Levels Of MDC Research Articles

Serum macrophage-derived chemokine and interleukin 18 concentrations are associated with disease severity in children with atopic dermatitis.

In the pathogenesis of AD, immunological disturbances play a central role. Immune dysregulation in AD includes increased production of inflammatory cytokines, elevated serum immunoglobulin E (IgE) levels and inflammatory cell infiltration. Defects in the innate and adaptive immune responses as well as T cell disturbances lead to and sustain allergic inflammation. Macrophage-derived chemokine (MDC/CCL22) and interleukin (IL)-18 are elements of both innate and adaptive immune responses whose role in the development of skin lesions is widely discussed. Serum MDC and IL-18 levels were assessed in 60 AD children (mean age 8.1 years) and 40 healthy controls (mean age 9.62 years) to investigate a possible associations between MDC and IL-18 concentrations and the clinical severity of the disease. The children were divided into two age groups (< 7 years old and ≥ 7 years old). We compared these groups for serum levels of MDC, IL-18, total IgE and blood eosinophils and analyzed their correlation with the severity of skin lesions, defined by the scoring atopic dermatitis (SCORAD) index.The serum concentrations of MDC and IL-18 in AD patients were significantly higher than in healthy controls. The serum MDC levels significantly correlated with the SCORAD and other markers of allergic inflammation such as total IgE and eosinophil count in peripheral blood in both age groups. Serum IL-18 levels correlated positively with SCORAD index in the young children. In conclusion MDC and IL-18 serum concentrations may be useful inflammatory markers for assessing AD severity in children.

Urinary cytokines in patients treated with intravesical mitomycin-C with and without hyperthermia.

To explore whether urinary cytokine and chemokine (CK) levels differed between cold mitomycin-C (cold-MMC)-treated patients and chemohyperthermia (C-HT)-treated patients, to shed light on the possible molecular mechanisms that might explain the superior outcome of C-HT. Furthermore, CK-differences were explored between C-HT responders and C-HT non-responders. Twelve NMIBC patients were included. Nine received six-weekly C-HT, and three received four-weekly cold-MMC instillations. Urine was collected on 8-12 time points before and after every treatment. MDC, IL-2, IL-6, IL-8, IP-10, MCP-1 and RANTES were determined by Luminex(®)-analysis. Elevated urinary CK levels were observed in both groups after treatment. In general, CK-peaks were lower in the cold-MMC group in comparison with levels in the C-HT group. Significant higher MCP-1 and IL-6 levels were observed in C-HT-treated patients. Additionally, significant cumulative effects were observed for IP-10 and IL-2. However, IP-10 and IL-2 levels did not significantly differ between treatments. MDC levels after the first week of treatment were significantly higher in the C-HT responders compared with the non-responders. MMC treatment leads to elevated urinary CK levels with significantly higher MCP-1 and IL-6 levels in C-HT-treated patients. Increased MDC levels after the first C-HT instillation appear to be related to good clinical outcome and might be of additional value to personalize treatment. Studies involving more patients and longer follow-up are needed to substantiate this observation.

Macrophage-derived chemokine (CCL22) is a novel mediator of lung inflammation following hemorrhage and resuscitation.

Resuscitation of patients after hemorrhage often results in pulmonary inflammation and places them at risk for the development of acute respiratory distress syndrome. Our previous data indicate that macrophage-derived chemokine (MDC/CCL22) is elevated after resuscitation, but its direct role in this inflammatory response is unknown. Macrophage-derived chemokine signaling through the C-C chemokine receptor type 4 (CCR4) is implicated in other pulmonary proinflammatory conditions, leading us to hypothesize that MDC may also play a role in the pathogenesis of lung inflammation following hemorrhage and resuscitation. To test this, C57BL/6 mice underwent pressure-controlled hemorrhage followed by resuscitation with lactated Ringer's solution. Pulmonary inflammation and inflammatory cell recruitment were analyzed with histological staining, and serum- and tissue-level cytokines were measured by enzyme-linked immunosorbent assay. Pulmonary inflammation and cell recruitment following hemorrhage and resuscitation were associated with systemic MDC levels. Inhibition of MDC via injection of a specific neutralizing antibody prior to hemorrhage and resuscitation significantly reduced pulmonary levels of the chemotactic cytokines keratinocyte-derived chemokine and macrophage inflammatory proteins 2 and 1α, as well as inflammatory cell recruitment to the lungs. Intravenous administration of recombinant MDC prior to resuscitation augmented pulmonary inflammation and cell recruitment. Histological evaluation revealed the expression of CCR4 within the bronchial epithelium, and in vitro treatment of activated bronchial epithelial cells with MDC resulted in production and secretion of neutrophil chemokines. The present study identifies MDC as a novel mediator of lung inflammation after hemorrhage and resuscitation. Macrophage-derived chemokine neutralization may provide a therapeutic strategy to mitigate this inflammatory response.

Dendritic cells in bone marrow at diagnosis and after chemotherapy in adult patients with acute myeloid leukaemia.

Dendritic cells (DCs) develop in the bone marrow from haematopoietic progenitor cells. Two subsets, plasmacytoid DCs (pDCs) and myeloid DCs (mDCs), have been identified. Little is known regarding DC levels in bone marrow of patients with acute myeloid leukaemia (AML) before and after chemotherapy. We investigated relative pDC and mDC levels in bone marrow from 37 hospital controls and 60 patients with AML [at diagnosis, complete remission (CR) and follow-up] using four-colour flow cytometry. The pDC immunophenotype was characterized as lin-/HLA-DR+/CD123+ and mDC as lin-/HLA-DR+/CD11c+. In 69% of patients with AML, no DCs were detected at diagnosis. At CR, mDC levels were the same in patients with AML and hospital controls while pDC levels were slightly lower. There was no association between minimal residual disease or survival rates and DC levels. Patients with low mDC levels at CR were more likely to suffer from complicated infections, although the difference was not statistically significant. Altogether, there was a profound decrease in DC levels in patients with AML at diagnosis. DC levels increased at CR and were higher than in hospital controls after post-remission therapy, suggesting that DCs recover after repeated chemotherapy. There may be an association between mDC levels and infectious complications.

226P - Clinical Significance of Elevated Serum Levels of Cc Thymus and Activation-Related Chemokine in Primary Gastric Cancer: Potential for a Serum Diagnostic and Prognostic Biomarker

ABSTRACT Aim: Chemokines and their receptors are involved in various processes of tumor cell biology. CC chemokine receptor 4 (CCR4), a receptor for thymus and activation-related chemokine (TARC, CCL17), is overexpressed in solid tumors including gastric cancer (GC), and its ligand, TARC, is considered to be a potent stimulator of GC cell proliferation and migration. Here, we evaluate the clinical significance of elevated serum TARC in GC patients and validate its potential as a biomarker for GC. Methods: Serum levels of TARC, MDC (CCL22), MCP-1 (CCL2), and SCF were measured by chemiluminescent immunoassay, and compared among 4 disease groups; normal, high-risk, early GC (EGC), and advanced GC (AGC) groups (each, n = 25) along the gastric carcinogenic sequences (one-way ANOVA) in training set. Serum levels of TARC and SCF were re-evaluated in following independent validation set (90 normal, 30 high-risk, 50 EGC, 50 AGC) and compared with serum carcinoembryonic antigen (CEA) levels. To evaluate the diagnostic potential of serum TARC for GC, receiver operating characteristic (ROC) curve was generated and logistic regression analysis was performed. Correlations of serum TARC with clinicopathological parameters of GC were evaluated by Spearman's correlation. Results: In training set, serum TARC and SCF were significantly correlated with each other and different between cancer and non-cancer conditions (each, p Conclusions: Serum TARC is a potential serologic biomarker for GC superior to CEA. Disclosure: All authors have declared no conflicts of interest.

Blood Dendritic Cell Levels Associated With Impaired IL-12 Production and T Cell Function in Kidney Disease: Implications for Post-Transplant Viral Infections.

Background: We previously demonstrated a reduction of blood myeloid dendritic cell (mDC) levels in patients with kidney disease pre-transplant that is associated statistically with the development of BK viremia and CMV disease post-transplant. However, the mechanisms by which mDC levels might influence these outcomes have not been elucidated. In the current report, we studied the association of mDC levels with mDC IL-12 production and T cell level/function. Methods: Peripheral blood was studied in 3 groups: (i) patients with end stage renal disease at hemodialysis units (HD; n=81); (ii) patients with kidney disease presenting for kidney transplant evaluation or transplantation (Eval/Tx; n=323); and (iii) healthy controls (HC; n=22). Peripheral blood mononuclear cell levels were measured by flow cytometry. IL-12 production was assessed by intracellular staining and multiplex assay. T cell IFN-g responses were measured by ELISpot assay. Results: Along with significant reduction in mDC levels, significantly reduced CD8+ T cell and monocyte levels were also demonstrated in the kidney disease groups compared to HC. Furthermore, levels of CD8+ T cells, which are stimulated by DCs and important to antiviral immunity, correlated positively with the deficient DC levels observed in kidney disease patients. Significantly reduced peripheral blood mDC and monocyte-derived DC (MoDC) IL-12 production was observed after in vitro LPS stimulation in the HD vs HC groups. Finally, addition of autologous MoDCs with or without CMV peptide to T cell culture failed to increase significantly the number of IFN-g responses in the HD group in contrast to that observed for the HC group. Conclusions: Peripheral blood mDC level deficiency in patients with kidney disease is associated with deficient IL-12 production and T cell level/function, which may explain the known correlation of CD8+ T cell lymphopenia with deficient antiviral responses following kidney transplantation.

Changes in dendritic cells and dendritic cell subpopulations in peripheral blood of recipients during acute rejection after kidney transplantation

Background Advances in transplantation immunology show that the balance between dendritic cells (DCs) and their subsets can maintain stable immune status in the induction of tolerance after transplantation. The aim of this study was to investigate if DCs and DC subpopulations in recipient peripheral blood are effective diagnostic indicators of acute rejection following kidney transplantation. Methods Immunofluorescent flow cytometry was used to classify white blood cells (WBCs), the levels of mononuclear cells and DCs (including the dominant subpopulations, plasmacytoid DC (pDC) and myeloid DC (mDC)) in peripheral blood at 0, 1, 7, and 28 days and 1 year after kidney transplantation in 33 patients. In addition, the blood levels of interleukin-10 (IL-10) and IL-12 were monitored before and after surgery. Fifteen healthy volunteers served as normal controls. Patients were undertaking hemodialysis owing to uremia before surgery. Results The total number of DCs, pDC, and mDC in peripheral blood and the pDC/mDC ratio were significantly lower in patients than controls (P &lt;0.05). Peripheral DCs suddenly decreased at the end of day 1, then gradually increased through day 28 but remained below normal levels. After 1 year, levels were higher than before surgery but lower than normal. The mDC levels were higher in patients with acute rejection before and 1 day after surgery (P &lt;0.005). There was no significant difference in IL-10 and IL-12 levels between patients with and without acute rejection. Conclusion The changes in DCs and DC subpopulations during the acute rejection period may serve as effective markers and referral indices for monitoring the immune state, and predicting rejection and reasonably adjusting immunosuppressants.

IVIg Immune Reconstitution Treatment Alleviates the State of Persistent Immune Activation and Suppressed CD4 T Cell Counts in CVID

Common variable immunodeficiency (CVID) is characterized by defective B cell function, impaired antibody production, and increased susceptibility to bacterial infections. Here, we addressed the hypothesis that poor antibody-mediated immune control of infections may result in substantial perturbations in the T cell compartment. Newly diagnosed CVID patients were sampled before, and 6–12 months after, initiation of intravenous immunoglobulin (IVIg) therapy. Treatment-naïve CVID patients displayed suppressed CD4 T cell counts and myeloid dendritic cell (mDC) levels, as well as high levels of immune activation in CD8 T cells, CD4 T cells, and invariant natural killer T (iNKT) cells. Expression of co-stimulatory receptors CD80 and CD83 was elevated in mDCs and correlated with T cell activation. Levels of both FoxP3+ T regulatory (Treg) cells and iNKT cells were low, whereas soluble CD14 (sCD14), indicative of monocyte activation, was elevated. Importantly, immune reconstitution treatment with IVIg partially restored the CD4 T cell and mDC compartments. Treatment furthermore reduced the levels of CD8 T cell activation and mDC activation, whereas levels of Treg cells and iNKT cells remained low. Thus, primary deficiency in humoral immunity with impaired control of microbial infections is associated with significant pathological changes in cell-mediated immunity. Furthermore, therapeutic enhancement of humoral immunity with IVIg infusions alleviates several of these defects, indicating a relationship between poor antibody-mediated immune control of infections and the occurrence of abnormalities in the T cell and mDC compartments. These findings help our understanding of the immunopathogenesis of primary immunodeficiency, as well as acquired immunodeficiency caused by HIV-1 infection.

Anti-Inflammatory Effect of Quercetagetin, an Active Component of Immature Citrus unshiu, in HaCaT Human Keratinocytes

Citrus fruit contain various flavonoids that have multiple biological activities. However, the content of these flavonoids are changed during maturation and immature Citrus is known to contain larger amounts than mature. Chemokines are significant mediators for cell migration, while thymus and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) are well known as the typical inflammatory chemokines in atopic dermatitis (AD), a pruritic and chronic inflammatory skin disease. We reported recently that the EtOH extract of immature Citrus unshiu inhibits TARC and MDC production. Therefore, we investigated the activity of flavonoids contained in immature Citrus on TARC and MDC levels. As a result, among the various flavonoids, quercetagetin has stronger inhibitory effects on the protein and mRNA expression of TARC and MDC than other flavonoids. Quercetagetin particularly has better activity on TARC and MDC level than quercetin. In HPLC analysis, the standard peak of quercetagetin matches the peaks of extract of immature C. unshiu. This suggests that quercetagetin is an anti-inflammatory component in immature C. unshiu.

Abnormally High Levels of Inflammatory Markers in Recipients of Continuous Flow Left Ventricular Assist Devices

Purpose Early activation of inflammatory markers immediately after implantation of left ventricular assist devices (LVADs) predicts multiple organ failure. Although the newer generation continuous flow (CF)-LVADs provide clinical advantages over the pulsatile pumps, the effects of low pulsatility on inflammation are not completely understood in this patient population. Under continuous-flow conditions, the levels of neurohormones, which are known to augment inflammation in heart failure (HF), are elevated. We hypothesize that in CF-LVAD recipients the levels of inflammatory markers are higher than both healthy controls and HF patients. The aim of our study was to compare the levels of inflammation in CF-LVAD recipients to those of healthy controls (HCs) and HF patients who were candidates for CF-LVADs. Methods and Materials The levels of the inflammatory markers were measured in 18 CF-LVAD recipients and compared to those of 14 HCs and 14 HF patients who were candidates for CF-LVADs. Results The levels of GM-CSF, MIP-1 beta and MDC were significantly higher in the CF-LVAD group compared to both the HC and the HF groups, whereas no significant differences were observed between the HC and the HF groups. Compared to the HC group, CRP, IP-10, MCP-1 and IL-8 levels were significantly higher in both the CF-LVAD and HF groups, while no significant differences were observed between the CF-LVAD recipients and the HF patients. [ figure 1 ] Conclusions We found that inflammation was high in CF-LVAD recipients. Further studies should investigate the time course of elevation of inflammatory mediators from pre- to post- CF-LVAD implantation and following long term CF-LVAD support.

Elevated Levels of Inflammation in Patients on Long-Term Continuous Flow Left Ventricular Assist Device (CF-LVAD) Support

Purpose Elevated levels of inflammatory markers shortly after implantation predict mortality in patients supported with left ventricular assist devices (LVADs). However, no study has investigated the long-term effects of continuous-flow LVAD (CF-LVAD) support on inflammation. CF-LVAD support may expose heart failure patients to high levels of inflammation either as a result of the non-physiological continuous-flow or as a result of the blood contact with an artificial surface. We hypothesize that in CF-LVAD recipients the levels of inflammatory markers are higher than those of healthy controls and these levels increase over time with long-term CF-LVAD support. Methods and Materials We examined the levels of inflammatory markers longitudinally at the heart failure baseline prior to CF-LVAD implantation and at 3, 6, and 9 months post-implantation and compared them to the levels of inflammation in healthy controls. Results The levels of MDC, MCP-1, TNF-alpha were significantly higher in the CF-LVAD recipients compared to the healthy controls at 3, 6, and 9 months post-implantation. Compared to healthy controls, IP-10 levels were higher at 3 and 9 months, while CRP levels were significantly higher only at 9 months post-implantation. [ figure 1 ] Conclusions Our study is the first to find that inflammation is high in patients on long term CF-LVAD support. Elevated levels of inflammation may be the results of both the heart failure condition prior to the implantation and the long term device support. Monitoring inflammatory markers and treatment to reduce inflammation may have clinical importance in these patients and should be further investigated.

Methyl 5-Chloro-4,5-didehydrojasmonate (J7) Inhibits Macrophage-Derived Chemokine Production &lt;i&gt;via&lt;/i&gt; Down-Regulation of the Signal Transducers and Activators of Transcription 1 Pathway in HaCaT Human Keratinocytes

Jasmonates are lipid-based stress hormones that are critical for the defense of plants against insects. Two naturally occurring jasmonates, jasmonic acid and methyl jasmonate, have recently been explored for their efficacy as anti-cancer agents. Furthermore, certain synthetic jasmonates (e.g., the cyclopentenone isoprostane J2) exert anti-inflammatory actions in lipopolysaccharide (LPS)-challenged murine macrophages via down-regulation of chemokines and other inflammatory mediators. Chemokines participate in the development and progression of many inflammatory disorders, such as atopic dermatitis (AD) and Crohn's disease, as exemplified by the role of macrophage-derived chemokine (MDC/CCL22) in the pathology of AD. The current study therefore investigated the impact of jasmonate derivatives (jasmonic acid and methyl jasmonate) and their synthetic analogues (J2 and J7) on the expression of MDC in interferon (IFN)-γ- and tumor necrosis factor (TNF)-α-stimulated HaCaT human keratinocytes, as well as the attendant mechanism of action. Jasmonic acid, methyl jasmonate, and J2 failed to inhibit the cytokine-stimulated production of MDC. By contrast, J7 suppressed the mRNA and protein expression levels of MDC in a dose-dependent manner. Moreover, J7 diminished the activation of signal transducers and activators of transcription 1 (STAT1), but had no inhibitory effect on the nuclear factor kappa B (NF-κB) or mitogen-activated protein kinase (MAPK) pathways. These results demonstrate that J7 impairs IFN-γ- and TNF-α-induced inflammatory chemokine production by targeting the STAT1 pathway.

Chemokine Binding Protein vCCI Attenuates Vaccinia Virus Without Affecting the Cellular Response Elicited by Immunization with a Recombinant Vaccinia Vector Carrying the HPV16 E7 Gene

Viral CC chemokine inhibitor (vCCI) of the clone P13 vaccinia virus (VACV) strain PRAHA lacks eight amino acids in the signal peptide sequence. To study the influence of vCCI on virus biology, a virus with the vCCI gene coding for a prolonged signal sequence was prepared. We found that secreted vCCI attenuated the virus in vivo, and that it correlated with decreased levels of RANTES, eotaxin, TARC, and MDC in the blood in comparison with the parental virus. We determined the influence of vCCI on the CTL response against VACV E3((140-148)) (VGPSNSPTF) and HPV16 E7((49-57)) (RAHYNIVTF) H-2D(b)-restricted epitopes. The examination of the specific CTL response elicited by immunization with the recombinant VACV-expressing tumor-associated HPV16 E7 antigen by IFN-γ ELISPOT showed that the immunogenicity of the recombinant VACV-producing secretory vCCI was similar to that of the parent virus or deletion mutant in the C23L/B29R locus. Immunization with the secretory vCCI-producing recombinant virus has a lower therapeutic anti-tumor effect against TC-1 tumors. Viral CCI downregulated the E7-specific response induced by gene gun immunization with the DNA vaccines pBSC-SigE7 LAMP and pBSC-vCCI. We also observed that the immune response against vCCI elicited by the DNA vaccine did not affect the multiplication of VACV in vivo.

Detection of HIV T-cell responses with polifunctionality and high plasma levels of the B-chemokine MDC in exposed HIV-seronegative individuals (ESN)

Background The mechanisms of protection from infection in ESN individuals are largely undefined and likely multifactorial. Our aim was to analyze HIV-specific T-cell responses and plasma cytokine levels in ESN and their respective HIV partners (HIV-P) in comparison with HIV chronically infected subjects (C) and healthy donors (HD). Methods Plasma levels of 37 cytokines were measured by Luminex technology in: 10 HD, 9 ESN and their HIV-P, and 12 C. All HIV patients were off-HAART. Frozen PBMCs from the same individuals were used to determine HIV-specific T-cell responses by IFN-g ELISPOT. Also, production of different cytokines was evaluated in certain samples by intracellular staining (ICS). Data was compared inter- and intra-groups and correlated to viral load (VL) and CD4 T cell counts, using parametric and non-parametric statistics.

Pre‐transplant myeloid dendritic cell deficiency associated with cytomegalovirus infection and death after kidney transplantation

Dendritic cells (DCs) are potent antigen-presenting cells critical for immunity. We previously demonstrated a significant association between pre-transplant blood myeloid dendritic cell (mDC) and plasmacytoid dendritic cell (pDC) deficiency and post-transplant BK viremia in renal transplant recipients. In the current post-hoc analysis, we studied the association of these same pre-transplant DC levels with other post-transplant outcomes. Pre-transplant peripheral blood mDC and pDC levels were quantified using flow cytometry in 78 patients undergoing kidney transplantation. Post-transplant outcomes were analyzed, including infection, rejection, and patient death, with a median follow-up of 5.3years. Associations between DC levels and outcomes were assessed using logistic regression analysis and Cox proportional hazards models. An independent association of mDC levels with post-transplant cytomegalovirus infection (adjusted odds ratio 7.0, P=0.01) and patient death (adjusted hazard ratio 13.0, P=0.015) was found. No associations were demonstrated between levels of either DC subtype and bacterial infections or rejection. Pre-transplant mDC deficiency is significantly associated with CMV infection and death after kidney transplantation.

Tocilizumab Treatment Decreases Circulating Myeloid Dendritic Cells and Monocytes, 2 Components of the Myeloid Lineage

Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) are proinflammatory cytokines involved in inflammatory response. Effective TNF-α blocker treatment is associated with an increase in circulating myeloid dendritic cells (mDC), suggesting their release from inflamed synovium. Currently, in vivo effects of IL-6 inhibition on DC are unknown. We monitored the changes in circulating mDC and plasmacytoid DC (pDC) during tocilizumab (TCZ) therapy in patients with rheumatoid arthritis (RA). DC subset levels were evaluated by flow cytometry in patients with RA (n = 43) and in healthy volunteers (n = 20). In patients with RA, these levels were measured before and during TCZ therapy (8 mg/kg every 4 weeks). Response to TCZ therapy was evaluated at 12 weeks. Statistical analysis was based on Mann-Whitney U tests or Wilcoxon signed-rank tests. At baseline, patients with active RA were characterized by a significantly lower level of circulating mDC and pDC compared to healthy donors. However, this difference did not correlate with any disease activity score. TCZ-treated patients who met the European League Against Rheumatism (EULAR) improvement criteria at Week 12 had significant reductions in mDC and monocyte levels as compared with EULAR nonresponders. Levels of pDC, CD4+ T cells, and CD8+ T cells remained stable during the TCZ courses, regardless of treatment response. Our study reveals an unexpected reduction of circulating mDC and monocytes in patients with RA in response to TCZ therapy. In accord with reports on neutrophils and platelets decreasing during TCZ therapy, our data suggest an effect of IL-6 inhibition on cells from myeloid lineage.

CCR4 Expression in Hodgkin Lymphoma

Abstract 2626In Hodgkin lymphoma (HL) a minority (<1%) of tumor cells is found in a reactive background. The tumor cells shape their environment by the production of several chemokines and cytokines. Production of the chemokine TARC will lead to the attraction of Th2 and Treg cells specifically to involved lymph nodes. Although Treg and Th2 cells are prominent in the infiltrate the expression of its receptor CCR4 is not very high in lymphocytes surrounding the tumor cells. We hypothesized that this is due to internalization of the receptor. We analyzed in which specific compartment of lymphocytes CCR4 is expressed and if CCR4 expression can be recovered when TARC and MDC levels are low.We analyzed 11 lymph node suspensions involved with HL and 11 with follicular hyperplasia (RLN) for expression of CD4, CD26, FoxP3 and CCR4 at t=0 and t=24 hours. We tested the effect of TARC and MDC on CCR4 expression and the mechanism of internalization on normal PBMCs.At t=0 the CD4+ T cell compartment was significantly lower for CD26 in HL (49% vs 68% in RLN, p<0.01) (Table 1). In the comparison between CD26+ and CD26- cells no difference was seen in CCR4 expression in HL but in RLN CCR4 in CD26- cells was significantly lower than in CD26+ cells (p<0.01).Recovery of CCR4 (in average %) after 24 hours was significantly lower in CD26+ cells and FoxP3+ cells in RLN (p<0.01 and p<0.001). Recovery of CCR4 as factor change is significantly higher in HL than in RLN for CCR4 as well as the 3 subpopulations (Table 2). Generally, internalization of CCR4 on lymphocytes is higher with MDC (54 to 90% internalization with 10 and 500ng/ml MDC) than TARC (28 to 48% with 10 and 1000ng/ml TARC) after 2 hours. Internalization occurs through the lipid raft/caveolae dependent pathway as filipin III could inhibit internalization completely and not by clathrin-mediated endocytosis since no effect of inhibition by sucrose was seen. Recovery of CCR4 back on the membrane is fast and by exocytosis since it can be disrupted by exocytosis inhibitor Brefeldin A.Table 1:Percentages subpopulations CD4+ T cells in Hodgkin lymphoma and reactive lymph node by flowcytometry on cell suspensionsHLRLNpCD264968<0.01CCR44137nsFoxP34.41.8nsCCR4/CD26+1925nsCCR4/CD26-2212nsCCR4/FoxP3+5560nsTable 2:Factor of CCR4 recovery after 24 hours in CD4+ subpopulations in Hodgkin lymphoma and reactive lymph node cell suspensionsHLRLNpCCR41.70.9<0.05CCR4 in CD26+1.30.8<0.05CCR4 in CD26-2.91.1<0.05CCR4 in FoxP3+1.60.8<0.05In conclusion, CD26 expression is significantly lower in HL than RLN. CCR4 expression is equally divided over CD26+ and CD26- cells in HL, but in RLN less is found in CD26-. Recovery of CCR4 expression after culture for 24 hours is lower in RLN than in HL. More CCR4 is recovered in HL especially in the CD26- CD4+ T cell population that is surrounding the tumor cells in HL. Disclosures:No relevant conflicts of interest to declare.

Increased prevalence of regulatory T cells in the lung cancer microenvironment: a role of thymic stromal lymphopoietin.

Expansion of CD4+CD25+ regulatory T cells (Tregs) in tumor microenvironment was one of the mechanisms by which cancer cells escaped host defense. Thymic stromal lymphopoietin (TSLP) contributes to the generation of natural Tregs in thymus. Therefore, the purpose of this report was to investigate the role of TSLP in the increasing prevalence of Tregs in lung cancer microenvironment. The expression ratio of TSLP protein in tumor tissues was significantly increased compared with that in benign lesion and non-cancer lung tissue. The prevalence of Tregs in tumor microenvironment was correlated with the expression of TSLP in lung cancer. Dendritic cells (DCs) were induced from peripheral blood mononuclear cells (PBMCs) collected from lung cancer patients and left unstimulated (imDCs) or exposed to hTSLP (TSLP-DCs) or LPS (LPS-DCs). TSLP-DCs expressed intermediate levels of CD83 and high levels of CD86, CD11C, and HLA-DR, which showed a characteristic of less mature DCs. TSLP-DCs secreted low levels of IL-6, IL-12, IL-10, TNF-α and IFN-γ, and high levels of TGF-β and MDC. The percentage of Tregs in CD4+CD25- T cells cocultured with TSLP-DCs group was statistically higher than that of LPS-DCs and imDCs. Transwell assays showed that TSLP-DCs exhibited increased ability to attract the migration of CD4+CD25- Tregs, when compared with imDCs. These results indicated that TSLP proteins were expressed in lung tumor tissue and correlated with the prevalence of Tregs. TSLP-DCs could induce CD4+CD25- T cells to differentiate into CD4+CD25+foxp3+ T cells and the migration of CD4+CD25+ T cells.

The Role of Plasmacytoid and Myeloid Dendritic Cells in Induction of Asthma in a Mouse Model and the Effect of a TLR9 Agonist on Dendritic Cells

PurposeTo determine the role of plasmacytoid dendritic cells (pDC) and myeloid dendritic cells (mDC) in priming effector T cells to induce allergy, and to evaluate the effect of immunostimulatory sequences (ISS, TLR9 agonist) on dendritic cells.MethodsCultured mDC and pDC with/without ISS were injected intratracheally into sensitized Balb/C mice. Mice were sacrificed, and then pulmonary function tests, bronchoalveolar lavage (BAL), cell counts, and cytokine levels were evaluated. Migration of dendritic cells was also evaluated after ISS administration.ResultsIn mice injected with mDC, airway hyperresponsiveness, eosinophil counts, and Th2 cytokine levels in BAL increased with increasing numbers of mDC injected. However, in mice injected with pDC, none of these changed, suggesting poor priming of T cells by pDC. In addition, mDC pulsed with ISS inhibited asthmatic reactions, and ISS administration inhibited migration of DC to the lung.ConclusionsWe suggest that pDC played a limited role in priming T cells in this asthma model and that mDC played a major role in inducing asthma. In addition, ISS inhibited migration of DC to the lung.

Characteristic Proinflammatory Serum Cytokine Profiles In Patients with B-Cell Chronic Lymphocytic Leukemia

AbstractAbstract 3595 Background:Cytokines are posited to play a critical regulatory role on the survival of the B-cell neoplastic clone in Chronic Lymphocytic Leukemia (CLL). AIM: The primary goals of this study were 1) to define additional relevant cytokines, growth factors and chemokines in CLL pathophysiology and 2) to correlate abnormal cytokine levels with disease stage, relevant hematological data and multiple prognostic factors. METHODS: A novel bead-based protein array system was employed to simultaneously measure 38 proinflammatory cytokines in the sera of CLL patients (N=116) and healthy age and sex matched controls (N=30). These results were correlated with Rai stage, β2-microglobulin level, LDH, CD38 expression and cytogenetic abnormalities. RESULTS: We first confirmed previous observations that TNFα, IL-1α, IL-1RA, IL-10, sIL-2Rα, VEGF and sCD40 ligand are significantly elevated in patients with CLL as compared with healthy controls. Expanding on the current literature, we demonstrated perturbations in an additional 15 serum cytokines in affected individuals. Compared to healthy controls, CLL patients had an increase in serum levels of IL-3 (p=0.002), IL-7 (p=0.008), INF-2α (p<0.0001), MCP-1 (p<0.0001), MIP-1β (p=0.002), MDC (p<0.0001), Fractakine (p<0.0001), EGF (p<0.0001), FGF-2 (p<0.0001), GRO (p<0.0001), Eotaxin (p<0.0001) and FLT-3 ligand (p<0.0001). Patients with CLL also exhibited significantly lower levels of INF-γ (p<0.01), IL-6 (p<0.005) and IL-8 (p<0.002) when compared to healthy individuals. Advanced Rai stage and high risk chromosomal abnormalities (del 11q and del 17p) strongly correlated with higher serum levels of TNFα, soluble IL-2Rα, IL-10, MCP-1, MIP-1α, MIP-1β and IP-10. Finally, serum levels of TNFα, MIP-1α and MIP-1β correlated with other adverse prognostic markers, including total white blood cell count, serum β2-microglobulin and LDH levels as well as CD38 expression. CONCLUSION: We have demonstrated numerous previously unrecognized cytokine abnormalities in patients with CLL and described a unique cytokine signature associated with advanced disease. Supported by the current understanding of cytokine biology and CLL pathophysiology, our observations suggest an important regulatory role for hematopoietic cytokines, such as IL-3 and IL-7, in promulgating survival of the aberrant B-cell clone. Likewise, the profoundly high levels of chemokines (i.e. MCP-1, MIP-1α, MIP-1β, IP-10) and their association with high risk prognostic factors argue for their role in sustaining the neoplastic microenvironment. Finally, the altered levels of IL-10, IL-6 and INF-γ observed in patients with CLL likely contribute to the immunosuppressive phenotype of the disease state. Disclosures:No relevant conflicts of interest to declare.