Impact of smoking on dendritic cell phenotypes in the airway lumen of patients with COPD

Background

Myeloid dendritic cells (DCs) are increased in the airway wall of patients with chronic obstructive pulmonary disease (COPD), and postulated to play a crucial role in COPD. However, DC phenotypes in COPD are poorly understood.

Methods

Function-associated surface molecules on bronchoalveolar lavage fluid (BALF) DCs were analyzed using flow cytometry in current smokers with COPD, in former smokers with COPD and in never-smoking controls.

Results

Myeloid DCs of current smokers with COPD displayed a significantly increased expression of receptors for antigen recognition such as BDCA-1 or Langerin, as compared with never-smoking controls. In contrast, former smokers with COPD displayed a significantly decreased expression of these receptors, as compared with never-smoking controls. A significantly reduced expression of the maturation marker CD83 on myeloid DCs was found in current smokers with COPD, but not in former smokers with COPD. The chemokine receptor CCR5 on myeloid DCs, which is also important for the uptake and procession of microbial antigens, was strongly reduced in all patients with COPD, independently of the smoking status.

Conclusion

COPD is characterized by a strongly reduced CCR5 expression on myeloid DCs in the airway lumen, which might hamper DC interactions with microbial antigens. Further studies are needed to better understand the role of CCR5 in the pathophysiology and microbiology of COPD.     

树突细胞上Toll样受体的介导作用

树突状细胞是体内最重要的抗原提呈细胞,它表面表达多种Toll样受体。Toll样受体可通过多条途径来激活树突细胞,介导树突细胞对抗原的摄取,递呈及生存与凋亡,促进T细胞增值和分化并参与免疫反应。     

Allergic airway inflammation induces the migration of dendritic cells into airway sensory ganglia

Background

A neuroimmune crosstalk between dendritic cells (DCs) and airway nerves in the lung has recently been reported. However, the presence of DCs in airway sensory ganglia under normal and allergic conditions has not been explored so far. Therefore, this study aims to investigate the localisation, distribution and proliferation of DCs in airway sensory ganglia under allergic airway inflammation.

Methods

Using the house dust mite (HDM) model for allergic airway inflammation BALB/c mice were exposed to HDM extract intranasally (25 μg/50 μl) for 5 consecutive days a week over 7 weeks. With the help of the immunohistochemistry, vagal jugular-nodose ganglia complex (JNC) sections were analysed regarding their expression of DC-markers (MHC II, CD11c, CD103), the neuronal marker PGP 9.5 and the neuropeptide calcitonin gene-related peptide (CGRP) and glutamine synthetase (GS) as a marker for satellite glia cells (SGCs). To address the original source of DCs in sensory ganglia, a proliferation experiment was also carried in this study.

Results

Immune cells with characteristic DC-phenotype were found to be closely located to SGCs and vagal sensory neurons under physiological conditions. The percentage of DCs in relation to neurons was significantly increased by allergic airway inflammation in comparison to the controls (HDM 51.38 ± 2.38% vs. control 28.16 ± 2.86%, p < 0.001). The present study also demonstrated that DCs were shown to proliferate in jugular-nodose ganglia, however, the proliferation rate of DCs is not significantly changed in the two treated animal groups (proliferating DCs/ total DCs: HDM 0.89 ± 0.38%, vs. control 1.19 ± 0.54%, p = 0.68). Also, increased number of CGRP-positive neurons was found in JNC after allergic sensitisation and challenge (HDM 31.16 ± 5.41% vs. control 7.16 ± 1.53%, p < 0.001).

Conclusion

The present findings suggest that DCs may migrate from outside into the ganglia to interact with sensory neurons enhancing or protecting the allergic airway inflammation. The increase of DCs as well as CGRP-positive neurons in airway ganglia by allergic airway inflammation indicate that intraganglionic DCs and neurons expressing CGRP may contribute to the pathogenesis of bronchial asthma. To understand this neuroimmune interaction in allergic airway inflammation further functional experiments should be carried out in future studies.     

Preparation of peptide-loaded dendritic cells for cancer immunotherapy

Dendritic cell-based vaccines are being evaluated in clinical trials to determine their ability to activate clinically relevant tumor antigen-specific immune responses. Although some groups isolate dendritic cells from peripheral blood, most have found it more efficient to generate large numbers from peripheral blood progenitors, particularly plastic adherent or CD14+ monocytes, in media supplemented with GM-CSF and IL-4. These DC may then be matured, if desired, and loaded with antigen, such as tumor-associated peptides, prior to administration. We describe the scheme that we are currently using to generate peptide-loaded dendritic cells for our clinical trials of cancer immunotherapy.     

Aspirin regulates SNARE protein expression and phagocytosis in dendritic cells

Abstract

Since being introduced globally as Aspirin in 1899, acetylsalicylic acid (ASA) has been widely used as an analgesic, immune-regulatory, anti-pyretic and anti-thrombotic drug. ASA and its metabolite, salicylate, were also reported to be able to modulate antigen presenting functions of dendritic cells (DC). However, the intracellular targets of ASA in DC are still poorly understood. Since phagocytosis is the initial step taken by antigen-presenting cells in the uptake of antigens for processing and presentation, ASA might exerts its immune-regulatory effects by regulating phagocytosis. Here we show that ASA inhibits phagocytosis and modulates expression of endosomal SNAREs, such as Vti1a, Vti1b, VAMP-3, VAMP-8 and Syn-8 (but not syn-6 and syn-16) in DC. We further show that the phagocytic inhibitory effect of ASA is dependent on the expression of Vti1a and Vti1b. Consistently, Vti1a and Vti1b localize to the phagosomes and up-regulation of Vti1a and Vti1b inhibits phagocytosis in DC. Our results suggest that ASA modulates phagocytosis in part through the control of endosomal SNARE protein expression and localization in DC. All experiments were performed using either a murine DC line (DC2.4) or primary DC derived from murine bone marrow cells.     

Ganglioside GD1a impedes lipopolysaccharide-induced maturation of human dendritic cells

Immunosuppressive membrane gangliosides are released by tumor cells and inhibit normal antigen presenting cell (APC) function. To better understand this process, we have studied the effect of gangliosides on lipopolysaccharide (LPS)-induced maturation of human dendritic cells (DCs). Immature DCs were generated in vitro from human peripheral blood monocytes and were exposed for 72 h to a highly purified ganglioside, G(D1a). During the last 24 h, LPS was added to effect maturation. As assessed by fluorescence activated cell sorting (FACS) analysis, incubation in 50 microM G(D1a) significantly blunted the LPS-induced maturation of the dendritic cells. The expected up-regulation of expression of the co-stimulatory molecules CD80 and CD86 was ablated and that of CD40 was reduced, as were surface CD83 expression and intracellular CD208 production. In addition, ganglioside pretreatment of DC markedly inhibited the allostimulatory capacity and partially prevented the down-regulation of FITC-dextran uptake characteristic of LPS-activated DC. Furthermore, ganglioside-exposed DC also evidenced a broad down-regulation of the cytokine release that is normally initiated by LPS exposure, i.e., there was no increase in IL-1 beta, IL-6, IL-10, IL-12, or tumor necrosis factor (TNF)-alpha release. That a common mechanism may underlie these defects was suggested by the finding that G(D1a) exposure of DC also inhibited the nuclear binding of NF-kappa B that is normally induced by LPS. These results suggest that tumor gangliosides may blunt the anti-tumor immune response in vivo by binding and interfering with dendritic cell maturation.     

消化道屏障与树突状细胞免疫研究进展

肠相关淋巴组织在防御病毒、细菌、寄生虫等有害物质入侵中发挥着重要作用,是消化道屏障的组成部分,其中肠道树突状细胞尤为重要,作为目前所知的机体内功能最强的专职性抗原呈递细胞,在肠道内,树突状细胞不但能对病原菌产生免疫应答,还能对肠腔内的正常菌群和各种食物蛋白产生免疫耐受,因此了解树突状细胞的功能及相关作用机制有着重要意义.现就对树突状细胞的生物学特性,以及其在肠道免疫中的作用进行综述.     

Financial incentives for smoking cessation in lowincome smokers: study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: Tobacco smoking is the leading avoidable cause of death in high-income countries. The smoking-related disease burden is borne primarily by the least educated and least affluent groups. Thus, there is a need for effective smoking cessation interventions that reach to, and are effective in this group. Research suggests that modest financial incentives are not very effective in helping smokers quit. What is not known is whether large financial incentives can enhance longer-term (1 year) smoking cessation rates, outside clinical and workplace settings. Trial design A randomized, parallel groups, controlled trial. METHODS: Participants: Eight hundred low-income smokers in Switzerland (the less affluent third of the population, based on fiscal taxation). Intervention: A smoking cessation program including: (a) financial incentives given during 6 months; and (b) Internet-based counseling. Financial rewards will be offered for biochemically verified smoking abstinence after 1, 2, and 3 weeks and 1, 3, and 6 months, for a maximum of 1,500 CHF (1,250 EUR, 1,600 USD) for those abstinent at all time-points. All participants, including controls, will receive Internet-based, individually-tailored, smoking cessation counseling and self-help booklets, but there will be no in-person or telephone counseling, and participants will not receive medications. The control group will not receive financial incentives. OBJECTIVE: To increase smoking cessation rates. Outcome: Smoking abstinence after 6 and 18 months, not contradicted by biochemical tests. We will assess relapse after the end of the intervention, to test whether 6-month effects translate into sustained abstinence 12 months after the incentives are withdrawn.Randomization: Will be done using sealed envelopes drawn by participants. Blinding: Is not possible in this context. DISCUSSION: Smoking prevention policies and interventions have been least effective in the least educated, low-income groups. Combining financial incentives and Internet-based counseling is an innovative approach that, if proven acceptable and effective, could be later implemented on a large scale at a reasonable cost, decrease health disparities, and save many lives. Trial registration Current Controlled Trials ISRCTN04019434.     

树突状细胞与Toll受体研究新进展

树突状细胞是最强的抗原提呈细胞,在免疫系统中发挥着着重要作用。Toll样受体是表达在树突状细胞上的一种PRRs,主要功能是通过识别病原体微生物所携带的病原相关分子模式激活DC,使其分泌各种免疫调节细胞因子,从而启动免疫应答。在肠道免疫中TLR信号的激活为肠道提供保护作用。本文简述了树突状细胞的生物特性、不同亚型。重点阐述了Toll样受体在肠道免疫中的作用及益生菌对肠道Toll样受体表达的影响。     

Fast generation of dendritic cells

Dendritic cells (DC) are potent antigen presenting cells capable of inducing immune responses. DC are widely used as vaccine adjuvant in experimental clinical settings. DC-based vaccines are normally generated using a standard 8 day DC protocol (SDDC). In attempts to shorten the vaccine production we have developed fast DC protocol by comparing two different fast DC protocols with SDDC. DC were evaluated by FACS analysis, and the optimal profile was considered: CD14^low, CD80^high, CD83^high, CD86^high, CCR7^high, HLA class I and II^high. FACS profiles were used as the selection criteria together with yield and morphology. Two fast DC protocols fulfilled these criteria and were selected for functional analysis. Our results demonstrate that DC generated within 5 days or 48 h are comparable with SDDC both phenotypically and functionally. However, we found that 48 h DC were more susceptible than SDDC to the IL-10 inducing stimulus of TLR ligands (R848 and LPS). Thus to determine the clinical relevance of fast DC protocols in cancer settings, small phase I trials should be conducted monitoring regulatory T cells carefully.     

Inefficient presentation of tumor-derived antigen by tumor-infiltrating dendritic cells

BACKGROUND: Transplantable B16 melanoma is widely used as a tumor model to investigate tumor immunity. We wished to characterize the leukocyte populations infiltrating B16 melanoma tumors, and the functional properties of tumor-infiltrating dendritic cells (TIDC). MATERIALS AND METHODS: We used the B16 melanoma cell line expressing ovalbumin protein (OVA) to investigate the phenotype and T cell stimulatory capacity of TIDC. RESULTS: The majority of leukocytes in B16 melanoma were macrophages, which colocalized with TIDCs, B and T cells to the peripheral area of the tumor. Both myeloid and plasmacytoid DC populations were present within tumors. Most of these DCs appeared immature, but about a third expressed a mature phenotype. TIDCs did not present tumor-derived antigen, as they were unable to induce the proliferation of tumor-specific CD4+ and CD8+ T cells in vitro unless in the presence of specific peptides. Some presentation of tumor-derived antigen could be demonstrated in the tumor-draining lymph node using in vivo proliferation assays. However, while proliferation of CD8+ T cells was reproducibly demonstrated, no proliferation of CD4+ T cells was observed. CONCLUSION: In summary, our data suggest that DCs in tumors have limited antigen-presenting function. Inefficient antigen presentation extends to the tumor-draining lymph node, and may affect the generation of antitumor immune responses.     

Comparative effectiveness of post-discharge interventions for hospitalized smokers: study protocol for a randomized controlled trial

ABSTRACT: BACKGROUND: A hospital admission offers smokers an opportunity to quit. Smoking cessation counseling provided in the hospital is effective, but only if it continues for more than one month after discharge. Providing smoking cessation medication at discharge may add benefit to counseling. A major barrier to translating this research into clinical practice is sustaining treatment during the transition to outpatient care. An evidence-based, practical, cost-effective model that facilitates the continuation of tobacco treatment after discharge is needed. This paper describes the design of a comparative effectiveness trial testing a hospital-initiated intervention against standard care. Methods/design: A 2-arm randomized controlled trial compares the effectiveness of standard post-discharge care with a multi-component smoking cessation intervention provided for 3 months after discharge. Current smokers admitted to Massachusetts General Hospital who receive bedside smoking cessation counseling, intend to quit after discharge and are willing to consider smoking cessation medication are eligible. Study participants are recruited following the hospital counseling visit and randomly assigned to receive Standard Care or Extended Care after hospital discharge. Standard Care includes a recommendation for a smoking cessation medication and information about community resources. Extended Care includes up to 3 months of free FDA-approved smoking cessation medication and 5 proactive computerized telephone calls that use interactive voice response technology to provide tailored motivational messages, offer additional live telephone counseling calls from a smoking cessation counselor, and facilitate medication refills. Outcomes are assessed at 1, 3, and 6 months after hospital discharge. The primary outcomes are self-reported and validated 7-day point prevalence tobacco abstinence at 6 months. Other outcomes include short-term and sustained smoking cessation, post-discharge utilization of smoking cessation treatment, hospital readmissions and emergency room visits, and program cost per quit. DISCUSSION: This study tests a potentially disseminable smoking intervention model for hospitalized smokers. If effective and widely adopted, it could help to reduce population smoking rates and thereby reduce tobacco-related mortality, morbidity, and health care costs.     

Oxysterols induce transition of monocytic cells to phenotypically mature dendritic cell-like cells

Dendritic cells (DCs) activate adaptive immune responses in atherosclerotic plaques; however, the origin of DCs is in question. We attempted to determine whether cholesterol or its oxide forms, which are detected in abundance in atheromatous lesions, could induce differentiation or transition of monocytic cells to DCs. Treatment of THP-1 cells with 27-hydroxycholesterol (27OH-Chol) and 7α-hydroxycholesterol (7αOH-Chol) resulted in an increase in the numbers of adherent cells, and, in contrast to PMA, decreased uptake of FITC-conjugated dextran. In addition, treatment with 27OH-Chol and 7αOH-Chol induced expression of mDC-specific molecules, including CD40, CD80, CD83, and CD88. Of the two oxysterols, 27OH-Chol enhanced expression of MHC class I and II molecules as well as CCR7. However, treatment with an identical concentration of cholesterol and 7-ketocholesterol did not influence adherence, uptake of FITC-conjugated dextran, and expression of the aforementioned molecules. This is the first study to report on change of monocytic cells by oxysterols to phenotypically atypical cells with some characteristics of mDCs detected in atherosclerotic lesions. We propose that a certain type of oxysterol would contribute to immune responses in atherosclerotic lesions by enhancing expression of multiple CD molecules as well as MHC molecules by monocytic cells.     

Immunoregulatory effects of freeze injured whole tumour cells on human dendritic cells using an in vitro cryotherapy model

Tumour cryotherapy has been described as both immunostimulatory and immunoinhibitory in previous studies. However, previous studies have not accurately reproduced the precise conditions of current clinical cryotherapy. The objective of this study is to assess the immunological effects of cryotreated whole tumour cells on dendritic cells (DC) maturation and function using an in vitro model. Prostate cancer cells were cooled using Endocare cryo-system to mimic temperatures achieved during clinical cryotherapy. Human DC were prepared from cluster of differentiation (CD) 14 monocytes and matured with lipopolysaccharide (LPS). Cryotreated cancer cells were added to DC on day 3. On day 7, DC were harvested and phenotyped. Cytokine gene expression was assessed using real time quantitative polymerase chain reaction (RT-PCR). Functional activity of DC was assessed in allogenic mixed lymphocyte reaction (MLR) and the molecular changes using gene microarray technology. There was statistically significant upregulation of costimulatory molecules and maturation markers (CD86, CD83, CD80 and CL II) in DC loaded with cryotreated whole tumour cells compared to both control DC and DC matured with LPS (P < 0.001). There was a significant increase in stimulatory cytokines gene expression (IL-2, IL-12, IL-15, IL-18 and IFN-γ). However, IL-10 and TGF-β expression reduced significantly. The effect of different freezing temperature was equal. cDNA microarray analysis showed upregulation of interleukin 1 (IL-1) and cycline dependent kinase inhibitor 1A (CDKN1A (p21) and downregulation of Caspase 8 and BCL2. Overall, our findings suggest that the effect of cryotherapy is generally stimulatory to DC which may enhance anti-tumour effects. Therefore, the combination of cryotherapy and DC vaccine may represent a novel method to increase the efficacy of cryotherapy especially at the peripheral zones of the prostate where cells are exposed to sub-lethal temperature.     

Listeria monocytogenes infection in pregnant mice: Abnormalities in the function of non-adherent accessory light density dendritic cells

Abstract Pregnant A/J mice were found to be more susceptible to the lethal effect of Listeria monocytogenes bacteria than virgin females. However, during the first four days of post-infection there was no difference in the elimination of Listeria from the spleens of pregnant and virgin mice. This suggests that the increase in the susceptibility of pregnant mice to pathogenic activity of L. monocytogenes was related to the diminution in Listeria -specific cellular reactions. Indeed, we found that non-adherent light density dendritic cells (DCs) from pregnant mice showed a marked reduction in the ability to form clusters with L. monocytogenes immune T lymphocytes and it is known that cell cluster formation between antigen presenting cells (APC) and responding T cells is required for antigen recognition as well as for cell proliferation. DCs from pregnant mice also demonstrated the decrease and an instability in the expression of H-2 class II molecules which play a crucial role in the recognition of exogenous antigens. The abnormalities demonstrated in the function of the light density dendritic cells from the spleens of pregnant mice could compromise cellular reactions to L. monocytogenes bacteria possibly resulting in increased susceptibility of pregnant mice to experimental listeriosis.     

Induction of JAM-A during differentiation of human THP-1 dendritic cells

Junctional adhesion molecule (JAM)-A is not only localized at tight junctions of endothelial and epithelial cells but is also expressed on circulating leukocytes and dendritic cells (DCs). In the present study, to investigate the regulation of JAM-A in DCs, mature DCs were differentiated from the human monocytic cell THP-1 by treatment with IL-4, GM-CSF, TNF-α, and ionomycin, and some cells were pretreated with the PPAR-γ agonists. In the THP-1 monocytes, mRNAs of tight junction molecules, occludin, tricellulin, JAM-A, ZO-1, ZO-2 and claudin-4, -7, -8, and -9 were detected by RT-PCR. In mature DCs that had elongated dendrites, mRNA and protein of JAM-A were significantly increased compared to the monocytes. PPAR-γ agonists prevented the elongation of dentrites but not upregulation of JAM-A in mature DCs. These findings indicated that the induction of JAM-A occurred during differentiation of human THP-1 DCs and was independent of PPAR-γ and the p38 MAPK pathway.     

Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels

Changes in gene expression occurring during differentiation of human monocytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase in mitochondrial function and in the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary.     

Phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells

Dendritic cells (DC) as key mediators of tolerance and immunity perform crucial immunosurveillance functions at epithelial surfaces. In order to induce an immune response, the DC have to gain access to antigens present at the luminal surface of mucosal epithelia. The mechanisms of this process are still largely unclear. We have therefore analysed the distribution of DC in the porcine intestinal and respiratory mucosa and their spatial relationship to epithelial cells by immunohistology. Immunofluorescence analysis of cryosections taken from jejunal Peyer’s patches and double-stained for DC and M cells (specialised for antigen uptake) have revealed that 35.2±3.9% of M cells are located directly adjacent to DC in the subepithelial domes, representing possible antigen transfer sites. In normal jejunal villi, a rare population of lamina propria DC extending cytoplasmic processes between enterocytes has been identified as a possible correlate for direct luminal antigen uptake. Like small intestinal DC, DC in the porcine trachea mostly co-express CD16 with MHC-II. Tracheal DC have been found at high densities both above and below the basement membrane (BM) of the tracheal epithelium, with 32.4 DC/mm BM and 23.0 DC/mm BM, respectively. The intraepithealial DC population forms a dense network, with many of the cytoplasmic processes being directed towards the tracheal lumen. Our morphological analyses indicate that DC at mucosal epithelial sites are ideally positioned for the uptake of luminal antigens.This work was supported by the EU (5th framework programme “Healthypigut” QLK5-CT-2000-00522 and 6th framework programme “Feed for Pig Health” FOOD-CT 2004-506144).     

Thrombin regulates the function of human blood dendritic cells

Thrombin is the key enzyme in the coagulation cascade and activates endothelial cells, neutrophils and monocytes via protease-activated receptors (PARs). At the inflammatory site, immune cells have an opportunity to encounter thrombin. However little is known about the effect of thrombin for dendritic cells (DC), which are efficient antigen-presenting cells and play important roles in initiating and regulating immune responses. The present study revealed that thrombin has the ability to stimulate blood DC. Plasmacytoid DC (PDC) and myeloid DC (MDC) isolated from PBMC expressed PAR-1 and released MCP-1, IL-10, and IL-12 after thrombin stimulation. Unlike blood DC, monocyte-derived DC (MoDC), differentiated in vitro did not express PAR-1 and were unresponsive to thrombin. Effects of thrombin on blood DC were significantly diminished by the addition of anti-PAR-1 Ab or hirudin, serine protease inhibitor. Moreover, thrombin induced HLA-DR and CD86 expression on DC and the thrombin-treated DC induced allogenic T cell proliferation. These findings indicate that thrombin plays a role in the regulation of blood DC functions.