Apoptosis-related mitochondrial dysfunction defines human monocyte-derived dendritic cells with impaired immuno-stimulatory capacities.

The death of dendritic cells (DCs) can potentially influence immune responses by affecting the duration of DC stimulation of lymphocytes. Here, we report that cultured mature monocyte-derived DCs manifest early mitochondrial damage (i.e. within 24 hrs), characterized by mitochondrial membrane potential (psi Delta m) disruption and mitochondrial release of pro-apoptotic factors, followed by reactive oxygen species (ROS) production and activation of caspases. Afterwards, DCs with mitochondrial alterations are condemned to undergo apoptosis and necrosis. Macroarray analysis results (validated by real time quantitative-PCR (QRT-PCR) and immunoblotting), showed up-regulation of the pro-apoptotic member of the Bcl-2 family, Bim, while expression of several anti-apoptotic molecules was down-regulated. Importantly, pre-apoptotic DCs (characterized by a low Delta psi m) showed a modified phenotype, with down-regulation of HLA-DR and of the co-stimulatory molecules CD80 and CD86. Moreover, sorted viable low psi Delta m DCs were unable to activate allogeneic T cells, indicating that pre-apoptotic DCs have already lost some of their immuno-stimulatory capabilities long before any detectable signs of death occur. Perturbations to mitochondrial respiration with rotenone identified the same modifications to DC immune functions. These data indicate a strong requirement for mitochondrial integrity for the immuno-stimulatory capacities of DC. Determining Delta psi m could be a useful parameter to select 'fully' functional DCs for anti-tumour vaccines.

Influence of heat stress on human monocyte-derived dendritic cell functions with immunotherapeutic potential for antitumor vaccines.

Mild heat stress can modulate the activities of immune cells, including dendritic cells (DC) and theoretically, would constitute an innovative approach capable of enhancing the antitumor functions of DC. Therefore, we tested the effects of mild heat stress on the physiology and viability of human monocyte-derived DC, the major type of DC used in tumor immunotherapy trials. We first designed a heat-stress protocol consisting of repetitive, sublethal heat shocks throughout the generation of DC. Using this protocol, we observed that heat stress did not perturb the morphology and the phenotype of immature or mature DC or the capacities of immature DC to uptake antigens efficiently. It is noteworthy that in response to heat stress, mature DC produced higher levels of IL-12p70 and TNF-alpha, which are two cytokines involved in the stimulation of inflammatory reaction, whereas IL-10 production remained low. After heat-stress exposure, mature DC have the full ability to stimulate naive T cells with Th1 response polarization (high IFN-gamma and low IL-4 production) in an allogeneic MLR. It is interesting that heat stress enhanced the migratory capacities of DC in response to MIP-3beta/CCL19. Finally, heat stress partly protected DC from apoptosis induced by cytokine withdrawal. Overall, these findings validate the feasibility of improving immune response by heating human monocyte-derived DC and provide a strong rationale for using mild heat stress in combination with DC vaccination to increase antitumor response.

Impact of bronchial epithelium on dendritic cell migration and function: modulation by the bacterial motif KpOmpA.

Mucosal immune response depends on the surveillance network established by dendritic cells (DC), APC localized within the epithelium. Bronchial epithelial cells (BEC) play a pivotal role both in the host defense and in the pathogenesis of inflammatory airway disorders. We previously showed that the outer membrane protein A from Klebsiella pneumoniae (KpOmpA), a pathogen-associated molecular pattern (PAMP) derived from Klebsiella pneumoniae, activates BEC. In this study, we evaluated the consequences of this activation on DC traffic and functions. KpOmpA significantly increased the production of CCL2, CCL5, CXCL10, and CCL20 by BEC. Stimulation of BEC increased their chemotactic activity for monocyte-derived DC (MDDC) precursors, through CCL5 and CXCL10 secretion. BEC/MDDC precursor coculture leads to an ICAM-1-dependent accelerated differentiation and enhanced maturation of MDDC. BEC/DC interactions did not affect the capacity of DC to induce T cell proliferation. However, DC preincubated with BEC increased significantly the IL-10 production by autologous T cells. Basolateral and intraepithelial DC differently enhance IL-4 and/or IL-10 synthesis according to the condition of stimulation. In vivo, intranasal injections of KpOmpA into BALB/c mice induced the recruitment of CD11c(+) and I-A(d+) myeloid DC associated with bronchial epithelium activation as evidenced by CCL20 expression. These data show that KpOmpA-exposed BEC participate in the homeostasis of myeloid DC network, and regulate the induction of local immune response.

Involvement of CCL18 in allergic asthma.

Allergic asthma is associated with a pulmonary recruitment of Th type 2 cells, basophils, and eosinophils, mainly linked to chemokine production. CCL18 is a chemokine preferentially expressed in the lung, secreted by APCs, induced by Th2-type cytokines, and only present in humans. Therefore, CCL18 may be involved in allergic asthma. PBMC from asthmatics allergic to house dust mite cultured in the presence of Dermatophagoides pteronyssinus 1 (Der p 1) allergen secreted CCL18, 48 and 72 h after stimulation, whereas those from healthy donors did not. Part of CCL18 was directly derived from Der p 1-stimulated plasmacytoid dendritic cells, whereas the other part was linked to monocyte activation by IL-4 and IL-13 produced by Der p 1-stimulated T cells. In bronchoalveolar lavages from untreated asthmatic allergic patients, CCL18 was highly increased compared with controls. Functionally, CCL18 preferentially attracted in vitro-polarized Th2 cells and basophils, but not eosinophils and Th1 cells, and induced basophil histamine and intracellular calcium release. These data show a new function for CCL18, i.e., the recruitment of Th2 cells and basophils, and suggest that CCL18 may play a predominant role in allergic asthma.

Asthmatic bronchial epithelium activated by the proteolytic allergen Der p 1 increases selective dendritic cell recruitment.

BACKGROUND: Airway dendritic cells (DCs) are crucial for allergen-induced sensitization and inflammation in allergic asthma. After allergen challenge, an increased number of DCs is observed in airway epithelium from patients with allergy. OBJECTIVE: Because Der p 1, a cysteine protease allergen from Dermatophagoides pteronyssinus , induces chemokine production by bronchial epithelial cells (BECs), the purpose of this investigation was to evaluate the capacity of BEC exposed to Der p 1 to recruit DCs. METHODS: Chemotactic activity of BEAS-2B, a bronchial epithelial cell line, and BECs from nonatopic controls and patients with allergic asthma was evaluated on the migration of precursors, immature and mature monocyte-derived DCs (MDDCs), and CD34 + -derived Langerhans cells (LCs). RESULTS: C-C chemokine ligand (CCL)-2, CCL5, and C-X-C chemokine ligand 10 production by BEAS-2B and BEC was increased after Der p 1 exposure, whereas the proenzyme proDer p 1 devoid of enzymatic activity had no effect. Der p 1 stimulation of BEAS-2B and BEC from both groups increased significantly the recruitment of MDDC precursors, depending on CCL2, CCL5, and C-X-C chemokine ligand 10 production. In a reconstituted polarized epithelium, apical application of Der p 1 enhanced MDDC precursor migration into the epithelial layer. Moreover, Der p 1 stimulation of BEC from patients with asthma but not from controls increased the migration of LC precursors, mainly dependent on CCL20 secretion. No migration of immature and mature DCs was observed. CONCLUSION: These data confirmed that BECs participate in the homeostasis of the DC network present within the bronchial epithelium through the secretion of chemokines. In allergic asthma, upregulation of CCL20 production induced LC recruitment, the role of which remains to be determined.

Balance between NF-kappaB and JNK/AP-1 activity controls dendritic cell life and death.

The life cycle of dendritic cells (DCs) must be precisely regulated for proper functioning of adaptive immunity. However, signaling pathways actively mediating DC death remain enigmatic. Here we describe a novel mechanism of hierarchical transcriptional control of DC life and death. Ligation of tumor necrosis factor receptor superfamily (TNFR-SF) members on DCs and cognate contact with T cells resulted in quantitatively balanced nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK)-mediated activator protein-1 (AP-1) induction and strongly enhanced DC longevity. Specific blockade of NF-kappaB in DCs induced strongly augmented JNK/AP-1 activity because of elevated levels of reactive oxygen species. In this scenario, DC activation by TNFR-SF members or T cells induced DC apoptosis. Specific inhibition of JNK/AP-1 rescued DCs from this activation-induced cell death program and restored TNFR-SF member- and T-cell-mediated survival. We conclude that JNK/AP-1 activity is under negative feedback control of NF-kappaB and can execute apoptosis in DCs. Thus, feedback-controlled signaling amplitudes of 2 transcriptional pathways decide the fate of a DC.

Activation of the D prostanoid receptor 1 regulates immune and skin allergic responses.

The mobilization of Langerhans cells (LCs) from epithelia to the draining lymph nodes is an essential process to initiate primary immune responses. We have recently shown that in mice, PGD2 is a potent inhibitor of epidermal LC emigration. In this study, we demonstrate that activation of the D prostanoid receptor 1 (DP1) impedes the TNF-alpha-induced migration of human LCs from skin explants and strongly inhibits the chemotactic responses of human LC precursors and of maturing LCs to CC chemokine ligands 20 and 19, respectively. Using a murine model of atopic dermatitis, a chronic Th2-type allergic inflammatory disease, we demonstrate that the potent DP1 agonist BW245C dramatically decreases the Ag-specific T cell activation in the skin draining lymph nodes and markedly prevents the skin lesions following repeated epicutaneous sensitization with OVA. Interestingly, analysis of the local response indicates that BW245C treatment strongly reduces the recruitment of inflammatory cells into the dermis and disrupts the Th1/Th2 balance, probably through the increased production of the immunoregulatory cytokine IL-10, in the skin of sensitized mice. Taken together, our results suggest a new function for DP1 in the regulation of the immune and inflammatory responses. We propose that DP1 activation by specific agonists may represent a strategy to control cutaneous inflammatory Th2-associated diseases.

Der p 1-pulsed myeloid and plasmacytoid dendritic cells from house dust mite-sensitized allergic patients dysregulate the T cell response.

Although reports suggest that dendritic cells (DC) are involved in the allergic reaction characterized by a T helper cell type 2 (Th2) profile, the role of myeloid (M-DC) and plasmacytoid DC (P-DC), controlling the balance Th1/Th2, remains unknown. Here, we showed that in Dermatophagoides pteronyssinus (Dpt)-sensitized allergic patients and in healthy donors, M-DC displayed a higher capacity to capture Der p 1, a major allergen of Dpt, than did P-DC. However, Der p 1-pulsed M-DC from healthy subjects overexpressed CD80 and secreted interleukin (IL)-10, whereas M-DC from allergic patients did not. In contrast, with Der p 1-pulsed P-DC from both groups, no increase in human leukocyte antigen-DR, CD80, and CD86 and no IL-10 secretion were detected. When cocultured with allogeneic naive CD4(+) T cells from healthy donors, Der p 1-pulsed M-DC from allergic patients favored a Th1 profile [interferon (IFN)-gamma(high)/IL-4(low)] and Der p 1-pulsed P-DC, a Th2 profile (IFN-gamma(low)/IL-4(high)). In healthy donors, no T cell polarization (IFN-gamma(low)/IL-4(low)) was induced by Der p 1-pulsed M-DC or P-DC, but in response to Der p 1-pulsed M-DC, T cells secreted IL-10. The neutralization of IL-10 produced by Der p 1-pulsed M-DC from healthy donors led to an inhibition of IL-10 production by T cells and a polarization toward a type 1. Thus, IL-10 produced by M-DC might be an essential mediator controlling the balance between tolerance and allergic status. In addition, P-DC could contribute to the steady state in healthy donors or to the development of a Th2 response in allergic donors.

Involvement of the mannose receptor in the uptake of Der p 1, a major mite allergen, by human dendritic cells.

BACKGROUND: Immature dendritic cells (DCs) take up antigens in peripheral tissues and, after antigen processing, mature to efficiently stimulate T cells in secondary lymph nodes. In allergic airway diseases DCs have been shown to be involved in the induction and maintenance of a T(H)2-type profile. OBJECTIVE: The present study was undertaken to determine pathways of Der p 1 (a house dust mite allergen) uptake by human DCs and to compare Der p 1 uptake between DCs from patients with house dust mite allergy and DCs from healthy donors. METHODS: Monocyte-derived DCs (MD-DCs) were obtained from patients with house dust mite allergy (n = 13) and healthy donors (n = 11). Der p 1 was labeled with rhodamine. Der p 1 uptake by MD-DCs was analyzed by means of flow cytometry and confocal microscopy. RESULTS: Rhodamine- labeled Der p 1 was demonstrated to be taken up by MD-DCs in a dose-, time-, and temperature- dependent manner. The involvement of the mannose receptor (MR) in the Der p 1 uptake was demonstrated by using (1) inhibitors of the MR- mediated endocytosis (mannan and blocking anti-MR mAb), which inhibited the Der p 1 uptake from 40 % to 50 %, and (2) confocal microscopy showing the colocalization of rhodamine-labeled Der p 1 with FITC-dextran. Interestingly, compared with DCs from healthy donors, DCs from allergic patients expressed more MR and were more efficient in Der p 1 uptake. CONCLUSION: These results suggest that the MR could play a key role in the Der p 1 allergen uptake by DCs and in the pathogenesis of allergic diseases in dust mite -sensitive patients.