Modulation of dendritic cell differentiation and cytokine secretion by the hydatid cyst fluid of Echinococcus granulosus

Chronic infection by Echinococcus granulosus results in establishment of fluid-filled cysts (hydatid cysts) in liver or lungs of infected hosts, which can escape destruction by the host immune system for long periods. This study explores the modulation by hydatid cyst fluid of the in vitro human monocyte to dendritic cell (DC) transition induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Addition of the fluid to adherent peripheral blood monocytes cultured in GM-CSF/IL-4 stimulates release of prostaglandin E2 (PGE2) and IL-6. Exposure of differentiating DC to the fluid during the 7-day culture in GM-CSF/IL-4 impairs their subsequent ability to secrete IL-12, IL-6 or PGE2 in response to lipopolysaccharide (LPS) stimulation. This inhibition is not dependent on the initial release of PGE2. The presence of hydatid cyst fluid also modulates the phenotype of the cells generated during culture, resulting in increased CD14 expression and decreased expression of CD1a. Finally, hydatid fluid can stimulate predifferentiated DC to mature, as evidenced by release of IL-12 and IL-6, and by up-regulation of class II major histocompatibility complex and CD86. The possible role of dendritic cell modulation in regulating the host immune response to hydatid cysts is discussed.     

Differential functional effects of biomaterials on dendritic cell maturation

The immunological outcome of dendritic cell (DC) treatment with different biomaterials was assessed to demonstrate the range of DC phenotypes induced by biomaterials commonly used in combination products. Immature DCs (iDCs) were derived from human peripheral blood monocytes, and treated with different biomaterial films of alginate, agarose, chitosan, hyaluronic acid (HA), or 75:25 poly(lactic-co-glycolic acid) (PLGA) and a comprehensive battery of phenotypic functional outcomes was assessed. Different levels of functional changes in DC phenotype were observed depending on the type of biomaterial films used to treat the DCs. Treatment of DCs with PLGA or chitosan films supported DC maturation, with higher levels of DC allostimulatory capacity, pro-inflammatory cytokine release, and expression of CD80, CD86, CD83, HLA-DQ and CD44 compared with iDCs, and lower endocytic ability compared with iDCs. Alginate film induced pro-inflammatory cytokine release from DCs at levels higher than from iDCs. Dendritic cells treated with HA film expressed lower levels of CD40, CD80, CD86 and HLA-DR compared with iDCs. They also exhibited lower endocytic ability and CD44 expression than iDCs, possibly due to an insolubilized (cross-linked) form of high molecular weight HA. Interestingly, treatment of DCs with agarose film maintained the DC functional phenotype at levels similar to iDCs except for CD44 expression, which was lower than that of iDCs. Taken together, these results can provide selection criteria for biomaterials to be used in immunomodulating applications and can inform potential outcomes of biomaterials within combination products on associated immune responses as desired by the application.     

Optimal dendritic cell differentiation in rpmi media requires the absence of HEPES buffer

Monocyte-derived dendritic cells (DCs) are considered an indispensible and one of primary tools for in vitro DC-based studies. For majority of in vitro DC-based studies the medium of choice is supplemented RPMI, with certain variable ingredients such as HEPES buffer or Phenol Red (PHR). In effort to identify potential obstruction of DC differentiation process due to presence of mentioned additives, we differentiated DCs using RPMI either with or without HEPES or PHR. Although PHR caused a certain down-regulation of immature DCs (iDCs) differentiation markers and lower expression of co-stimulatory molecules on mature DCs, these changes were not significant. In contrast, use of RPMI also containing HEPES resulted in significantly lower CD1a and DC-SIGN expression on iDCs and extensively lowered co-stimulatory molecule expression after DC activation (HEPES-DCs). Furthermore, DCs differentiated in HEPES-free RPMI possessed more genuine immature/mature DC characteristics in context of Th1 polarization. Additionally, during classical differentiation procedure, fewer DCs remained adherent and possessed better overall morphology in HEPES-free medium. In summary our study clarifies a seemingly minor, but a very important issue, that will most likely facilitate lab work for many scientists dealing with monocyte-derived DCs.     

DC-SIGN ligation greatly affects dendritic cell differentiation from monocytes compromising their normal function

DC-SIGN is a C-type lectin selectively expressed by certain types of DCs, including monocyte-derived DCs. Many reports have described the impact of DC-SIGN engagement with concomitant TLR signaling in tailoring of the DC maturation process, but so far, none has addressed the importance of DC-SIGN engagement during their differentiation from blood progenitors. We therefore examined the role of DC-SIGN engagement limited to the stage of IL-4-guided differentiation of DCs from human peripheral blood monocytes but not during maturation. We used two different anti-DC-SIGN antibodies with reported DC-SIGN-engaging activities. In cultures with DC-SIGN ligands, the resulting iDCs displayed abrogated expression of differentiation markers CD1a and DC-SIGN. Without further DC-SIGN activation, such DCs matured with low CD80/CD86 and high ILT3 expression, along with the appearance of macrophage marker CD14. Additionally, treated DCs indicated a tolerogenic potential by possessing a low, allostimulatory capacity and inducing na?ve, allogeneic CD4(+) T cells to produce low levels of IFN-γ. Upon activation, IL-10 production was greatly increased by such DCs; however, the use of IL-10-blocking antibodies could not completely reverse alternative DC activation. This suggests an alternative activation response that is a result of a different elementary state of DCs generated with concomitant ligation of DC-SIGN. During differentiation, IL-4-induced pSTAT6 was reduced by DC-SIGN ligands. Furthermore, during LPS-induced maturation, treated DCs displayed lowered activation levels of p38 MAPK, STAT1, as well as STAT6, compared with controls. Collectively, evidence is presented confirming a crucial role for DC-SIGN signaling in DC generation from monocytes.     

Differential levels of dendritic cell maturation on different biomaterials used in combination products

Immature dendritic cells (iDCs) were derived from human peripheral blood monocytes, and treated with films of biomaterials commonly used in combination products (e.g., tissue engineered constructs or vaccines) to assess the resultant dendritic cell (DC) maturation compared to positive control of lipopolysaccharide (LPS) treatment for DC maturation or negative control of untreated iDCs. The following biomaterials were tested: alginate, agarose, chitosan, hyaluronic acid, 75:25 poly(lactic-co-glycolic acid) (PLGA). The effect of DC culture on these films was undertaken to identify biomaterials which support DC maturation and those biomaterials that did not. Dendritic cells treated with chitosan or PLGA (agarose to a lesser extent) films increased expression levels of CD86, CD40, and HLA-DQ, compared to control iDCs, similar to LPS-matured DCs, whereas DCs treated with alginate or hyaluronic acid films decreased their expression levels of these same molecules. In summary, a differential effect of the biomaterial on which iDCs were cultured was observed as far as the extent of induced DC maturation. The effect of biomaterials on DC maturation, and the associated adjuvant effect, is a novel biocompatibility selection and design criteria for biomaterials to be used in combination products in which immune consequences are potential complications or outcomes.     

Impaired dendritic cell differentiation and maturation in the absence of C3

Human monocytes can be differentiated into immature dendritic cells (DCs) in the presence of serum and cytokines. One of the main functions of immature DCs is to capture and process antigens. Following maturation, they differentiate into antigen presenting cells. The role of complement in the differentiation process from monocytes towards immature DCs remains elusive. Here we demonstrate that complement 3 (C3) has a regulatory impact on the expression of specific DC surface molecules and DC-derived cytokine production during DC differentiation. We isolated human adherent peripheral blood mononuclear cells, which were cultured in the presence of GM-CSF plus IL-4 in medium supplemented with normal human serum or C3 deficient serum. The lack of C3 during DC differentiation negatively impacted the expression of C-type lectin receptor DC-SIGN, the antigen presenting molecules HLA-DR and CD1a, and the costimulatory molecules CD80 and CD86. Further, the spontaneous production of IL-6 and IL-12 was reduced in the absence of C3. Moreover, the maturation of immature DCs in response to LPS challenge was impaired in the absence of C3 as evidenced by reduced MHC-II, co-stimulatory molecule expression as well as modulated IL-12 and TNF-alpha production. Collectively, our results provide evidence for a novel role of C3 as a critical cofactor in human DC differentiation and maturation.     

Thymosin-alpha1 modulates dendritic cell differentiation and functional maturation from human peripheral blood CD14+ monocytes

Although thymosins have been demonstrated to have immunomodulatory effects, it is still not clear whether they could affect dendritic cells (DCs), the most professional antigen-presenting cells. The objective of this study was to determine the effect and potential mechanisms of thymosin-alpha1 (Talpha1) on DC differentiation and functional maturation. Human peripheral blood CD14(+) monocytes were purified by using a magnetic separation column and cultured with GM-CSF and IL-4 to differentiate into immature DCs (iDCs). In the presence of Talpha1, iDC surface markers CD40, CD80, MHC class I and class II molecules were significantly upregulated as measured by flow cytemotry analysis. However, Tbeta4 or Tbeta10 did not show these effects on iDCs. There was an approximately 30% reduction in antigen (FITC-conjugated dextran)-uptake by Talpha1-treated iDCs as compared with non-Talpha1-treated iDCs. In addition, Talpha1-treated matured DCs (mDCs) showed an increased stimulation of allogeneic CD3(+) T-cell proliferation as measured by a mixed-lymphocyte reaction assay. Talpha1-treated mDCs also increased the production of several Th1- and Th2-type cytokines as measured by a Bio-Plex cytokine assay. Furthermore, rapid activation of p38 MAPK and NFkappaB was seen in Talpha1-treated iDCs as measured by a Bio-Plex phosphoprotein assay. Thus, Talpha1 significantly enhances DC differentiation, activation, and functions from human peripheral blood CD14(+) monocytes possibly through a mechanism of the activation of p38 MAPK and NFkappaB pathways. This study provides a basis to further evaluate Talpha1 as a possible adjuvant for a DC-directed vaccine or therapy.     

Cord blood mesenchymal stem cells propel human dendritic cells to an intermediate maturation state and boost interleukin‐12 production by mature dendritic cells

Pathogen‐derived entities force the tissue‐resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC [unrestricted somatic stem cells (USSCs)]. The maturation to mature DCs (mDCs) was enhanced when DCs were co‐cultured with USSC, as evidenced by the up‐regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin‐12 production. So, USSCs mature iDCs, thereby redirecting the antigen‐uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.     

Distinct compartmental distribution of mature and immature dendritic cells in esophageal squamous cell carcinoma

Dendritic cells (DCs) play a critical role in generating anti-tumor immunity. DC functional defect has been related to the growth and progression of various human cancers. In esophageal squamous cell carcinoma (ESCC), the examination of DCs using immunohistochemistry (IHC) with anti-S100 antibody has demonstrated an increased infiltration of DCs into the tumor mass, however, the distribution patterns of DCs at different maturation states in ESCC are not fully evaluated. In this study, we immunohistochemically analyzed the DC maturation status by examining the S100-positive DCs, CD1α-positive immature DCs (iDCs), and CD208-positive mature DCs (mDCs) and their distribution patterns in 45 ESCCs and 10 control tissues. The IHC analysis showed that the number of S100-positive DCs was increased in both the cancer epithelium and tumor stroma. Further phenotypic analyses revealed that intraepithelial DCs in the cancer mass were predominantly CD1α-positive iDCs. Whereas DCs presented in the tumor stroma were exclusively CD208-positive mDCs, CD208-positive mDCs were particularly dense in the margin of cancerous lesions and formed clusters with CD3-positive lymphocytes. The number of CD208-positive mDCs in the tumor mass was significantly lower than the number of CD1α-positive iDCs. The current results suggest that ESCC tissue comprises a high frequency of iDCs in the cancerous epithelium and a low density of mDCs in the tumor stroma. Such a distinct distribution pattern may reflect the ongoing DC tracking in ESCCs.     

Neem leaf glycoprotein partially rectifies suppressed dendritic cell functions and associated T cell efficacy in patients with stage IIIB cervical cancer

Myeloid-derived dendritic cells (DCs) generated from monocytes obtained from stage IIIB cervical cancer (CaCx IIIB) patients show dysfunctional maturation; thus, antitumor T cell functions are dysregulated. In an objective to optimize these dysregulated immune functions, the present study is focused on the ability of neem leaf glycoprotein (NLGP), a nontoxic preparation of the neem leaf, to induce optimum maturation of dendritic cells from CaCx IIIB patients. In vitro NLGP treatment of immature DCs (iDCs) obtained from CaCx IIIB patients results in upregulated expression of various cell surface markers (CD40, CD83, CD80, CD86, and HLA-ABC), which indicates DC maturation. Consequently, NLGP-matured DCs displayed balanced cytokine secretions, with type 1 bias and noteworthy functional properties. These DCs displayed substantial T cell allostimulatory capacity and promoted the generation of cytotoxic T lymphocytes (CTLs). Although NLGP-matured DCs derived from CaCx monocytes are generally subdued compared to those with a healthy monocyte origin, considerable revival of the suppressed DC-based immune functions is noted in vitro at a fairly advanced stage of CaCx, and thus, further exploration of ex vivo and in vivo DC-based vaccines is proposed. Moreover, the DC maturating efficacy of NLGP might be much more effective in the earlier stages of CaCx, where the extent of immune dysregulation is less and, thus, the scope of further investigation may be explored.     

Suppression of dendritic cell activation by diabetes autoantigens linked to the cholera toxin B subunit

Antigen presenting cells, specifically dendritic cells (DCs) are a focal point in the delicate balance between T cell tolerance and immune responses contributing to the onset of type I diabetes (T1D). Weak adjuvant proteins like the cholera toxin B subunit when linked to autoantigens may sufficiently alter the balance of this initial immune response to suppress the development of autoimmunity. To assess adjuvant enhancement of autoantigen mediated immune suppression of Type 1 diabetes, we examined the cholera toxin B subunit (CTB)-proinsulin fusion protein (CTB-INS) activation of immature dendritic cells (iDC) at the earliest detectable stage of the human immune response. In this study, Incubation of human umbilical cord blood monocyte-derived immature DCs with CTB-INS autoantigen fusion protein increased the surface membrane expression of DC Toll-like receptor (TLR-2) while no significant upregulation in TLR-4 expression was detected. Inoculation of iDCs with CTB stimulated the biosynthesis of both CD86 and CD83 co-stimulatory factors demonstrating an immunostimulatory role for CTB in both DC activation and maturation. In contrast, incubation of iDCs with proinsulin partially suppressed CD86 co-stimulatory factor mediated DC activation, while incubation of iDCs with CTB-INS fusion protein completely suppressed iDC biosynthesis of both CD86 and CD83 costimulatory factors. The incubation of iDCs with increasing amounts of insulin did not increase the level of immune suppression but rather activated DC maturation by stimulating increased biosynthesis of both CD86 and CD83 costimulatory factors. Inoculation of iDCs with CTB-INS fusion protein dramatically increased secretion of the immunosuppressive cytokine IL-10 and suppressed synthesis of the pro-inflammatory cytokine IL12/23 p40 subunit protein suggesting that linkage of CTB to insulin (INS) may play an important role in mediating DC guidance of cognate naïve Th0 cell development into immunosuppressive T lymphocytes. Taken together, the experimental data suggests Toll like receptor 2 (TLR-2) plays a dominant role in CTB mediated INS inhibition of DC induced type 1 diabetes onset in human Type 1 diabetes autoimmunity. Further, fusion of CTB to the autoantigen was found to be essential for enhancement of immune suppression as co-delivery of CTB and insulin did not significantly inhibit DC costimulatory factor biosynthesis. The experimental data presented supports the hypotheses that adjuvant enhancement of autoantigen mediated suppression of islet beta cell inflammation is dependent on CTB stimulation of dendritic cell TLR2 receptor activation and co-processing of both CTB and the autoantigen in the same dendritic cell.     

Cryopreservation of monocytes or differentiated immature DCs leads to an altered cytokine response to TLR agonists and microbial stimulation

Literature on the effects of cryopreservation and thawing of monocytes or monocyte-derived immature dendritic cells (iDCs) on the subsequent functional capacities of the DCs is limited to a few specific maturation stimuli and is focused on applications in clinical immunotherapy. Given the cardinal role of DCs in regulating tolerance and immunity at mucosal surfaces there is a growing interest in understanding the effect of stromal, microbial and probiotic signals on DC function. Therefore our aim was to investigate the effects of cryopreservation on the functional properties of DCs stimulated with bacteria or the bacterial components using a standardized method. Surface markers CD83 and CD86 were expressed at similar levels on iDCs generated from cryopreserved or freshly isolated monocytes. Cryopreservation of iDCs led to slightly decreased expression of CD86 and CD83 compared to freshly generated iDCs prepared from unfrozen cells but this did not affect the capacity of DCs to acquire fully mature characteristics after stimulation. In contrast the cytokine response to lipoteichoic acid and bacterial stimulation was altered by cryopreservation of monocytes or iDCs, particularly for IL-12p70 which was decreased up to 250 fold or not detected. Cryopreservation also decreased TNF-α and IL-1β production in stimulated iDCs but to a lesser extent than for IL-12p70, depending on the maturation factors used. The amounts of IL-10 produced by stimulated iDCs were increased up to 3.6 fold when iDCs were cryopreserved, but decreased up to 90 fold when generated from cryopreserved monocytes. Immature DCs are often used to investigate the immunomodulatory properties of probiotics and here we show for the first time that cryopreserved monocytes and cryopreserved iDCs have a skewed cytokine response to microbial stimulation. These findings have implications for the methods used in bacterial-DC immune assays and highlight the importance of comparing different cytokines and stimuli in immune cell cryopreservation protocols.     

Tick saliva inhibits differentiation, maturation and function of murine bone-marrow-derived dendritic cells

Haematophagous arthropod vectors such as mosquitoes, tsetse flies, sandflies and ticks have evolved salivary immunomodulatory factors that prevent the vertebrate host from rejecting them meanwhile enhancing pathogen transmission. As dendritic cells (DC) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC differentiation and maturation. Flow cytometry analysis revealed that the addition of saliva to bone marrow cells inhibits the differentiation of DC and decreased the population of differentiated immature DC, increasing the levels of major histocompatibility complex (MHC) class II while not altering the expression of costimulatory (CD40, CD80 and CD86) and adhesion (CD54) molecules. Furthermore, maturation of DC stimulated by lipopolysaccharide (LPS) in the presence of saliva resulted in a lower expression of costimulatory molecules, but did not alter the up-regulation of MHC class II and CD54. The lipopolysaccharide (LPS)-matured DC cultured with saliva also presented reduced production of interleukin-12, whereas interleukin-10 production was unaltered. Assessment of the function of DC cultured with tick saliva revealed them to be poor stimulators of cytokine production by antigen-specific T cells. Our data indicate a novel modulatory role for the saliva of arthropod vectors at an initial step of the immune response through the inhibition of differentiation and maturation of DC into functional antigen-presenting cells.     

The role of heat shock protein (hsp70) in dendritic cell maturation: hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors

Members of the heat shock protein (hsp70) family are either constitutively expressed (hsc70) or can be induced by hyperthermic stress (hsp70). Recombinant hsp70 (rhsp70) stimulates cytokine production from monocytes and enhances NK cell proliferation and cytotoxicity. Here we demonstrate that rhsp70 binds to immature dendritic cells (DC) derived from monocyte precursors and induces their maturation as evidenced by an increase in CD40, CD86 and CD83 expression. Immature DC stimulated to mature with rhsp70 show an enhanced ability to present tyrosinase peptide to specific CTL. Mature DC did not bind rhsp70, suggesting a down-regulation in the expression of its receptor. When rhsp70 was added to monocyte precursors at the same time as GM-CSF and IL-4 it reduced the differentiation of monocytes into DC as shown by a decrease in the level of CD40, CD83, CD86 and HLA-DR expression and an increase in CD14 expression. The constitutively expressed hsc70 had neither a stimulatory effect on the maturation of immature DC nor did it reduce the differentiation of monocytes into DC. These findings demonstrate the specific ability of rhsp70 to induce the maturation of immature DC. Therefore rhsp70 may be useful for its adjuvant like properties in DC based immunotherapy of certain tumors.     

Lipopolysaccharide-induced expression of TRAIL promotes dendritic cell differentiation

Tumour necrosis factor‐related apoptosis inducing ligand (TRAIL) is a death‐inducing cytokine whose physiological function is not well understood. Here, we show that TRAIL has a role in programming human dendritic cell (DC) differentiation. TRAIL expression was strongly induced in DCs upon stimulation with lipopolysaccharide (LPS) or Polyinosine‐polycytidylic acid (poly(I:C)) stimulation. Blockade of TRAIL with neutralizing antibody partially inhibited LPS‐induced up‐regulation of co‐stimulatory molecules and the expression of inflammatory cytokines including interleukin‐12 (IL‐12) p70. In addition, neutralization of TRAIL in LPS‐treated DCs inhibited the DC‐driven differentiation of T cells into interferon‐γ (IFN‐γ) ‐producing effectors. The effects of TRAIL neutralization in poly(I:C)‐treated DCs were similar, except that IL‐12 production and the differentiation of effector T cells into IFN‐γ producers were not inhibited. Strikingly, TRAIL stimulation alone was sufficient to induce morphological changes resembling DC maturation, up‐regulation of co‐stimulatory molecules, and enhancement of DC‐driven allogeneic T‐cell proliferation. However, TRAIL alone did not induce inflammatory cytokine production. We further show that the effects of TRAIL on DC maturation were not the result of the induction of apoptosis, but may involve p38 activation. Hence, our data demonstrate that TRAIL co‐operates with other cytokines to facilitate DC functional maturation in response to Toll‐like receptor activation.     

Inhibitory effects of suplatast tosilate on the differentiation and function of monocyte-derived dendritic cells from patients with asthma

Background Dendritic cells (DCs) are antigen‐presenting cells that efficiently activate T cells. Objective We examined the effects of suplatast tosilate, which prevents T‐helper type 2 responses, on the differentiation and function of monocyte‐derived DCs (moDCs). Methods DCs were differentiated in vitro from peripheral monocytes from patients with asthma by the addition of granulocyte macrophage colony‐stimulating factor and IL‐4 in the presence or absence of suplatast tosilate. Cell surface molecules (CD1a, CD14, CD80, CD83, CD86, HLA‐DR) on immature and mature DCs were analysed with flow cytometry, and the secretion of CC chemokine ligand (CCL)17 (thymus and activation‐regulated chemokine), IL‐12p70, IL‐12p40, and IL‐10 was measured with an ELISA. We also studied the proliferative responses of allogeneic CD4⁺ T cells from healthy subjects to DCs differentiated in the presence of suplatast tosilate. In addition, the production of IFN‐γ and IL‐5 by CD4⁺ T cells after coculture with untreated DCs or suplatast tosilate‐treated DCs was measured with ELISA. Results Suplatast tosilate significantly inhibited the expression of CD1a, CD80, and CD86 on immature DCs and of CD1a, CD80, CD83, and CD86 on mature DCs. Suplatast tosilate also significantly inhibited the secretion of CCL17, IL‐12p70, and IL‐12p40; however, the secretion of IL‐10 was not affected. The proliferative responses of allogeneic CD4⁺ T cells to suplatast tosilate‐treated DCs were suppressed. Moreover, suplatast tosilate‐treated DCs had an impaired capacity to stimulate CD4⁺ T cells to produce IFN‐γ and IL‐5. Conclusion Suplatast tosilate inhibits the differentiation, maturation, and function of moDCs.     

Quantitative analysis of chemokine expression by dendritic cell subsets in vitro and in vivo

Upon maturation, dendritic cells (DCs) have to adjust their chemokine expression to sequentially attract different leukocyte subsets. We used real-time quantitative polymerase chain reaction analysis to study in detail the expression of 12 chemokines involved in the recruitment of leukocytes into and inside secondary lymphoid organs, by DCs in distinct differentiation stages, both in vitro and in vivo. Monocyte-derived immature DCs expressed high levels of DC chemokine 1 (DC-CK1), EBI1-ligand chemokine (ELC), macrophage-derived chemokine (MDC), macrophage-inflammatory protein (MIP)-1alpha, and thymus and activation-regulated chemokine (TARC). Upon maturation, DCs up-regulated the expression of DC-CK1 (60-fold), ELC (7-fold), and TARC (10-fold). Activation of DCs by CD40 ligand further up-regulated the expression of ELC (25-fold). We found that freshly isolated blood DCs expressed only low levels of interleukin-8, lymphotactin, and MIP-1alpha. It is interesting that the chemokine profile expressed by activated CD11c(-) lymphoid-like as well as CD11c(+) myeloid blood DCs mimics that of monocyte-derived DCS: Additionally, purified Langerhans cells that had migrated out of the epidermis expressed a similar chemokine pattern. These data indicate that different DC subsets in vitro and in vivo can express the same chemokines to attract leukocytes.     

Pimecrolimus does not affect the differentiation,maturation and function of human monocyte-derived dendritic cells,in contrast to corticosteroids

Clinically, corticosteroids (CS) are among the first line drugs in the therapy of autoimmune and allergic diseases and potently inhibit the activation of immune cells. However, due to their pleiotropic mode of action, the prolonged use of CS is generally associated with a range of undesirable side‐effects. In this study, we compared the activity of pimecrolimus, a novel immunomodulatory drug for the treatment of inflammatory skin disorders, and the CS dexamethasone (Dex) and beta‐methasone‐valerate (β‐MSV) in different in vitro assays addressing the cytokine‐induced differentiation and maturation of monocyte‐derived dendritic cells (M‐DC), the susceptibility of M‐DC to drug‐induced apoptosis and the potency of differentiated M‐DC to induce primary T cell activation. In contrast to pimecrolimus, Dex and β‐MSV strongly induced apoptosis of M‐DC precursors if added at the start of the DC differentiation culture. Flow cytometric analysis of surviving cells on day 6 of culture showed that the expression of several DC‐specific antigens such as CD1a, CD40 and CD80 was inhibited by 50% to 80% at concentrations between 1 nm and 10 nm of either Dex or β‐MSV. Furthermore, the presence of CS during the final maturation of M‐DC inhibited the synthesis of IL‐12p70, the expression of critical DC costimulatory molecules, such as CD83 and CD86 and impaired their ability to activate primary CD4⁺ T cell proliferation. In contrast, pimecrolimus did not inhibit the LPS‐induced secretion of IL‐12, surface expression of costimulatory molecules or the maturation of M‐DC into potent stimulators of T cells. Taken together, these data indicate that pimecrolimus does not interfere with the differentiation and viability of dendritic cells and their precursors or with the function of mature M‐DC to prime naïve T lymphocytes, and thus may have a lower potential than CS to interfere with DC‐mediated immunosurveillance.