Skullcapflavone II Suppresses TNF-α/IFN-γ-Induced TARC, MDC, and CTSS Production in HaCaT Cells.

Skullcapflavone II (SFII), a flavonoid derived from Scutellaria baicalensis, has been reported to have anti-inflammatory properties. However, its therapeutic potential for skin inflammatory diseases and its mechanism are unknown. Therefore, this study aimed to investigate the effect of SFII on TNF-α/IFN-γ-induced atopic dermatitis (AD)-associated cytokines, such as thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC). Co-stimulation with TNF-α/IFN-γ in HaCaT cells is a well-established model for induction of pro-inflammatory cytokines. We treated cells with SFII prior to TNF-α/IFN-γ-stimulation and confirmed that it significantly inhibited TARC and MDC expression at the mRNA and protein levels. Additionally, SFII also inhibited the expression of cathepsin S (CTSS), which is associated with itching in patients with AD. Using specific inhibitors, we demonstrated that STAT1, NF-κB, and p38 MAPK mediate TNF-α/IFN-γ-induced TARC and MDC, as well as CTSS expression. Finally, we confirmed that SFII significantly suppressed TNF-α/IFN-γ-induced phosphorylation of STAT1, NF-κB, and p38 MAPK. Taken together, our study indicates that SFII inhibits TNF-α/IFN-γ-induced TARC, MDC, and CTSS expression by regulating STAT1, NF-κB, and p38 MAPK signaling pathways.

Epstein-Barr virus-positive pyothorax-associated lymphoma expresses CCL17 and CCL22 chemokines that attract CCR4-expressing regulatory T cells.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas associated with chronic inflammation (DLBCL-CI) develop in patients with chronic inflammation but without any predisposing immunodeficiency. Given the expression of the EBV latent genes, DLBCL-CI should have mechanisms for evasion of host antitumor immunity. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and may provide a valuable model for the study of immune evasion by DLBCL-CI. This study demonstrates that PAL cell lines express and secrete CCL17 and/or CCL22 chemokines, the ligands of C-C motif chemokine receptor 4 (CCR4), in contrast to EBV-negative DLBCL cell lines. Accordingly, culture supernatants of PAL cell lines efficiently attracted CCR4-positive regulatory T (Treg) cells in human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CCR4-expressing Treg cells. Furthermore, this study confirmed that CCR4-expressing Treg cells were abundantly present in primary PAL tissues. Collectively, these findings provide new insight into the mechanisms of immune evasion by PAL, and further studies are warranted on whether such mechanisms eventually lead to the development of DLBCL-CI.

SCRG1 suppresses LPS-induced CCL22 production through ERK1/2 activation in mouse macrophage Raw264.7 cells.

Recently, we identified the scrapie responsive gene 1 (SCRG1) secreted from mesenchymal stem cells (MSCs) and its receptor bone marrow stromal cell antigen 1 (BST1) as positive regulators of stem cell qualities such as self­renewal, migration abilities, and osteogenic differentiation potential. Here, we examined the effect of the paracrine activity of SCRG1 in macrophages. The mouse macrophage­like cell line Raw264.7 expressed BST1/ß1 or BST1/ß2 integrin as possible SCRG1 receptors. Unexpectedly, recombinant SCRG1 did not enhance cell proliferation, migration, or adhesion in these macrophages. However, further examination of the effect of SCRG1 in Raw264.7 cells did reveal a potent anti­inflammatory effect whereby SCRG1 suppressed LPS­induced CCL22 production. SCRG1 also induced the phosphorylation of extracellular signal­regulated kinase 1/2 (ERK1/2) in these cells and, moreover, a mitogen­activated protein kinase (MAPK)/ERK kinase inhibitor U0126 significantly suppressed the effect of SCRG1 on LPS­induced chemokine CCL22 production. Taken together, these data indicate that SCRG1 signals through the MAPK pathway and suppresses the LPS signaling pathway. CCL22 is generally known to be chemotactic for monocytes, dendritic cells, natural killer cells and chronically activated T lymphocytes, suggesting that MSC­derived SCRG1 may block infiltration of these cells. A mechanism is proposed by which MSCs play their immunosuppressive role through suppressing chemokine expression in monocyte/macrophage lineage cells.

Radiotherapy suppressed tumor-specific recruitment of regulator T cells via up-regulating microR-545 in Lewis lung carcinoma cells.

OBJECTIVE: Radiotherapy is an important treatment for cancer. The main irradiated action is thought to be the irreversible damage to tumor cell DNA, but recent studies showed that high dose radiotherapy related to the tumor immune response. This study was designed to determine the relationship between Lewis lung tumor radiosensitivity and CD4+CD25+ regulatory T cells (Tregs) infiltration and elucidate the underlying mechanisms in vitro. METHODS: With tumor transplantation method to establish mice Lewis lung tumor mice model, to observe the inhibition rate of radiotherapy to tumor growth. Proliferation profiles of CD4+CD25+ Tregs and CD4+ T cells were assessed by flow cytometry. MiR-545 and CCL-22 mRNA were determined by Quantitative Real-Time PCR. CCL-22 protein was determined by western blot assay. RESULTS: Radiotherapy caused a time-dependent inhibition of tumor growth as well as a decrease in the percentage of tumor-infiltrating CD4+CD25+ Tregs of CD4+ T cells compared with no treatment group. And the miR-545 was significantly upregulated and CCL-22 was significantly down-regulated in irradiated tumor and Lewis lung cancer cells. In Lewis lung cancer cell transfection experiments, mimic or inhibitor for miR-545 negatively regulated CCL-22 expression when cells treated or treated without irradiation. Silenced miR-545 promotes CD4+CD25+ Treg proliferation. Additionally, silenced miR-545 reversed radiosensitivity of Lewis lung cancer. CONCLUSION: Radiotherapy suppressed specific recruitment of regulator CD4+CD25+ Treg cells in Lewis lung carcinoma via up-regulating microR-545.

Mesenchymal stem cells abrogate experimental asthma by altering dendritic cell function.

Mesenchymal stem cells (MSCs) have been investigated in the treatment of numerous autoimmune diseases. However, the immune properties of MSCs on the development of asthma have remained to be fully elucidated. Airway dendritic cells (DCs) have an important role in the pathogenesis of allergic asthma, and disrupting their function may be a novel therapeutic approach. The present study used a mouse model of asthma to demonstrate that transplantation of MSCs suppressed features of asthma by targeting the function of lung myeloid DCs. MSCs suppressed the maturation and migration of lung DCs to the mediastinal lymph nodes, and thereby reducing the allergen-specific T helper type 2 (Th2) response in the nodes. In addition, MSC-treated DCs were less potent in activating naive and effector Th2 cells and the capacity of producing chemokine (C-C motif) ligand 17 (CCL17) and CCL22, which are chemokines attracting Th2 cells, to the airways was reduced. These results supported that MSCs may be used as a potential treatment for asthma.

Macrophage-derived chemokine CCL22 and regulatory T cells in ovarian cancer patients.

The study was undertaken to evaluate macrophage-derived chemokine (CCL22) levels in the peritoneal fluid (PF) and plasma of patients with ovarian cancer (n = 93) in relation to regulatory T cells (Tregs; n = 75). The peritoneal fluid CCL22 concentrations were significantly higher in epithelial ovarian cancer (EOC) patients than in patients with benign tumors-serous cystadenoma (n = 32). There was no difference in plasma levels of CCL22 in EOC patients compared with the non-cancer and healthy volunteers (n = 10). There were no significant differences in the plasma and PF CCL22 levels based on tumor grade. However, women with stage IV FIGO (International Federation of Gynecologists and Obstetricians) had significantly higher plasma CCL22 levels than patients with stages I and III. Women with stage I FIGO had significantly higher PF CCL22 levels than patients with stages II and III. Women with endometrioid cystadenocarcinoma had higher PF CCL22 levels than women with undifferentiated carcinoma. The percentage of tumor-infiltrating Tregs (11.06 %) was significantly higher compared to PF (3.05 %) and peripheral blood (PB) (2.01 %). Moreover, the percentage of Tregs was higher in the PF than in the PB of EOC patients. There were no significant differences in the PB, PF, and tumor-infiltrating Tregs percentage based on tumor stage, grade, or histology. Elevated levels of CCL22 found in the ascites could create a chemokine gradient aiding in Treg cells migration. Increased Tregs percentage in the local microenvironment of ovarian cancer might be an important mechanism of immunosuppression.

(+)-Nootkatone inhibits tumor necrosis factor α/interferon γ-induced production of chemokines in HaCaT cells.

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.

Cleavage of the T cell protein tyrosine phosphatase by the hepatitis C virus nonstructural 3/4A protease induces a Th1 to Th2 shift reversible by ribavirin therapy.

Ribavirin has proven to be a key component of hepatitis C therapies both involving IFNs and new direct-acting antivirals. The hepatitis C virus-mediated interference with intrahepatic immunity by cleavage of mitochondrial antiviral signaling protein (MAVS) and T cell protein tyrosine phosphatase (TCPTP) suggests an avenue for compounds that may counteract these effects. We therefore studied the effects of ribavirin, with or without inhibition of the nonstructural (NS)3/4A protease, on intrahepatic immunity. The intrahepatic immunity of wild-type and NS3/4A-transgenic mice was determined by Western blot, ELISA, flow cytometry, and survival analysis. Various MAVS or TCPTP constructs were injected hydrodynamically to study their relevance. Ribavirin pretreatment was performed in mice expressing a functional or inhibited NS3/4A protease to analyze its effect on NS3/4A-mediated changes. Intrahepatic NS3/4A expression made mice resistant to TNF-α-induced liver damage and caused an alteration of the intrahepatic cytokine (IFN-γ and IL-10) and chemokine (CCL3, CCL17, CCL22, CXCL9, and CXCL11) profiles toward an anti-inflammatory state. Consistent with this, the number of intrahepatic Th1 cells and IFN-γ(+) T cells in NS3/4A-transgenic mice decreased, whereas the amount of Th2 cells increased. These effects could be reversed by injection of uncleavable TCPTP but not uncleavable MAVS and were absent in a mouse expressing a nonfunctional NS3/4A protease. Importantly, the NS3/4A-mediated effects were reversed by ribavirin treatment. Thus, cleavage of TCPTP by NS3/4A induces a shift of the intrahepatic immune response toward a nonantiviral Th2-dominated immunity. These effects are reversed by ribavirin, supporting that ribavirin complements the effects of direct-acting antivirals as an immunomodulatory compound.

The lymphoid chemokine CCL21 enhances the cytotoxic T lymphocyte-inducing functions of dendritic cells.

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.

Tumor secretion of CCL22 activates intratumoral Treg infiltration and is independent prognostic predictor of breast cancer.

It has been reported that dense intratumoral infiltration of Foxp3 (+)Tregs (Tregs) was an independent factor for poor prognosis of breast cancer (BC) patients. However, the cytokines activating the Treg infiltration are not known. This study was undertaken to evaluate the role of CCL22 and TGF-ß1 in this cascade and their prognostic significance for BC patients. 417 cases of invasive breast cancer were selected from the prior study cohort and the expressions of CCL22 and TGF-ß1 were assessed by immunohistochemistry. It was identified that tumor secretion of CCL22 was positively correlated with the intratumoral Treg infiltration (P<0.0001), but its association with lymphoid aggregates surrounding the tumor was not proven to be significant (P=0.056). Moreover, CCL22 expression was found to be associated with the tumor histological features known to be related with unfavorable prognosis of patients, including high histological grade (P<0.0001), negative ER (P<0.0001), negative PR (P=0.001), and HER2 amplification (P=0.028). Similar to intratumoral Treg infiltrates, CCL22 tumor secretion correlated with the prognosis of the molecular subtypes of breast carcinoma (P<0.0001). Univariate analysis revealed CCL22 to be an independent prognostic factor for overall survival (OS, P<0.0001) and progression-free survival (PFS, P<0.0001) of BC patients that were confirmed by multivariate analysis (P=0.011 and P=0.010 respectively). In contrast, although TGF-ß1 expression was positively correlated with both Tregs infiltrates into the tumor bed and lymphoid aggregates surrounding the tumor (P=0.023; P=0.046, respectively), its expression was not significantly associated with the molecular subtypes of breast carcinoma and the prognosis of the patients. Our study indicates that both CCL22 and TGF-ß1 are candidate chemoattractants for intratumoral Foxp3 (+)Tregs infiltration; however, unlike the later, CCL22 is an independent prognostic predictor of BC patients, and it therefore may have the potential to serve as a target for immunotherapeutic strategy of BC.

Acceleration of bone repair in NOD/SCID mice by human monoosteophils, novel LL-37-activated monocytes.

BACKGROUND: An incomplete understanding of bone forming cells during wound healing and ectopic calcification has led to a search for circulating cells that may fulfill this function. Previously, we showed that monoosteophils, a novel lineage of calcifying/bone-forming cells generated by treatment of monocytes with the natural peptide LL-37, are candidates. In this study, we have analyzed their gene expression profile and bone repair function. METHODS AND FINDINGS: Human monoosteophils can be distinguished from monocytes, macrophages and osteoclasts by their unique up-regulation of integrin α3 and down-regulation of CD14 and CD16. Monoosteophils express high mRNA and protein levels of SPP1 (osteopontin), GPNMB (osteoactivin), CHI3L1 (cartilage glycoprotein-39), CHIT1 (Chitinase 1), MMP-7, CCL22 and MAPK13 (p38MAPKδ). Monocytes from wild type, but not MAPK13 KO mice are also capable of monoosteophil differentiation, suggesting that MAPK13 regulates this process. When human monoosteophils were implanted in a freshly drilled hole in mid-diaphyseal femurs of NOD/SCID mice, significant bone repair required only 14 days compared to at least 24 days in control treated injuries. CONCLUSION: Human derived monoosteophils, characterized as CD45(+)α3(+)α3ß(+)CD34(-)CD14(-)BAP (bone alkaline phosphatase)(-) cells, can function in an animal model of bone injury.

The antagonism of histamine H1 and H4 receptors ameliorates chronic allergic dermatitis via anti-pruritic and anti-inflammatory effects in NC/Nga mice.

BACKGROUND: Although histamine H1 receptor (H1R) antagonists are commonly used to treat atopic dermatitis, the treatment is not always effective. The histamine H4 receptor (H4R) was recently described as important to the pruritus in dermatitis. Here, we investigated whether the combination of a H1R antagonist plus a H4R antagonist attenuates chronic dermatitis in NC/Nga mice. METHODS: Chronic dermatitis was developed by repeated challenges with picryl chloride on the dorsal back and ear lobes. The therapeutic effects of the H1R antagonist olopatadine and H4R antagonist JNJ7777120 on scratching and the severity of dermatitis were evaluated. In addition, the mechanisms responsible for the anti-allergic effects of H1R and/or H4R antagonism were examined using bone marrow-derived mast cells (BMMC) and keratinocytes. RESULTS: JNJ7777120 attenuated scratching behavior after a single administration and improved dermatitis, as assessed with clinical scores, pathology, and cytokine levels in skin lesions when administered repeatedly. These effects were augmented by combined treatment with olopatadine, having a similar therapeutic efficacy to prednisolone. JNJ7777120 inhibited dose-dependently the production of thymus and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 from antigen-stimulated BMMC. In addition, olopatadine reversed the histamine-induced reduction of semaphorin 3A mRNA in keratinocytes. CONCLUSION: Combined treatment with H1R and H4R antagonists may have a significant therapeutic effect on chronic dermatitis through the synergistic inhibition of pruritus and skin inflammation.

Hepatocellular carcinoma and macrophage interaction induced tumor immunosuppression via Treg requires TLR4 signaling.

AIM: To investigated the interaction between toll-like receptor 4 (TLR4)-activated hepatoma cells and macrophages in the induction of tumor-immune suppression mediated by CD4+CD25(high) family of transcription factor P3 (FOXP3) regulatory T cells (Tregs). METHODS: The proportion of FOXP3+ Tregs was identified in peripheral blood and tumor tissues of 60 hepatocellular carcinoma (HCC) patients. TLR4 expression was examined in tumor tissues and cell lines. The correlation was examined between FOXP3+ Tregs in peripheral blood and TLR4 expression of HCC tissues. Following activation of TLR4 in H22 murine hepatoma cells pre-incubated with lipopolysaccharide (LPS) and co-cultured with macrophage cell line RAW246.7, the synthesis of cytokines tumor necrosis factor-α, CCL22, and interleukin (IL)-10 by the two cell lines was detected and analyzed. RESULTS: FOXP3+ Tregs were enriched in tumor sites, and circulating FOXP3+ Tregs were increased in HCC patients in correlation with multiple tumor foci and up-regulated TLR4 expression in HCC tissues. Semi-quantitative analysis indicated that TLR4 was over-expressed in HCC compared with the matched normal tissues. Cell cultivation experiments indicated that the mRNAs of IL-10 and CCL22 were significantly up-regulated in the RAW246.7 cell line when co-cultured with LPS pre-incubated H22 cells. CONCLUSION: In hepatoma cell lines, TLR4 may indirectly facilitate the recruitment of Tregs to the tumor site and promote intrahepatic metastasis through its interaction with macrophages.

Casuarinin suppresses TARC/CCL17 and MDC/CCL22 production via blockade of NF-κB and STAT1 activation in HaCaT cells.

Casuarinin is a naturally occurring tannin that is isolated from the leaves of Hippophae rhamnoides. It has been shown to have anti-oxidant, anti-cancer, anti-viral, and anti-inflammatory activities. The aim of this study was to investigate the possible mechanism by which casuarinin inhibits TNF-α/IFN-γ-induced Th2 chemokines expression in the human keratinocytes cell line HaCaT. We found that casuarinin suppressed TNF-α/IFN-γ-induced expression of TARC and MDC mRNA and protein in HaCaT cells. Casuarinin significantly inhibited TNF-α/IFN-γ-induced activation of NF-κB, STAT1, and p38 MAPK. Furthermore, we observed that p38 MAPK contributes to inhibition of TNF-α/IFN-γ-induced TARC and MDC production by blocking NF-κB and STAT1 activation in HaCaT cells. Taken together, these results suggest that casuarinin may exert anti-inflammatory responses by suppressing TNF-α/IFN-γ-induced expression of TARC and MDC via blockage of p38 MAPK activation and subsequent activation of NF-κB and STAT1. We propose that it could therefore be used as a therapeutic agent against inflammatory skin diseases.

Early detection of tumor cells by innate immune cells leads to T(reg) recruitment through CCL22 production by tumor cells.

In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (T(reg)) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1ß], contrasting with CCL17. Primary breast tumors and CD45(+) infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1ß and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ-producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1ß and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of T(reg). As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits T(reg) to evade this early antitumor immune response.

Human T-lymphotropic virus type 1-induced CC chemokine ligand 22 maintains a high frequency of functional FoxP3+ regulatory T cells.

We recently reported that human T-lymphotropic virus type 1 (HTLV-1) infection is accompanied by a high frequency of CD4(+)FoxP3(+) cells in the circulation. In asymptomatic carriers of HTLV-1 and in patients with HTLV-1-associated inflammatory and malignant diseases, a high FoxP3(+) cell frequency correlated with inefficient cytotoxic T cell-mediated killing of HTLV-1-infected cells. In adult T cell leukemia/lymphoma (ATLL), the FoxP3(+) population was distinct from the leukemic T cell clones. However, the cause of the increase in FoxP3(+) cell frequency in HTLV-1 infection was unknown. In this study, we report that the plasma concentration of the chemokine CCL22 is abnormally high in HTLV-1-infected subjects and that the concentration is strongly correlated with the frequency of FoxP3(+) cells, which express the CCL22 receptor CCR4. Further, we show that CCL22 is produced by cells that express the HTLV-1 transactivator protein Tax, and that the increased CCL22 enhances the migration and survival of FoxP3(+) cells in vitro. Finally, we show that FoxP3(+) cells inhibit the proliferation of ex vivo, autologous leukemic clones from patients with ATLL. We conclude that HTLV-1-induced CCL22 causes the high frequency of FoxP3(+) cells observed in HTLV-1 infection; these FoxP3(+) cells may both retard the progression of ATLL and HTLV-1-associated inflammatory diseases and contribute to the immune suppression seen in HTLV-1 infection, especially in ATLL.

Titanium induced production of chemokines CCL17/TARC and CCL22/MDC in human osteoclasts and osteoblasts.

There is increasing evidence that titanium (Ti(IV)) ions are released from orthopedic implants and play a role in aseptic loosening. This study aimed to investigate whether titanium induces expression of chemokines and cytokines that are important in osteoclastogenesis in human osteoclasts and osteoblasts. Incubation of those cells with 1 muM Ti(IV) significantly upregulated expression of CCL17/TARC and CCL22/MDC, RANK-L, M-CSF and pro-inflammatory cytokines as determined by quantitative real-time PCR and ELISA assays. Additionally, flow cytometry was used to show Ti(IV) related increased expression of CCR4, the cognate receptor for CCL17 and CCL22 in challenged osteoclast precursors. These results strongly suggest that Ti(IV) ions play a role in the recruitment of osteoclast precursors to the bone-implant interface by increasing CCL17 and CCL22 expression and by upregulating their cognate receptor. Moreover the increased expression of RANK-L and M-CSF by osteoblasts together with increased levels of pro-inflammatory cytokines may enhance osteoclast differentiation and activity, and subsequently contribute to the pathomechanism of aseptic loosening.

The adenylyl cyclase-cAMP system suppresses TARC/CCL17 and MDC/CCL22 production through p38 MAPK and NF-kappaB in HaCaT keratinocytes.

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.

Human memory CCR4+CD8+ T cell subset has the ability to produce multiple cytokines.

The CC chemokine receptor (CCR)4 is associated with trafficking of specialized cutaneous memory type 2 T(h) cells in the skin. However, a CD8(+) T cell population expressing CCR4 still remains uncharacterized. In the present study, we investigated the expression and function of CCR4 on human CD8(+) T cells and characterized CCR4(+)CD8(+) human T cells. Multi-color flow cytometric analysis revealed that CCR4(+)CD8(+) T cells were predominantly found in the CD27(+)CD28(+)CD45RA(-) memory subset and expressed the CCR7(+/-)CCR5(-) phenotype. CCR4(+)CD8(+) T cells expressed neither perforin (Per) nor granzymes (Gra) A/B, suggesting that they were more immature memory T cells than the CCR6(+)CD8(+) early effector memory T cells that express GraA and Per. CCR4(+)CD8(+) T cells effectively produced IL-4, IFN-gamma, IL-2 and tumor necrosis factor-alpha, indicating that they are memory T cells having the ability to secrete type 1 and type 2 cytokines. These cells also showed chemotaxic activity in response to CC chemokine receptor ligand (CCL)17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine. These results suggest that CCR4(+)CD8(+) T cells are in an immature memory T cell subset in the differentiation pathway of human CD8(+) T cells and that they migrate to inflammatory sites in the skin where they are involved in cutaneous immunity.

NK-dependent increases in CCL22 secretion selectively recruits regulatory T cells to the tumor microenvironment.

Tumor-induced immune suppression involves the accumulation of immune-suppressive infiltrates in the microenvironment. This study demonstrates increased numbers of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) in the lungs of C57BL/6 mice bearing a metastatic Lewis lung carcinoma (LLC) variant. These Tregs suppressed the proliferation of endogenous CD4(+)CD25(-) cells and expressed higher levels of the chemokine receptor CCR4 than other types of T cells. LLC-bearing lungs secreted elevated levels of the CCR4-associated chemokine CCL22 compared with normal lungs. However, CCL22 was not secreted by LLC or normal epithelial controls, suggesting that CCL22 is secreted by a nonepithelial component of the microenvironment. Migration assays revealed that medium conditioned by LLC-bearing lungs selectively recruited Tregs at higher frequencies than did medium conditioned by normal lungs. Neutralization of CCL22 significantly reduced this selective recruitment toward both conditioned media. A series of immunomagnetic isolations, FACS, and flow cytometric analyses were used to isolate different cellular fractions from both normal and LLC-bearing lungs. When isolated, only the NK-containing fractions secreted CCL22, and the same fraction isolated from LLC-bearing lungs secreted higher levels. Depletion of NK cells from both normal and LLC-bearing lung tissue significantly reduced CCL22 secretion, suggesting that a large portion of secreted CCL22 is NK cell dependent. Flow cytometric analysis of the lung NK compartments revealed no significant increase in NK cell numbers across LLC-bearing lung tissue as a whole as compared with normal tissue. However, immunofluorescent staining revealed an increased frequency of NK cells at the tumor periphery that were closely associated with the elevated FoxP3(+) infiltrate.