3-Hydroxy-4,7-megastigmadien-9-one, isolated from Ulva pertusa, attenuates TLR9-mediated inflammatory response by down-regulating mitogen-activated protein kinase and NF-κB pathways.

CONTEXT: Seaweeds are rich in bioactive compounds in the form of vitamins, phycobilins, polyphenols, carotenoids, phycocyanins and polysaccharides; many of these are known to have advantageous applications in human health. 3-Hydroxy-4,7-megastigmadien-9-one (comp) was isolated from Ulva pertusa (U. pertusa) Kjellman (Ulvaceae), which is a familiar edible green seaweed. OBJECTIVE: This study evaluates the anti-inflammatory activity of comp in CpG DNA-stimulated bone marrow-derived dendritic cells (BMDCs). MATERIALS AND METHODS: For evaluating the effect of comp on cytokines production, BMDCs were treated with doses of comp (0, 0.5, 1, 2, 5, 10, 25 and 50 µM) for 1 h before stimulation with CpG DNA (1 µM). Cytokine production was measured by ELISA. Western blotting was conducted for evaluating effect of comp (50 µM) on MAPKs and NF-κB pathways. Luciferase reporter gene assay was conducted for effect of comp (0, 5, 10 and 25 µM) on transcriptional activity of AP-1 and NF-κB. RESULTS: Comp exhibited strong inhibition of interleukin (IL)-12 p40, IL-6 and TNF-α cytokine production with IC50 values of 6.02 ± 0.35, 27.14 ± 0.73, and 7.56 ± 0.21 µM, respectively. It blocked MAPKs and NF-κB pathways by inhibiting the phosphorylation of ERK1/2, JNK1/2, p38 and IκBα. In addition, it strongly inhibited the transcriptional activity of AP-1 and NF-κB with IC50 values of 8.74 ± 0.31 and 12.08 ± 0.24 µM, respectively. DISCUSSION AND CONCLUSION: Taken together, these data suggest that comp has a significant anti-inflammatory property and warrants further studies concerning the potential of comp for medicinal use.

Small-molecule screening identifies inhibition of salt-inducible kinases as a therapeutic strategy to enhance immunoregulatory functions of dendritic cells.

Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs-MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow-derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1ß, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs-MΦs and anti-inflammatory CD11c(+) CX3CR1(hi) cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors.

Rhamnogalacturonan II is a Toll-like receptor 4 agonist that inhibits tumor growth by activating dendritic cell-mediated CD8+ T cells.

We evaluated the effectiveness of rhamnogalacturonan II (RG-II)-stimulated bone marrow-derived dendritic cells (BMDCs) vaccination on the induction of antitumor immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). BMDCs treated with RG-II had an activated phenotype. RG-II induced interleukin (IL)-12, IL-1ß, tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) production during dendritic cell (DC) maturation. BMDCs stimulated with RG-II facilitate the proliferation of CD8+ T cells. Using BMDCs from the mice deficient in Toll-like receptors (TLRs), we revealed that RG-II activity is dependent on TLR4. RG-II showed a preventive effect of immunization with OVA-pulsed BMDCs against EG7 lymphoma. These results suggested that RG-II expedites the DC-based immune response through the TLR4 signaling pathway.

Immunological effects of Oenothein B, an ellagitannin dimer, on dendritic cells.

Oenothein B is a unique macrocyclic ellagitannin dimer that has been found in various medicinal plants belonging to Onagraceae, Lythraceae, and Myrtaceae, with diverse biological activities. The immunological effects of tannins in terms of cytokine-release from macrophages and monocytes have been discussed, while the effects on other immunocompetent cells have been the subject of minimal investigation. We evaluated the immunomodulatory effects induced by tannin treatment in human dendritic cells (DCs), which play a critical role in the initial immune response, by measuring the changes in cytokine production, cell differentiation, and cell viability. Oenothein B showed significant down-regulation of the expression of cell surface molecules, CD1a and CD83, suggesting the inhibition of DC differentiation and/or maturation. The suppressive effect on DCs was associated with the induction of apoptosis without the activation of caspase-3/7, 8, and 9, and this was supported by the morphological features indicating significant nuclear condensation. Oenothein B also markedly suppressed the production of inflammatory cytokines, such as IL-1ß and IL-6, in a dose-dependent manner. These data may, in part, be able to explain the traditional use of tannin-containing medicinal plants for the treatment of a variety of inflammatory diseases, including inflammatory bowel disease, celiac disease, and rheumatoid arthritis.

Inhibition of fms-like tyrosine kinase 3 alleviates experimental arthritis by reducing formation of dendritic cells and antigen presentation.

TKs are intracellular signaling molecules essential for cell homeostasis. Inhibition of TKs is used in treatment of malignancies and diabetes mellitus. The present study evaluated the role of Flt3 in antigen-induced arthritis. Mice were immunized with mBSA, and arthritis was induced by an i.a. injection of mBSA. Treatment with the Flt3 inhibitor sunitinib was started together with mBSA immunization or together with the induction of arthritis. The mBSA-injected joints were evaluated morphologically for signs of synovitis and bone/cartilage destruction. Markers of bone metabolism and antibody responses were measured by ELISA. Maturation of DCs in the bone marrow and spleen was evaluated by flow cytometry. Sunitinib treatment reduced the intensity of synovitis and the incidence of bone destruction. The reduction in bone destruction was seen when the treatment was started at the time of immunization or at the time of arthritis induction. The antiarthritic effect was achieved by inhibition of DCs, reduction of antibody production, and bone metabolism. Inhibition of Flt3 is a potent antiarthritic mechanism reducing antigen presentation, synovial inflammation, and bone resorption. Down-regulation of TKs may be a useful tool in the treatment of human RA.

Honokiol inhibits LPS-induced maturation and inflammatory response of human monocyte-derived dendritic cells.

Honokiol (HNK) is a phenolic compound isolated from the bark of houpu (Magnolia officinalis), a plant widely used in traditional Chinese and Japanese medicine. While substantial evidence indicates that HNK possesses anti-inflammatory activity, its effect on dendritic cells (DCs) during the inflammatory reaction remains unclear. The present study investigates how HNK affects lipopolysaccharide (LPS)-stimulated human monocyte-derived DCs. Our experimental results show that HNK inhibits the inflammatory response of LPS-induced DCs by (1) suppressing the expression of CD11c, CD40, CD80, CD83, CD86, and MHC-II on LPS-activated DCs, (2) reducing the production of TNF-α, IL-1ß, IL-6, and IL-12p70 but increasing the production of IL-10 and TGF-ß1 by LPS-activated DCs, (3) inhibiting the LPS-induced DC-elicited allogeneic T-cell proliferation, and (4) shifting the LPS-induced DC-driven Th1 response toward a Th2 response. Further, our results show that HNK inhibits the phosphorylation levels of ERK1/2, p38, JNK1/2, IKKα, and IκBα in LPS-activated DCs. Collectively, the findings show that the anti-inflammatory actions of HNK on LPS-induced DCs are associated with the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways.

Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens.

Under physiological conditions the gut-associated lymphoid tissues not only prevent the induction of a local inflammatory immune response, but also induce systemic tolerance to fed antigens. A notable exception is coeliac disease, where genetically susceptible individuals expressing human leukocyte antigen (HLA) HLA-DQ2 or HLA-DQ8 molecules develop inflammatory T-cell and antibody responses against dietary gluten, a protein present in wheat. The mechanisms underlying this dysregulated mucosal immune response to a soluble antigen have not been identified. Retinoic acid, a metabolite of vitamin A, has been shown to have a critical role in the induction of intestinal regulatory responses. Here we find in mice that in conjunction with IL-15, a cytokine greatly upregulated in the gut of coeliac disease patients, retinoic acid rapidly activates dendritic cells to induce JNK (also known as MAPK8) phosphorylation and release the proinflammatory cytokines IL-12p70 and IL-23. As a result, in a stressed intestinal environment, retinoic acid acted as an adjuvant that promoted rather than prevented inflammatory cellular and humoral responses to fed antigen. Altogether, these findings reveal an unexpected role for retinoic acid and IL-15 in the abrogation of tolerance to dietary antigens.

Identification of crassin acetate as a new immunosuppressant triggering heme oxygenase-1 expression in dendritic cells.

By screening 720 natural compounds in a standard 2-way allogeneic mixed leukocyte reaction assay, we identified a potent immunosuppressive capacity of crassin acetate (CRA), a coral-derived cembrane diterpenoid. CRA efficiently inhibited allogeneic mixed leukocyte reaction as well as antigen-specific activation of CD4 T cells by bone marrow-derived dendritic cells (DCs). With regard to cellular targets, CRA suppressed not only mitogen-triggered T-cell activation, but also lipopolysaccharide-induced DC maturation, indicating dual functionality. Treatment with CRA at nontoxic doses induced heme oxygenase-1 (HO-1) mRNA/protein expression and HO-1 enzymatic activity in DCs, suggesting a unique mechanism of action. In fact, lipopolysaccharide-induced DC maturation was also inhibited by structurally unrelated compounds known to induce HO-1 expression or carbon monoxide (CO) release. Allergic contact hypersensitivity response to oxazolone and oxazolone-induced Langerhans cell migration from epidermis were both prevented almost completely by systemic administration of CRA. Not only do our results support the recent concept that HO-1/CO system negatively regulates immune responses, they also form both conceptual and technical frameworks for a more systematic, large-scale drug discovery effort to identify HO-1/CO-targeted immunosuppressants with dual target specificity.

Cyclooxygenase inhibition during allergic sensitization increases STAT6-independent primary and memory Th2 responses.

Immune sensitization and memory generation are required for the development of allergic inflammation. Our previous studies demonstrate that the cyclooxygenase (COX) metabolic pathway is actively involved in allergic responses and COX inhibition increases allergic airway inflammation in a STAT6-independent fashion. To test the hypothesis that COX inhibition augments allergic inflammation by enhancing immune sensitization and memory, we sensitized STAT6 knockout mice with an i.p. injection of OVA with aluminum hydroxide as an adjuvant and treated the mice with the COX inhibitor indomethacin or vehicle for analyses of the primary and memory immune responses. We found that COX inhibition during immune sensitization, but not the allergic challenge phase, was necessary and sufficient to increase allergic inflammation. COX inhibition during sensitization increased the numbers of mature dendritic cells and activated CD4 T cells in the spleen and augmented OVA-specific IL-5 and IL-13 responses of the splenic CD4 T cells at day 5 after sensitization. COX inhibition during sensitization also augmented allergic Th2 response to OVA challenge 90 days after the sensitization. Therefore, COX inhibition during allergic sensitization augments allergic responses by enhancing Th2 cell activation and memory generation and the proallergic effect is STAT6-independent. These findings provide a mechanistic explanation for the increased allergic inflammation previously shown in the mice treated with COX inhibitors and in COX-deficient mice and suggest that use of COX-inhibiting drugs during initial allergen exposure may increase the risk of developing allergic responses.

All-trans retinoic acid enhances murine dendritic cell migration to draining lymph nodes via the balance of matrix metalloproteinases and their inhibitors.

Cancers escape immune surveillance through the manipulation of the host's immune system. Sequestration of dendritic cells (DCs) within tumor tissues and the subsequent inhibition of their migration is one of the several mechanisms by which tumors induce immunosuppression. In view of recent findings depicting the improvement of tumor immune responses in cancer patients following all-trans retinoic acid (ATRA) treatment, we sought to identify the effects of ATRA on DC mobility in the context of tumor immunotherapy. Our results demonstrate that ATRA, added to differentiating murine bone marrow progenitor cells, enhances the invasive capacity of the resulting DCs. Immature DCs injected intratumorally in mice show increased accumulation in draining lymph nodes, but not in nondraining lymph nodes and spleens, when differentiated in the presence of ATRA. The in vitro migration of mature DCs through the basement membrane matrix toward the lymphoid chemokines CCL19 and CCL21 is enhanced in these cells, albeit not in the presence of a matrix metalloproteinase (MMP) inhibitor. An increase in MMP production with a simultaneous decrease in the production of their inhibitors (tissue inhibitors of matrix metalloproteinase or TIMPs) is provoked by ATRA. This affects the MMP/TIMP balance in DCs, in particular that of MMP-9 and TIMP-1, favoring protease activity and thus allowing for enhanced DC mobilization. In conclusion, this study demonstrates that ATRA is capable of improving DC trafficking in a tumor milieu and, in view of the encouraging results obtained in the clinic, further supports the notion that ATRA might be a valuable chemical adjuvant to current immunotherapeutic strategies for cancer.

[Protective effect of puerarin on endothelial dysfunction of heat shock protein 60 induced specific immunity in apolipoprotein E-null mice].

OBJECTIVE: To investigate the influence of endothelial dysfunction induced by inoculated dendritic cells (DCs) loaded heat shock protein 60 (HSP60) in apolipoprotein (Apo) E-null mice, and the effect of Puerarin on it. METHODS: HSP60 DC (DChsp) acquired after prepared bone marrow-derived DCs of ApoE-null mice and treated with HSP60. In vitro, the function of DCs and the effect of Puerarin were detected. While in vivo, ApoE-null mice fed with high-cholesterol forage were divided into two groups and intravenous inoculated with DCh-sp or normal saline via vein twice respectively. The mice in the two groups were subdivided into the Puerarin group and non-treated group, and they were injected intraperitoneally with Puerarin and normal saline at the beginning of inoculation and the following 3 weeks, respectively. In addition, C57BL/6 mice without inoculation were taken as the normal control group. Two weeks after the last time inoculated, the response of T lymphocytes to HSP60 and endothelial-dependent diastolic function of aortic ring were detected. RESULTS: HSP60 could promote DCs expressed CD86 and stimulate T lymphocytes proliferation in vitro, while Puerarin had significantly inhibitory effect. After inoculated, DChsp activated inflammatory response in vivo and aggravated endothelium-dependent dilation in mice. Puerarin could significantly inhibit inflammatory reaction caused by DChsp and improve endothelium dilation. CONCLUSION: Hsp60 could activate DCs in vitro and in vivo, Puerarin could significantly inhibit specific immunity induced by HSP60 and improve vascular endothelium-dependent dilation.

Conjugated linoleic acid suppresses NF-kappa B activation and IL-12 production in dendritic cells through ERK-mediated IL-10 induction.

Polyunsaturated fatty acids (PUFA) have been shown to modulate immune responses and have therapeutic effects in inflammatory disorders. However, the influence of PUFA on dendritic cells (DC), key cells of the innate immune system in shaping adaptive immune responses, has not yet been defined. In this study, we examine the effects of the cis-9, trans-11 isomer of conjugated linoleic acid (c9, t11-CLA), a dietary PUFA found in meat and dairy products, on murine DC activation. Treatment of DC with c9, t11-CLA suppressed LPS-induced IL-12, enhanced IL-10R expression, and enhanced IL-10 production at the transcriptional and protein level. The suppression of IL-12 by c9, t11-CLA was found to be IL-10 dependent. We investigated the involvement of the MAPK, ERK, and the transcription factor, NF-kappaB, in this IL-10-mediated effect. c9, t11-CLA enhanced ERK activation after LPS stimulation, and inhibition of ERK resulted in abrogation of IL-10 and recovery of IL-12 production. c9, t11-CLA decreased NF-kappaB:DNA binding after LPS stimulation, which was concomitant with delayed translocation of NF-kappaBp65 into the nucleus and an increase in IkappaBalpha. These effects were reversed by addition of a neutralizing anti-IL-10 Ab. Our findings demonstrate that c9, t11-CLA suppresses IL-12 production by LPS-stimulated DC by ERK mediated IL-10-induction. Furthermore, these IL-10-mediated effects are dependent on inhibition of NF-kappaB activation. This is the first study to demonstrate that c9, t11-CLA can enhance transcription and production of the anti-inflammatory cytokine IL-10, while inhibiting the Th1-promoting cytokine IL-12, and may explain certain of its immunosuppressive properties.

Natural killer cell-dendritic cell crosstalk in the initiation of immune responses.

Dendritic cells (DCs) and natural killer (NK) cells play a critical role in early defences against cancer and infections. They specialise in complementary functions, including IL-12 or IFN-alpha/beta secretion and antigen presentation for the former, and IFN-gamma secretion and killing of infected or tumour cells for the latter. Both DCs and NK cells are also sensors of the immune system that have developed different, but partially overlapping, systems to identify pathology associated danger signals. Evidence of NK-DC interaction has accumulated recently. This interaction may lead to NK cell activation, DC activation, or apoptosis depending on the activation status of both cell types. Thus, the outcome of NK-DC crosstalk is likely to influence both innate and adaptive immune responses. This review addresses the molecular mechanisms under-lying the different NK-DC interactions, and their in vivo significance in anti-tumour or antimicrobial immunity. Finally, we discuss the potential clinical implications of this new field.

Epigallocatechin-3-gallate, constituent of green tea, suppresses the LPS-induced phenotypic and functional maturation of murine dendritic cells through inhibition of mitogen-activated protein kinases and NF-kappaB.

The effects of epigallocatechin-3-gallate (EGCG) on dendritic cells (DC) maturation were investigated. EGCG, in a dose-dependent manner, profoundly inhibited CD80, CD86, and MHC class I and II expression on bone marrow-derived murine myeloid DC. EGCG restored the decreased dextran-FITC uptake and inhibited enhanced IL-12 production by LPS-treated DC. EGCG-treated DC were poor stimulators of nai;ve allogeneic T-cell proliferation and reduced levels of IL-2 production in responding T cells. EGCG-pretreated DC inhibited LPS-induced MAPKs, such as ERK1/2, p38, JNK, and NF-kappaB p65 translocation. Therefore, the molecular mechanisms by which EGCG antagonized LPS-induced DC maturation appeared to involve the inhibition of MAPK and NF-kappaB activation. These novel findings provide new insight into the immunopharmacological role of EGCG and suggest a novel approach to the manipulation of DC for therapeutic application of autoimmune and allergic diseases.

Pertussis toxin enhances Th1 responses by stimulation of dendritic cells.

Pertussis toxin (PTX) has been widely used as an adjuvant to induce Th1-mediated organ-specific autoimmune diseases in animal models. However, the cellular and molecular mechanisms remain to be defined. In this study, we showed that dendritic cells (DC) stimulated with PTX (PTX-DC) were able to substitute for PTX to promote experimental autoimmune uveitis (EAU). EAU induced by PTX-DC revealed a typical Th1 response, characterized by high uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP)-specific IFN-gamma and IL-12 production in the draining lymph nodes, as well as increased levels of anti-IRBP IgG2a and decreased levels of anti-IRBP IgG1 in the serum of IRBP-immunized mice. Furthermore, PTX-DC preferentially induced T cells to produce the Th1 cytokine, IFN-gamma. After being stimulated with PTX, DC exhibited up-regulation of MHC class II, CD80, CD86, CD40, and DEC205. PTX-DC had also increased allostimulatory capacity and IL-12 and TNF-alpha production. Serum IL-12 was increased in naive mice that received PTX-DC i.p. In addition, PTX activated extracellular signal-regulated kinase in DC. Following the inhibition of extracellular signal-regulated kinase signaling, the maturation of PTX-DC was reduced. Subsequently, the ability of PTX-DC to promote IFN-gamma production by T cells in vitro and to induce EAU in vivo was blocked. The results suggest that PTX might exert an adjuvant effect on DC to promote their maturation and the production of proinflammatory cytokines, thereby eliciting a Th1 response.

Prostaglandins inhibit 5-lipoxygenase-activating protein expression and leukotriene B4 production from dendritic cells via an IL-10-dependent mechanism.

PGs produced from arachidonic acid by the action of cyclooxygenase enzymes play a pivotal role in the regulation of both inflammatory and immune responses. Because leukotriene B4 (LTB4), a product of 5-lipoxygenase (5-LO) pathway, can exert numerous immunoregulatory and proinflammatory activities, we examined the effects of PGs on LTB4 release from dendritic cells (DC) and from peritoneal macrophages. In concentration-dependent manner, PGE1 and PGE2 inhibited the production of LTB4 from DC, but not from peritoneal macrophage, with an IC50 of 0.04 microM. The same effect was observed with MK-886, a 5-LO-activating protein (FLAP)-specific inhibitor. The decreased release of LTB4 was associated with an enhanced level of IL-10. Furthermore, the inhibition of LTB4 synthesis by PGs was significantly reversed by anti-IL-10, suggesting the involvement of an IL-10-dependent mechanism. Hence, we examined the effects of exogenous IL-10 on the 5-LO pathway. We demonstrate that IL-10 suppresses the production of LTB4 from DC by inhibiting FLAP protein expression without any effect on 5-LO and cytosolic phospholipase A2. Taken together, our results suggest links between DC cyclooxygenase and 5-LO pathways during the inflammatory response, and FLAP is a key target for the PG-induced IL-10-suppressive effects.

CD26 is expressed on a restricted subpopulation of dendritic cells in vivo.

Two major sub-populations of dendritic cells (DC) are present in afferent lymph draining the skin of cattle distinguished by expression of signal regulator protein alpha (SIRPalpha). The SIRPalpha(-) population expresses the uncharacterized bovine WC10 antigen (Ag). Initial N-terminal sequencing of the WC10 protein purified by affinity chromatography showed significant homology with human CD26. A cDNA encoding bovine CD26 was cloned and the recombinant molecule expressed in COS-7 cells. Transfectants abrogated the ability of macrophage-derived chemokine (MDC) to cause a calcium flux in bovine PBMC indicating enzymatic activity characteristic of CD26. They also stained with WC10 monoclonal antibody confirming that the Ag is CD26. This is the first description of CD26 expression by DC in vivo or in vitro. It is expressed on a sub-population of ex vivo DC in afferent lymph draining the skin and on sub-populations of DC isolated from prescapular and mesenteric lymph nodes draining the skin or intestine, respectively. CD26 is an exopeptidase with specificity for motifs within the receptor-binding domain of several chemokines including MDC. CD26 mediated truncation of MDC affects the Th cell response effected by the chemokine and may produce a Th1 bias. Transcripts for MDC were present in both CD26(+) and CD26(-) DC, thus CD26 mediated modification of MDC may bias the immune response induced in naive T cells by DC.

Monophosphoryl lipid A activates both human dendritic cells and T cells.

The induction of dendritic cell (DC) maturation is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. In this study, we have investigated the effects of monophosphoryl lipid A (MPL) on human monocyte-derived DC as well as peripheral blood T cells. Calcium mobilization, mitogen-activated protein kinase activation, and the NF-kappaB transcription factor were induced after MPL stimulation of DC and required high doses of MPL (100 microg/ml). Maturation parameters such as production of IL-12 and increases in cell surface expression of HLA-DR, CD80, CD86, CD40, and CD83 were observed following DC treatment with MPL. However, lower levels of IL-12 were induced by MPL when compared with lipopolysaccharide. This is likely to be related to differences in the kinetics of extracellular signal-related kinase 1/2 and p-38 phosphorylation induced by both molecules. Although maturation induced by MPL was weaker when compared with lipopolysaccharide, it appeared to be sufficient to support optimal activation of allogeneic naive CD45RA(+) T cell and anti-tetanus toxoid CD4 T cells. MPL at low doses (5 microg/ml) had no impact on DC maturation, while its addition to DC-T cell cocultures induced full T cell activation. The observed effect was related to the fact that MPL also acts directly on T cells, likely through their Toll-like receptors, by increasing their intracellular calcium and up-regulating their CD40 ligand expression. Together, these data support a model where MPL enhances T cell responses by having an impact on DC and T cells.

Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production.

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE(2) production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE(2) synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-gamma present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE(2) synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-gamma. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-gamma in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.

Polymerase chain reaction selects a novel disintegrin proteinase from CD40-activated germinal center dendritic cells.

To identify genes expressed by a specific subset of dendritic cells found in vivo a polymerase chain reaction-based cDNA subtraction technique was applied to the recently described germinal center dendritic cells. A novel member of the disintegrin metalloproteinase family was cloned which comprises a not typical zinc-chelating catalytic site most similar to a bacterial metalloproteinase. Dendritic cell precursors or immature dendritic cells express no or low levels of the message. It is induced to high levels upon spontaneous or CD40-dependent maturation and in a mixed lymphocyte reaction. In situ hybridization showed distinct expression of this gene in the germinal center. This, together with the findings that certain disintegrin metalloproteinases regulate the activity of tumor necrosis factor alpha and that metalloproteinases have also been implicated in FasL processing, suggest that this novel molecule may play an important role in dendritic cell function and their interactions with germinal center T cells.