22 production by MoDCs in AD patients, psoriasis vulgaris (PsV) patients and healthy controls (HC).

METHODS

MoDCs were obtained from AD patients, PsV patients or HC and were cultured. In addition, the chemokine levels were measured in the supernatants.

RESULTS

We found that the CCL22 levels produced by MoDCs in AD patients to be significantly higher than those of PsV patients and HC. There was a significant correlation between the CCL22 levels produced by MoDCs and the SCORAD index. No significant difference in the CCL17 levels produced by MoDCs was detected among AD patients, PsV patients or HC. Immunosuppressive drugs such as dexamethasone (Dex), tacrolimus and cyclosporine (Cys) inhibited the CCL22 production by MoDCs in the AD patients.

CONCLUSIONS

These data suggest that the CCL22 level produced by MoDCs thus reflects the disease activity of AD and it may also play an important role regarding the production of CCL22 in the pathogenesis of AD.

We have previously described the human osteoclast associated receptor (hOSCAR), expressed in all cells of the myeloid lineage, and its immune functions. This receptor, which associates with the FcRgamma chain to transduce an activating signal, induces calcium flux in monocytes and dendritic cells, and modulates specific responses of dendritic cells. In this study, we have examined the effects of hOSCAR ligation on various proinflammatory responses of monocytes and neutrophils. Monocytes stimulated via hOSCAR ligation released IL-8/CXCL8 and other chemokines such as epithelial neutrophil-activating peptide-78/CXCL5, macrophage-derived chemokine/CCL22, and MCP-1/CCL2 and up-regulated markers involved in cell adhesion and costimulatory functions. Monocytes stimulated via hOSCAR in the absence of survival factors had an increased life span. Although the life span of neutrophils was unaffected, these cells, when stimulated via hOSCAR, rapidly released reactive oxygen intermediates, degranulated lactoferrin, myeloperoxidase, and matrix metalloproteinase-9 and also secreted IL-8/CXCL8. Neutrophils also underwent changes in cell surface molecule expression with the cleavage of CD62L and increased expression of CD11b and CD66b after 2-h stimulations. Finally, we demonstrated synergy between hOSCAR and TLR ligands on both monocytes and neutrophils, with up to 8-fold increases in cytokine secretion when hOSCAR was cross-linked in the presence of LPS or R-848. Overall, our data demonstrate that hOSCAR is a functional receptor on monocytes and neutrophils, involved in the induction of the primary proinflammatory cascade and the initiation of downstream immune responses.

Immunocompromised individuals, including those infected with human immunodeficiency virus (HIV), are at increased risk of Epstein-Barr virus (EBV)-associated aggressive B cell malignancies such as Burkitt's lymphoma (BL) or diffuse large B cell lymphoma (DLBCL). Differential diagnosis of these lymphomas requires histopathological, immunohistochemical and cytogenetic assessments. Rapid, less invasive approaches to the diagnosis of EBV-associated B cell lymphomas are needed. Here, high-throughput cytokine profiling of BL cell lines and EBV-transformed B lymphoblastoid cell lines (B-LCL), representing DLBCL, was carried out. By monitoring the production of 42 different cytokines, unique cytokine signatures were identified for BL and B-LCL/DLBCL. The BL cells produced interleukin (IL)-10, 10 kDa interferon gamma-induced protein (IP-10)/CXCL10, macrophage-derived chemokine (MDC)/CCL22, macrophage inflammatory protein (MIP)-1α/CCL3 and MIP-1β/CCL4. In addition to these five cytokines, the cytokine signature of B-LCL/DLBCL cells included IL-8/CXCL8, IL-13, platelet-derived growth factor (PDGF)-AA, and regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5. Epstein-Barr virus latency was responsible for the increased production of IL-10, MDC/CCL22 and MIP-1α/CCL3 in BL cells, suggesting that EBV-mediated BL-genesis involves these three cytokines. These results suggest that high-throughput cytokine profiling might be a valuable tool for the differential diagnosis and might deepen our understanding of the pathogenesis of EBV-associated B cell malignancies.

We explore potential dysregulation of macrophage phenotypes in COPD pathogenesis through integrated study of human small airway tissue, bronchoalveolar lavage (BAL) and an experimental murine model of COPD. We evaluated human airway tissue and BAL from healthy controls, normal lung function smokers (NLFS), and COPD subjects. Both small airways and BAL cells were immunohistochemically stained with anti-CD68 for total macrophages and with anti-CD163 for M2, and anti-iNOS for M1 macrophages. Multiplex ELISA measured BAL cytokines. Comparable cigarette smoke-induced experimental COPD mouse model was assessed for relevant mRNA profiles. We found an increase in pro-inflammatory M1s in the small airways of NLFS and COPD compared to controls with a reciprocal decrease in M2 macrophages, which remained unchanged among pathological groups. However, luminal macrophages showed a dominant M2 phenotype in both NLFS and COPD subjects. BAL cytokine skewed towards an M2 profile with increase in CCL22, IL-4, IL-13, and IL-10 in both NLFS and COPDs. The mouse-model of COPD showed similar increase in mRNA for M2 markers. Our finding suggests abnormal macrophage switching in both mucosal and luminal areas of COPD patients, that strongly associated with cytokine balance. There may be potential for beneficial therapeutic cytokine manipulation of macrophage phenotypes in COPD.

Atopic dermatitis is a chronic or chronically relapsing inflammatory skin disease with a prevalence ranging from 10% to 20% in children and 1% to 3% in adults of developed countries. Skin-infiltrating leukocytes play a pivotal role in the initiation and amplification of atopic skin inflammation. Recent studies demonstrated that infiltration of inflammatory cells into tissues is regulated by chemokines. A subset of chemokines including CCL27, CCL17, CCL22, CCL18, CCL11, and CCL13 are highly expressed in atopic dermatitis. The corresponding chemokine receptors are found on the main leukocyte subsets involved in allergic skin inflammation, such as T cells, eosinophils, and dendritic cells. In this article, we provide an overview of the role of chemokines in the complex immunopathogenesis of atopic dermatitis, highlighting potential areas for therapeutic intervention.

Condylomata acuminata derived from the infection of human papillomavirus is a common sexually transmitted disease. Although T cell-mediated cellular immunity is considered as the main arm against such infection, the regulation of T cell immune responses in genital condylomata is unclear to date. In this study, we analyzed FOXP3(+) regulatory T cells in genital condylomata of patients. The results show that FOXP3(+) regulatory T cells with suppressive function accumulated in large warts. Consistently, the immunosuppressive milieu in large warts was characterized by high expression of IL-10 and TGF-beta1 and low expression of IL-2 and IFN-gamma. The responsiveness of wart-infiltrating T cells both in vitro and in vivo can be increased by depleting FOXP3(+) T cells. The accumulation of FOXP3(+) regulatory T cells in large warts can be partly ascribed to the chemotaxis of CCL17 and CCL22, derived from Langerhans cells and macrophages in wart. Although such accumulation favors the local immunosuppression, it seems not to influence the systemic immunity. In conclusion, these findings demonstrate that FOXP3(+) regulatory T cells play an important role in genital condylomata, which has multiple implications in the comprehensive treatment of condylomata acuminata.

We previously reported the inhibitory effect of chrysin, a natural flavonoid plentifully contained in propolis, vegetables and fruits, on the mast cell-mediated allergic reaction. In this study, we evaluated the effect of chrysin on atopic dermatitis (AD) and defined underlying mechanisms of action. We used an AD model in BALB/c mice by the repeated local exposure of 2,4-dinitrochlorobenzene (DNCB) and house dust mite (Dermatophagoides farinae extract, DFE) to the ears. Repeated alternative treatment of DNCB/DFE caused AD-like skin lesions. Oral administration of chrysin diminished AD symptoms such as ear thickness and histopathological analysis, in addition to serum IgE and IgG2a levels. Chrysin decreased infiltration of mast cells, and reduced serum histamine level. Chrysin also suppressed AD by inhibiting the inflammatory responses of Th1, Th2, and Th17 cells in mouse lymph node and ear. Interestingly, chrysin significantly inhibited the production of cytokines, Th2 chemokines, CCL17 and CCL22 by the down-regulation of p38 MAPK, NF-κB, and STAT1 in tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated human keratinocytes (HaCaT). Chrysin also inhibited TNF-α/IFN-γ-stimulated IL-33 expression in HaCaT cells and mouse primary keratinocytes. Taken together, the results indicate that chrysin suppressed AD symptoms, suggesting that chrysin might be a candidate for the treatment of AD and skin allergic diseases.

Age-related Epstein-Barr virus-positive (EBV(+)) B-cell lymphoproliferative disorder (ALPD) is a disease entity identified from a large-scale re-survey of cases diagnosed as diffuse large B-cell lymphoma. ALPD is a group of EBV(+) polymorphic B-cell lymphoma typically seen in elderly patients. An age-associated decline in host immunity against EBV might be partly responsible for the pathogenesis of ALPD. Histologically, ALPD is often characterized by a minor proportion of EBV-encoded RNA-positive tumor cells in a background of extensive cellular infiltration, similar to that of classical Hodgkin's lymphoma. In contrast to Hodgkin and Reed-Sternberg cells, ALPD tumor cells are clearly positive for B cell markers CD20 and/or CD79a. Hodgkin and Reed-Sternberg cells produce various chemokines, including CCL17 and CCL22, that attract chemokine receptor CCR4-expressing Th2 cells and regulatory T cells. Previously, we have shown that EBV-immortalized B cells also produce CCL17 and CCL22 through latent membrane protein 1 (LMP1)-mediated activation of nuclear factor kappaB. Here we examined expression of CCL17 and CCL22 in ALPD. ALPD tumor cells were often heterogeneous in size in accordance with the differential expression of EBV latent genes at the single cell level. LMP1-expressing tumor cells were typically large in size and selectively positive for CCL17 and CCL22. CCR4(+) cells and forkhead box protein 3(+) regulatory T cells were abundantly present, and the majority of forkhead box protein 3(+) cells were CCR4(+). Collectively, our data show production of CCL17 and CCL22 by LMP1(+) large-sized tumor cells and accumulation of CCR4-expressing cells including regulatory T cells in ALPD.

Chemokines have crucial roles in organ development and orchestration of leukocyte migration. The chemokine CCL22 is expressed constitutively at high levels in the lymph node, but the functional significance of this expression is so far unknown. Studying a newly established CCL22-deficient mouse, we demonstrate that CCL22 expression by dendritic cells (DCs) promotes the formation of cell-cell contacts and interaction with regulatory T cells (T reg) through their CCR4 receptor. Vaccination of CCL22-deficient mice led to excessive T cell responses that were also observed when wild-type mice were vaccinated using CCL22-deficient DCs. Tumor-bearing mice with CCL22 deficiency showed prolonged survival upon vaccination, and further, CCL22-deficient mice had increased susceptibility to inflammatory disease. In conclusion, we identify the CCL22-CCR4 axis as an immune checkpoint that is crucial for the control of T cell immunity.

Cytokines and chemokines contribute to the pathogenesis of acute disseminated encephalomyelitis (ADEM). Using a multiplex immunochemiluminescence ELISA, we measured 8 Th1/Th2 cytokines and 18 chemokines in the cerebrospinal fluid (CSF) and serum of 17 ADEM patients, 14 multiple sclerosis (MS) patients, and 7 healthy controls (HCs). Relative to HCs, ADEM patients had significantly high mean CSF concentrations of chemokines with attractant/activating properties towards neutrophils (CXCL1 and CXCL7), monocytes/T cells (CCL3 and CCL5), Th1 cells (CXCL10), and Th2 cells (CCL1, CCL22, and CCL17). Mean CSF concentrations of CXCL7, CCL1, CCL22, and CCL17 were higher in ADEM than in MS, whereas those of CCL11 were lower in MS than in ADEM and HCs. CSF pleocytosis correlated with CSF concentrations of CXCL1, CXCL10, CCL1, CCL17, and CCL22. Most of the functionally homologous chemokines correlated with each other. CSF Th1/Th2 cytokines were not detectable in most samples. Their mean concentrations did not differ in the three groups, and the same held for serum cytokines and chemokines. Our data suggest that the upregulation of chemokines active on neutrophils and Th2 cells differentiates ADEM from MS inflammation, and that both Th1 and Th2 chemokines might be produced in ADEM. Chemokines upregulated in ADEM could become CSF biomarkers after a posteriori evaluations in unselected case series.

CCR4 is purported to be a Th type 2 (Th2) cell-biased receptor but its functional role is unclear. Recent studies suggest that chemokine receptor expression and function are more complex in vivo and raise doubts regarding restricted CCR4 expression by Th2 cells. To address these issues, we analyzed the role of CCR4 in highly polarized models of Th type 1 (Th1) and Th2 cell-mediated pulmonary granulomas, respectively, elicited by i.v. challenge of primed mice with either mycobacterial purified protein derivative or schistosomal egg Ag-coated beads. CCR4 agonists were expressed during both responses, correlating with a shift of CCR4+ CD4+ T cells from blood to lungs. CCL22 dominated in draining nodes during the Th1 response. Analysis of CD4+ effector T cells revealed CCR4 expression and CCR4-mediated chemotaxis by both IFN-gamma and IL-4 producers. Studies of CCR4 knockout (CCR4(-/-)) mice showed partial impairment of the local type-2 cytokine response and surprisingly strong impairment of the Th1 response with abrogated IFN-gamma production during secondary but not primary challenge. Adoptive transfer indicated CCR4(-/-)CD4+ Th1 cell function was defective but this could not be reconstituted with wild-type (CCR4(+/+)) CD4+ T cells indicating involvement of another CCR4+ population. Coculture of CCR4(+/+)CD4+ T cells and CCR4(-/-) dendritic cells revealed intact IL-2 but impaired IFN-gamma production, pointing to a role for CCR4+ dendritic cells in effector cell expression. Therefore, CCR4 is not Th2-restricted and was required for sustenance and expression of the Th1 effector/memory response to mycobacterial Ags.

Patients with atopic dermatitis (AD) have significantly reduced plasma cAMP levels, and the cAMP level is correlated with the immunopathogenesis of AD. The production of thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) in keratinocytes is significantly enhanced in patients with AD. In the present study, we investigated the in vitro effects of the adenylyl cyclase-cAMP system on IFN-gamma and TNF-alpha-stimulated production of TARC and MDC in human HaCaT keratinocytes. Both forskolin (a direct activator of adenylyl cyclase) and dibutyryl-cAMP (DBcAMP, a permeable analog of cAMP) suppressed production of TARC and MDC in parallel with the activation of NF-kappaB in IFN-gamma and TNF-alpha-stimulated HaCaT cells. Moreover, inhibition of NF-kappaB suppressed TARC and MDC production induced by IFN-gamma plus TNF-alpha. However, dideoxyforskolin, a forskolin derivative that does not activate cAMP, failed to suppress the secretion of these chemokines. An inhibitor of p38 MAPK suppressed the production of TARC and MDC in parallel to the activation of NF-kappaB in HaCaT cells. Of note, the IFN-gamma plus TNF-alpha-stimulated activation of p38 MAPK was suppressed following incubation with forskolin or DBcAMP alone. These results indicate that the adenylyl cyclase-cAMP system has an inhibitory role in IFN-gamma plus TNF-alpha-stimulated production of TARC and MDC in HaCaT keratinocytes by inhibiting NF-kappaB activation through p38 MAPK pathway, implying that the adenylyl cyclase-cAMP system could be a candidate therapeutic target of Th2-skewed skin inflammation such as AD.

Systemic inflammation is a major factor influencing the outcome and quality of patient with chronic obstructive pulmonary disease (COPD) and acute exacerbations (AECOPD). Because of the inflammatory complexity, a great challenge is still confronted to optimize the identification and validation of disease-specific biomarkers. This study aimed at developing a new protocol of specific biomarker evaluation by integrating proteomic profiles of inflammatory mediators with clinical informatics in AECOPD patients, understand better their function and signal networks. Plasma samples were collected from healthy non-smokers or patients with stable COPD (sCOPD) or AECOPD on days 1 and 3 of the admission and discharging day (day 7-10). Forty chemokines were measured using a chemokine multiplex antibody array. Clinical informatics was achieved by a Digital Evaluation Score System (DESS) for assessing severity of patients. Chemokine data was compared among different groups and its correlation with DESS scores was performed by SPSS software. Of 40 chemokines, 30 showed significant difference between sCOPD patients and healthy controls, 16 between AECOPD patients and controls and 13 between AECOPD patients and both sCOPD and controls, including BTC, IL-9, IL-18Bpa, CCL22,CCL23, CCL25, CCL28, CTACK, LIGHT, MSPa, MCP-3, MCP-4 and OPN. Of them, some had significant correlation with DESS scores. There is a disease-specific profile of inflammatory mediators in COPD and AECOPD patients which may have a potential diagnostics together with clinical informatics of patients. Our preliminary study suggested that integration of proteomics with clinical informatics can be a new way to validate and optimize disease-special biomarkers.

Skin inflammation in atopic dermatitis (AD) is characterized by the predominant infiltration of T-helper (Th)2-cells in lesional skin. However, the mechanism of recruitment of these cells in lesional skin of AD is not yet fully elucidated. In this study, we investigated the role of IL-13-stimulated human primary keratinocytes (HPKs) in the recruitment of lymphocytes and further delineated the mechanism of enrichment of these cells. In the migration assays, we observed preferential enrichment of CD4(+)CCR4(+) T cells towards IL-13-stimulated HPKs. Interestingly, CD4(+)CCR4(+) T cells from AD showed a higher chemotactic response than those from healthy individuals. We observed a significant increase in the expression of CCL22 in IL-13-stimulated HPKs as compared to unstimulated cells. Blocking of CCL22 in IL-13-stimulated HPKs by a neutralizing antibody resulted in 70-90% inhibition in migration of CD4(+)CCR4(+) T cells. Moreover, IL-13 upregulated IFN-gamma-induced chemokines, CCL2 and CCL5, in HPKs. Taken together, our data suggest that IL-13-stimulated HPKs participate in a positive feedback loop by preferentially enriching Th2-cells in lesional skin of acute AD patients. However, in chronic phase, IL-13 may act in synergy with IFN-gamma resulting in lymphocytes recruitment of a mixed phenotype at the site of inflammation, thus contributing to the chronification of eczema.

BACKGROUND

Sensitization to protease allergens, such as papain, or helminth infection is associated with basophil recruitment to draining lymph nodes (LNs). Basophils have the capacity to present antigen to naive T cells and promote T(H)2 differentiation directly or indirectly through IL-4 production.

OBJECTIVE

We studied how papain induces basophil migration to LNs and the contribution of various leukocytes to papain-induced immune responses.

METHODS

We immunized mice in the footpad with papain and studied leukocyte recruitment and inflammatory cytokine and chemokine production in the draining popliteal LNs.

RESULTS

Papain directly activated naive T cells through protease-activated receptor (PAR) 2 to initiate a chemokine/cytokine program that includes CCL17, CCL22, and IL-4. Papain-triggered innate immune responses were dependent on both CD4 T cells and PAR2 and were strongly reduced in the absence of CCR4, the primary receptor for CCL17/CCL22.

CONCLUSIONS

These results elucidate a novel innate allergen-recognition pathway mediated by naive T cells through PAR2, which provide an immediate source of chemokines and IL-4 upstream of basophils and antigen-restricted T(H)2 differentiation. PAR2 antagonism might thus hold promise for the treatment of allergic disease.

Increased levels of chemokines and prostaglandins have been reported in patients with inflammatory bowel disease, although their changes during disease development are less understood. The aim of this study was to investigate the local production of nine selected chemokines and prostaglandin E(2) (PGE(2)) to elucidate their role in colitis progression in BALB/c and C57BL/6 mice exposed to dextran sulphate sodium. The acute inflammation in both strains was accompanied by a significant up-regulation of CXCL1, CXCL2/3, CXCL10, CCL2, CCL4 and CCL22 and a downregulation of PGE(2). In the recovery phase in BALB/c, one-week post-DSS, PGE(2) levels were significantly increased with a concomitant downregulation of CXCL1, CXCL2/3, CXCL10, CCL2, and CCL4. In contrast, in C57BL/6 mice CXCL1, CXCL2/3, CXCL10, CCL2, CCL3 and CCL4 production remained high during the chronic phase, without any up-regulation of PGE(2). In addition, CCL5 was significantly increased at d26 and 33 compared to d5. Interestingly, the number of macrophages was significantly increased during the acute phase, whereas T cells were significantly increased in both the acute and chronic phase in C57BL/6 mice. Thus, our results show that chemokines are produced in a dynamic manner during colitis progression.

The innate immune and acquired immune responses are not separate, parallel systems but form interdependent components of a single integrated immune response. This is nicely highlighted by an expanding database demonstrating that the innate immune response provides the acquired immune response with information about the origin of the antigen and the type of response required via pattern recognition receptors (PRRs). Aspergillus is among a growing list of allergens that can aggravate asthmatic responses. Significant pulmonary pathology is associated with Aspergillus-induced allergic and asthmatic lung disease characterized by increased Th2 cytokine generation, IgE and IgG, eosinophilia, airway hyper-responsiveness and airway remodeling. Experimental data from a model of chronic fungal asthma demonstrate that thymus associated and regulated chemokine (TARC/CCL17) and macrophage derived chemokine (MDC/CCL22), working via CCR4, directly impair the innate anti-fungal immune response, thereby promoting the maintenance of acquired Th2-mediated asthmatic disease. Both chemokines appear to accomplish this by regulating the expression of PRRs such as toll like receptors (TLRs) and triggering receptor expressed on myeloid cells (TREM-1) by immune cells. Thus, the link between Aspergillus and asthma appears to reside in the magnitude and appropriateness of the host innate immune response, and ongoing research is revealing promising targets for therapy.

There are immediate, late-phase, and delayed-type reactions to exogenous agents. In IFN-gamma-knockout (IFN-gamma(-/-)) and wild-type B6 mice, we examined the response to picryl chloride (PCl) for assessing delayed-type reactions, and the responses to repeatedly challenged FITC for immediate and late-phase reactions. The delayed-type hypersensitivity was depressed in IFN-gamma(-/-) mice, and the immediate and late-phase reactions were enhanced in IFN-gamma(-/-) mice. As skin-infiltrating lymphocytes were scarce at the PCl-challenged site of IFN-gamma(-/-) mice, we investigated chemokine production by keratinocytes and Langerhans cells (LCs). A real-time PCR analysis demonstrated that Th1 chemokines (CXCL9 and CXCL10) and Th2 chemokines (CCL17 and CCL22) were derived mainly from keratinocytes and LCs, respectively. Challenge with PCl or FITC augmented keratinocyte expression of Th1 chemokines in wild-type but not in IFN-gamma(-/-) mice, and Th2 chemokine production by LCs was induced by repeated FITC in IFN-gamma(-/-) mice. Finally, transfer of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled draining lymph node cells from hapten-sensitized B6 mice or lymph node cells from sensitized green fluorescent protein (GFP) mice to naive IFN-gamma(-/-) mice revealed less infiltration of CFSE(+) or GFP(+) lymphocytes at the challenged site. Our study suggests that one of the crucial actions of IFN-gamma is upregulation of keratinocyte production of Th1 chemokines and downregulation of LC production of Th2 chemokines.