Early Induction of Human Regulatory Dermal Antigen Presenting Cells by Skin-Penetrating Schistosoma Mansoni Cercariae.

Following initial invasion of Schistosoma mansoni cercariae, schistosomula reside in the skin for several days during which they can interact with the dermal immune system. While murine experiments have indicated that exposure to radiation-attenuated (RA) cercariae can generate protective immunity which is initiated in the skin stage, contrasting non-attenuated cercariae, such data is missing for the human model. Since murine skin does not form a reliable marker for immune responses in human skin, we used human skin explants to study the interaction with non-attenuated and RA cercariae with dermal innate antigen presenting cells (APCs) and the subsequent immunological responses. We exposed human skin explants to cercariae and visualized their invasion in real time (initial 30 min) using novel imaging technologies. Subsequently, we studied dermal immune responses and found an enhanced production of regulatory cytokine interleukin (IL)-10, pro-inflammatory cytokine IL-6 and macrophage inflammatory protein (MIP)-1α within 3 days of exposure. Analysis of dermal dendritic cells (DDCs) for their phenotype revealed an increased expression of immune modulators programmed death ligand (PD-L) 1 and 2, and increased IL-10 production. Ex vivo primed DDCs suppress Th1 polarization of naïve T-cells and increase T-cell IL-10 production, indicating their regulatory potential. These immune responses were absent or decreased after exposure to RA parasites. Using transwells, we show that direct contact between APCs and cercariae is required to induce their regulatory phenotype. To the best of our knowledge this is the first study that attempts to provide insight in the human dermal S. mansoni cercariae invasion and subsequent immune responses comparing non-attenuated with RA parasites. We reveal that cercariae induce a predominantly regulatory immune response whereas RA cercariae fail to achieve this. This initial understanding of the dermal immune suppressive capacity of S. mansoni cercariae in humans provides a first step toward the development of an effective schistosome vaccine.

Myc Cooperates with Ras by Programming Inflammation and Immune Suppression.

The two oncogenes KRas and Myc cooperate to drive tumorigenesis, but the mechanism underlying this remains unclear. In a mouse lung model of KRasG12D-driven adenomas, we find that co-activation of Myc drives the immediate transition to highly proliferative and invasive adenocarcinomas marked by highly inflammatory, angiogenic, and immune-suppressed stroma. We identify epithelial-derived signaling molecules CCL9 and IL-23 as the principal instructing signals for stromal reprogramming. CCL9 mediates recruitment of macrophages, angiogenesis, and PD-L1-dependent expulsion of T and B cells. IL-23 orchestrates exclusion of adaptive T and B cells and innate immune NK cells. Co-blockade of both CCL9 and IL-23 abrogates Myc-induced tumor progression. Subsequent deactivation of Myc in established adenocarcinomas triggers immediate reversal of all stromal changes and tumor regression, which are independent of CD4+CD8+ T cells but substantially dependent on returning NK cells. We show that Myc extensively programs an immune suppressive stroma that is obligatory for tumor progression.

Fenretinide inhibits macrophage inflammatory mediators and controls hypertension in spontaneously hypertensive rats via the peroxisome proliferator-activated receptor gamma pathway.

Fenretinide is a novel anticancer agent reported to exhibit anti-invasive and antimetastatic activities. It has also been shown to improve obesity and diabetes, although the effects of fenretinide on hypertension are still unknown, and the detailed mechanisms remain unclear. In this study, we have shown that treatment with lipopolysaccharide (LPS) decreased the expression of peroxisome proliferator-activated receptor γ (PPARγ) in RAW264.7 macrophages, and pretreatment with fenretinide reversed the effect of LPS on PPARγ expression. In addition, LPS-induced pro-inflammatory cytokine production, including tumor necrosis factor-α, interleukin 6, and monocyte chemoattractant protein 1 were dose-dependently reversed by fenretinide, and the effects of fenretinide on LPS-induced pro-inflammatory cytokine production were blocked by treatment with PPARγ antagonist. Moreover, fenretinide decreased LPS-induced inducible nitric oxide synthase expression and nitrogen oxide production. These effects were blocked by the pretreatment with PPARγ antagonist in a dose-dependent manner, indicating fenretinide activated PPARγ to exert anti-inflammation activity. In view of the role of inflammation in hypertension and the anti-inflammatory action of fenretinide, we found that administration of fenretinide in spontaneously hypertensive rats significantly decreased blood pressure. Taken together, these results indicate that fenretinide might be a potent antihypertensive agent that works by suppressing inflammation via activating PPARγ.

Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches.

The microfold (M) cell residing in the follicle-associated epithelium is a specialized epithelial cell that initiates mucosal immune responses by sampling luminal antigens. The differentiation process of M cells remains unclear due to limitations of analytical methods. Here we found that M cells were classified into two functionally different subtypes based on the expression of Glycoprotein 2 (GP2) by newly developed image cytometric analysis. GP2-high M cells actively took up luminal microbeads, whereas GP2-negative or low cells scarcely ingested them, even though both subsets equally expressed the other M-cell signature genes, suggesting that GP2-high M cells represent functionally mature M cells. Further, the GP2-high mature M cells were abundant in Peyer's patch but sparse in the cecal patch: this was most likely due to a decrease in the nuclear translocation of RelB, a downstream transcription factor for the receptor activator of nuclear factor-κB signaling. Given that murine cecum contains a protrusion of beneficial commensals, the restriction of M-cell activity might contribute to preventing the onset of any excessive immune response to the commensals through decelerating the M-cell-dependent uptake of microorganisms.

Response profiles of cytokines and chemokines against avian H9N2 influenza virus within the mouse lung.

The circulation of H9N2 viruses throughout the world, along with their expanded host range, poses a potential health risk to the public, but the host responses to H9N2 virus in mammals were little known. To obtain insight into the host immune responses to the avian H9N2 virus, the expressions of both cytokines and chemokines in the lungs of infected mice were examined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. We found that interferon gamma (IFN-γ) was the dominant antiviral component, and IFN-γ-induced protein 10 kDa, interleukin 6, chemokine (C-C motif) ligand 5 and macrophage inflammatory protein-1 alpha all played a role in pro-inflammatory responses to H9N2 viruses. In conclusion, this research can make us further understand the infection characteristics of H9N2 virus in mammalian host by providing the data on mice lung immune responses to the avian H9N2 virus.

Efeito imunomodulador de vesículas de membrana obtidas de macrófagosinfectados por leishmania sobre macrófagos não infectados / Immunomodulatory Effect of membrane vesicles obtained from macrophages Leishmania infection of uninfected macrophages

Vesículas de membrana (VMs) derivadas de macrófagos infectados com microorganismos intracelulares têm capacidade inflamatória. Estas vesículas podem conter antígenos do patógeno, carrear moléculas de MHC II e componentes celulares que podem atuar como PAMPs ou DAMPs induzindo resposta imune. Na infecção por Leishmania, a indução de uma resposta do tipo Th1 é crucial para promoção de proteção contra o parasito. O objetivo do trabalho foi avaliar a capacidade imunomoduladora de VMs derivadas de macrófagos infectados com L. amazonensis sobre a produção de citocinas por outros macrófagos. VMs foram visualizadas por microscopia eletrônica tanto em preparações celulares como no precipitado obtido por sucessivas centrifugações de sobrenadantes de cultivos celulares. Foi observada por citometria de fluxo a presença de marcadores celulares específicos (F4/80 e CD11b) nas VMs, bem como MHC II. O tratamento de macrófagos não infectados com VMs derivadas de macrófagos infectados com L. amazonensis ocasionou aumento consistente da produção de IL-12p70 e IL-1β. Estas vesículas poderiam, portanto, favorecer a modulação da resposta imune em favor do combate ao parasito.

Membrane vesicles (MV) derived macrophages infected with intracellular microbes are proinflammatory. These vesicles contain antigens of the pathogen, carry MHC II molecules and cellular components that can act as PAMPs or DAMPs inducing immune responses. In Leishmania infection the induction of a Th1 response is crucial for the protection against the parasite. The aim of the study was to evaluate whether vesicles derived from macrophages infected with L. amazonensis had the capacity to modulate the response of other macrophages. MV were visualized by electron microscopy in cellular preparations as well in the precipitate obtained by centrifugation of cell supernatants. Flow cytometry revealed the presence of specific cellular markers (F4/80 and CD11b) in the MV, as well as MHC II. Treatment of noninfected macrophages with MV derived from L. amazonensis-infected macrophages consistently caused increased production of IL-12p70 and IL-1β. These vesicles can favor the modulation of the immune response in favor of combating the parasite.

Stress-evoked sterile inflammation, danger associated molecular patterns (DAMPs), microbial associated molecular patterns (MAMPs) and the inflammasome.

Since the inception of the field of psychoneuroimmunolology research, there has been an appreciation that the physiological response to stressors includes modulation of immune function. Investigators initially focused on the effect of stress on cellular migration and immunosuppression and the resultant decreases in tumor surveillance, anti-viral T cell immunity and antigen-specific antibody responses. More recently, it has become clear that exposure to stressors also potentiate innate immune processes. Stressor exposure, for example, can change the activation status of myeloid lineage cells such as monocytes, macrophages, neutrophils, and microglia, leading to a primed state. In addition, stressor exposure increases the synthesis and release of a vast cadre' of inflammatory proteins both in the blood and within tissues (i.e., spleen, liver, adipose, vasculature and brain). The mechanisms for stress-evoked innate immune 'arousal' remain unknown. The goals of this presidential address are the following: (1) offer a personalized, brief overview of stress and immunity with a focus on 'aroused' innate immunity; (2) describe sterile inflammatory processes and the role of the inflammasome; and (3) suggest that these same processes likely contribute to primed myeloid cells and inflammatory protein responses (systemic and tissue) produced by stress in the absence of pathogens.

CCR1-mediated STAT3 tyrosine phosphorylation and CXCL8 expression in THP-1 macrophage-like cells involve pertussis toxin-insensitive Gα(14/16) signaling and IL-6 release.

Agonists of CCR1 contribute to hypersensitivity reactions and atherosclerotic lesions, possibly via the regulation of the transcription factor STAT3. CCR1 was demonstrated to use pertussis toxin-insensitive Gα(14/16) to stimulate phospholipase Cß and NF-κB, whereas both Gα(14) and Gα(16) are also capable of activating STAT3. The coexpression of CCR1 and Gα(14/16) in human THP-1 macrophage-like cells suggests that CCR1 may use Gα(14/16) to induce STAT3 activation. In this study, we demonstrated that a CCR1 agonist, leukotactin-1 (CCL15), could indeed stimulate STAT3 Tyr(705) and Ser(727) phosphorylation via pertussis toxin-insensitive G proteins in PMA-differentiated THP-1 cells, human erythroleukemia cells, and HEK293 cells overexpressing CCR1 and Gα(14/16). The STAT3 Tyr(705) and Ser(727) phosphorylations were independent of each other and temporally distinct. Subcellular fractionation and confocal microscopy illustrated that Tyr(705)-phosphorylated STAT3 translocated to the nucleus, whereas Ser(727)-phosphorylated STAT3 was retained in the cytosol after CCR1/Gα(14) activation. CCL15 was capable of inducing IL-6 and IL-8 (CXCL8) production in both THP-1 macrophage-like cells and HEK293 cells overexpressing CCR1 and Gα(14/16). Neutralizing Ab to IL-6 inhibited CCL15-mediated STAT3 Tyr(705) phosphorylation, whereas inhibition of STAT3 activity abolished CCL15-activated CXCL8 release. The ability of CCR1 to signal through Gα(14/16) provides a linkage for CCL15 to regulate IL-6/STAT3-signaling cascades, leading to expression of CXCL8, a cytokine that is involved in inflammation and the rupture of atherosclerotic plaque.

Expression and regulation of CCL15 by human airway smooth muscle cells.

BACKGROUND: Structural cells are an important reservoir of chemokines that coordinate the influx of various immune cells to the lungs of asthmatics. Airway smooth muscle cells (ASMC) are an important source of these chemokines. CCL15 is a recently described chemo-attractant for neutrophils, eosinophils, monocytes and lymphocytes. OBJECTIVE: To determine the production and the regulation of CCL15 by ASMC and to investigate its production in asthmatic airways. METHODS: Human ASMC were obtained from main bronchial airway segments of patients with mild, moderate and severe asthma. To induce chemokine production, cells were incubated with IL-4, IL-13, TNF-α or IFN-γ in presence or absence of dexamethasone, mithramycin A (SP-1 inhibitor) or the IKK-2 inhibitor, AS602868. CCL15 mRNA expression was evaluated by real-time PCR. Immunoreactive CCL15 was detected by immuno-fluorescence and CCL15 protein concentration in the supernatant was measured using ELISA. RESULTS: CCL15 is constitutively expressed in human ASMC and is strongly up-regulated by TNF-α. This up-regulation is inhibited by dexamethasone, mithramycin A and AS602868. TNF-α-induced CCL15 levels can be synergistically enhanced by the presence of IFN-γ, at both the transcriptional and translation level. This synergism is NF-κB-dependent. Asthmatic biopsies demonstrated higher expression of CCL15 compared with non-asthmatic controls. CONCLUSION AND CLINICAL RELEVANCE: Our results show that ASMC are a potent source of CCL15 in the airways and may directly participate in the recruitment of inflammatory cells to asthmatic airways. Targeting the production of CCL15 by ASMC might reduce the inflammatory response within the airways of asthmatic patients.

Antitumor activity mediated by CpG: the route of administration is critical.

Unmethylated CpG oligodeoxynucleotides (CpG) are synthetic toll-like receptor 9 agonists that activate innate immune cells and which have been tested as an immune therapy in a number of cancer clinical trials. Although some antitumor immune responses have been reported, so far the majority of studies have failed to show significant clinical responses to CpG. Here we showed that the route of administration is critical to the antitumor activity of CpG. Although intravenous (i.v.) injection of CpG was capable of inducing the activation and expansion of tumor antigen-specific T cells, most of these activated T cells failed to migrate to tumor sites. By contrast, intratumoral (i.t.) injection of CpG led to extensive tumor infiltration of antigen-specific T cells and subsequent tumor suppression. We further showed that very high levels of inflammatory chemokines [regulated upon activation, normal T-cell expressed, and secreted (RANTES), interferon-inducible protein-10 (IP-10), monocyte chemoattractant protein-1, monocyte chemotactic protein (MCP5), macrophage inflammatory proteins (MIP1α, and MIP1ß)] were induced in the tumor microenvironment after i.t. CpG injection, compared with administration by the i.v. route. It is interesting to note that, in vivo depletion of plasmacytoid dendritic cells greatly reduced the levels of chemokines induced; also, T-cell accumulation and antitumor effect were impaired. We also showed that i.t. but not i.v. CpG injection induced a broad antigen-specific T-cell response against tumor-derived antigens. Collectively, our data provides evidence that the route of CpG administration is a critical factor in mediating antitumor activity. By inducing localized inflammatory signals at tumor sites, i.t. CpG effectively promotes the migration, activation and function of immune cells, ultimately leading to improved tumor control.

Importance of macrophage inflammatory protein-1α and splenic macrophages in neurodegeneration induced by PVC-211 murine leukemia virus.

We recently reported that infection of rats with the neurodegenerative disease-causing retrovirus PVC-211 MuLV results in elevated levels of the chemokine MIP-1α followed by the accumulation of activated microglia in the brain. To investigate the importance of MIP-1α in recruitment of microglia to the brain, we treated rats with MIP-1α antibodies before and after PVC-211 MuLV infection. This caused a delay in the development of paralysis which was associated with a decrease in activated microglia without affecting virus expression. To determine the source of activated microglia, rats were splenectomized 4 days after virus infection. Splenectomized rats showed a delay in disease development that was associated with decreased numbers of activated microglia without affecting virus expression. Together, these results suggest that MIP-1α is directly involved in the neurodegeneration induced in rats by PVC-211 MuLV by recruiting macrophages/microglia from the periphery into regions of the brain that eventually become diseased.

Levels of transforming growth factor-beta are low in systemic lupus erythematosus patients with active disease.

OBJECTIVE: Cytokines are central regulators of the immune response but the workings of this complex network in systemic lupus erythematosus (SLE) are not fully understood. We investigated a range of inflammatory and immune-modulating cytokines to determine their value as biomarkers for disease subsets in SLE. METHODS: This was a cross-sectional study in 102 patients with SLE (87% women, disease duration 10.6 yrs). Circulating concentrations of interleukin 1ß (IL-1ß), IL-4, IL-6, IL-10, IL-12, IL-17, monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 (MIP-1α), MIP-1ß, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and total transforming growth factor-ß1 (TGF-ß1) were related to disease activity (SLE Disease Activity Index; SLEDAI), lymphocyte subsets, autoantibody levels, accrued damage (Systemic Lupus International Collaborating Clinics/ACR Damage Index; SDI), and concomitant treatment. RESULTS: Patients with SLE had lower levels of TGF-ß1 (p = 0.01) and IL-1ß (p = 0.0004) compared to controls. TGF-ß1 levels were lower in patients with SLEDAI scores 1-10 and SDI > 3; and were correlated with CD4+, CD8+, and natural killer cell counts; and were independent of steroid or cytotoxic drug use. Treatment with cardiovascular drugs was associated with lower IL-12 levels. No consistent disease associations existed for the other cytokines investigated. CONCLUSION: Lower TGF-ß1 was the most consistent cytokine abnormality in patients with SLE. The associations with disease activity, lymphocyte subsets, and damage suggest that TGF-ß1 may be a therapeutic target of interest in SLE.

Retinoid X receptor alpha controls innate inflammatory responses through the up-regulation of chemokine expression.

The retinoid X receptor alpha (RXRalpha) plays a central role in the regulation of many intracellular receptor signaling pathways and can mediate ligand-dependent transcription by forming homodimers or heterodimers with other nuclear receptors. Although several members of the nuclear hormone receptor superfamily have emerged as important regulators of macrophage gene expression, the existence in vivo of an RXR signaling pathway in macrophages has not been established. Here, we provide evidence that RXRalpha regulates the transcription of the chemokines Ccl6 and Ccl9 in macrophages independently of heterodimeric partners. Mice lacking RXRalpha in myeloid cells exhibit reduced levels of CCL6 and CCL9, impaired recruitment of leukocytes to sites of inflammation, and lower susceptibility to sepsis. These studies demonstrate that macrophage RXRalpha plays key roles in the regulation of innate immunity and represents a potential target for immunotherapy of sepsis.

Mycobacterium tuberculosis-induced expression of Leukotactin-1 is mediated by the PI3-K/PDK1/Akt signaling pathway.

Chemokines function in the migration of circulating leukocytes to regions of inflammation, and have been implicated in chronic inflammatory conditions including mycobacterial infection. We investigated whether Leukotactin-1 (Lkn-1), a novel member of the CC-chemokines, is involved in the immune response of macrophages against Mycobacterium tuberculosis (MTB). In PMA-differentiated THP-1 cells, MTB infection increased mRNA expression of Lkn-1 in a dose-dependent manner. Lkn-1 induction peaked 12 h after infection, then declined gradually and returned to its basal level at 72 h. Secretion of Lkn-1 was elevated by MTB infection. The increase in expression and secretion of Lkn-1 caused by MTB was reduced in cells treated with inhibitors of phosphatidylinositol 3-kinase (PI3-K), 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt. MTB-induced Akt phosphorylation was blocked by treatment with a PI3-K inhibitor or a PDK1 inhibitor, implying that PI3-K, PDK1, and Akt are associated with the signaling pathway that up-regulates Lkn-1 in response to MTB. These results suggest that Lkn-1 is novel member of the group of chemokines that is released by macrophages infected with MTB.

Mouse macrophages primed with alendronate down-regulate monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) production in response to Toll-like receptor (TLR) 2 and TLR4 agonist via Smad3 activation.

Alendronate is one of the nitrogen-containing bisphosphonates (NBPs) used as anti-bone resorptive drugs. However, NBPs have inflammatory side effects including osteomyelitis and osteonecrosis of the jaw. In the present study, we examined the effects of alendronate on chemokine production by the macrophage-like cell line, J774.1, when incubated with Pam(3)CSK(4) (a Toll-like receptor (TLR) 2 agonist) and Lipid A (a TLR4 agonist). Pretreatment of J774.1 cells with alendronate decreased the production of TLR ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) but did not influence nuclear factor-kappaB (NF-kappaB) activation. While this agent induced caspase-8 activation, a caspase-8 inhibitor did not affect the decrease in MCP-1 production by alendronate and TLR ligands. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-kappaB and caspase-8 activation. Although transforming growth factor-beta1 (TGF-beta1) is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-beta1 production by J774.1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3. These results suggest that by down-regulating MCP-1 and MIP-1alpha production via Smad3, long-term use of alendronate might inhibit normal activation and migration of osteoclasts and cause osteonecrosis.

ICSBP-mediated immune protection against BCR-ABL-induced leukemia requires the CCL6 and CCL9 chemokines.

Interferon (IFN) is effective at inducing complete remissions in patients with chronic myelogenous leukemia (CML), and evidence supports an immune mechanism. Here we show that the type I IFNs (alpha and beta) regulate expression of the IFN consensus sequence-binding protein (ICSBP) in BCR-ABL-transformed cells and as shown previously for ICSBP, induce a vaccine-like immunoprotective effect in a murine model of BCR-ABL-induced leukemia. We identify the chemokines CCL6 and CCL9 as genes prominently induced by the type I IFNs and ICSBP, and demonstrate that these immunomodulators are required for the immunoprotective effect of ICSBP expression. Insights into the role of these chemokines in the antileukemic response of IFNs suggest new strategies for immunotherapy of CML.

RAGE signaling sustains inflammation and promotes tumor development.

A broad range of experimental and clinical evidence has highlighted the central role of chronic inflammation in promoting tumor development. However, the molecular mechanisms converting a transient inflammatory tissue reaction into a tumor-promoting microenvironment remain largely elusive. We show that mice deficient for the receptor for advanced glycation end-products (RAGE) are resistant to DMBA/TPA-induced skin carcinogenesis and exhibit a severe defect in sustaining inflammation during the promotion phase. Accordingly, RAGE is required for TPA-induced up-regulation of proinflammatory mediators, maintenance of immune cell infiltration, and epidermal hyperplasia. RAGE-dependent up-regulation of its potential ligands S100a8 and S100a9 supports the existence of an S100/RAGE-driven feed-forward loop in chronic inflammation and tumor promotion. Finally, bone marrow chimera experiments revealed that RAGE expression on immune cells, but not keratinocytes or endothelial cells, is essential for TPA-induced dermal infiltration and epidermal hyperplasia. We show that RAGE signaling drives the strength and maintenance of an inflammatory reaction during tumor promotion and provide direct genetic evidence for a novel role for RAGE in linking chronic inflammation and cancer.

Chemokine redundancy in BOS pathogenesis. A possible role also for the CC chemokines: MIP3-beta, MIP3-alpha, MDC and their specific receptors.

Bronchiolitis obliterans syndrome (BOS) is one of the most important factors limiting the long-term survival of lung transplant recipients (LTR), however its pathogenesis still remains unclear. We hypothesized that an increased production of certain specific proinflammatory mediators in the first post-transplant year would predispose to BOS. We retrospectively evaluated temporal kinetics of some CC chemokines that have not yet been evaluated, including CCL3/MIP1-alpha, CCL4/MIP1-beta, CCL17/TARC, CCL19/MIP3-beta, CCL20/MIP3-alpha, CCL22/MDC and CCL26/eotaxin, in broncho-alveolar lavage fluid (BAL-f) in the first post-transplant year in a cohort of 8 LTR before the development of BOS (pre-BOS LTR) and 8 LTR with long-term stable clinical conditions (stable LTR). Chemokine levels were assayed by means of a multiplex sandwich ELISA. Furthermore, for those ligands which resulted significantly predictive of BOS onset, we analyzed the expression of specific receptors (CCR) on BAL cells. The proportion of CCR-expressing BAL cells was assessed by flow cytometry. We demonstrated that MIP3-beta/CCL19, MIP3-alpha/CCL20, MDC/CCL22 levels at 6 months post-transplant significantly predicted BOS onset. In addition, the temporal behavior of these factors resulted significantly different in pre-BOS patients as compared to stable LTR. Finally the expression of CCR was documented on BAL lymphocytes and macrophages, and, in some cases, their expression was found to vary between the two groups. Within the complexity of the chemokine network, these three CCL factors could play an additive role in the pathogenesis of the inflammatory process leading to bronchiolar fibro-obliteration.

Allergen-IgE complexes trigger CD23-dependent CCL20 release from human intestinal epithelial cells.

BACKGROUND & AIMS: In food allergic individuals, exposure to food allergens by the oral route can trigger immediate (within minutes) local hypersensitivity reactions in the intestine followed by a late-phase inflammatory response. Previous work has shown that CD23 is constitutively expressed by human intestinal epithelial cells and mediates the uptake of allergen-IgE complexes. We hypothesized that allergen-IgE complexes could also signal via CD23 to trigger an inflammatory cascade in the local environment. METHODS: Caco-2 monolayers were stimulated with human IgE-antigen (Ag) complexes. IL-8 and CCL20 mRNA and protein were determined by RT-PCR and ELISA, respectively. Signaling pathways were assessed by immunoblotting. Endogenous CD23 expression was knocked down by stable transfection with CD23 shRNA retroviral plasmid. Migration assays were performed using human monocyte-derived dendritic cells. RESULTS: Stimulation of Caco-2 cells with IgE-Ag complexes triggered upregulation of IL-8 and CCL20 at the mRNA and protein level. Allergen complexes induced phosphorylation of ERK and JNK, but not p38 MAP kinase or NK-kappaB, and resulted in AP-1 activation. Cross-linking of CD23 replicated the findings with IgE-Ag complexes, and silencing of CD23 expression abrogated the response to allergen-IgE complexes. Supernatant from IgE-Ag-stimulated epithelial cells induced migration of dendritic cells in a CCL20-dependent manner. Finally, immunostaining of duodenal biopsies demonstrated that CCL20 was constitutively expressed by epithelial cells in vivo. CONCLUSIONS: Signaling via epithelial CD23 may participate in the late-phase inflammatory response by the release of chemokines capable of recruiting antigen presenting cells and effector cells of allergic inflammation.

Memory CD8+ T cells mediate antibacterial immunity via CCL3 activation of TNF/ROI+ phagocytes.

Cytolysis, interferon gamma and tumor necrosis factor (TNF) alpha secretion are major effector mechanisms of memory CD8+ T cells that are believed to be required for immunological protection in vivo. By using mutants of the intracellular bacterium Listeria monocytogenes, we found that none of these effector activities is sufficient to protect against secondary infection with wild-type (WT) bacteria. We demonstrated that CCL3 derived from reactivated memory CD8+ T cells is required for efficient killing of WT bacteria. CCL3 induces a rapid TNF-alpha secretion by innate inflammatory mononuclear phagocytic cells (MPCs), which further promotes the production of radical oxygen intermediates (ROIs) by both MPCs and neutrophils. ROI generation is the final bactericidal mechanism involved in L. monocytogenes clearance. These results therefore uncover two levels of regulation of the antibacterial secondary protective response: (a) an antigen-dependent phase in which memory CD8+ T cells are reactivated and control the activation of the innate immune system, and (b) an antigen-independent phase in which the MPCs coordinate innate immunity and promote the bactericidal effector activities. In this context, CCL3-secreting memory CD8+ T cells are able to mediate "bystander" killing of an unrelated pathogen upon antigen-specific reactivation, a mechanism that may be important for the design of therapeutic vaccines.