Activation Of Immature Dendritic Cells Research Articles

Lack of differences in radiation-induced immunogenicity parameters between HPV-positive and HPV-negative human HNSCC cell lines

Background and purposeClinical studies indicate that patients with HPV/p16-associated head & neck squamous cell carcinoma (HNSCC) represent a subgroup with a better prognosis and improved response to conventional radiotherapy. Involvement of immune-based factors has been hypothesized. In the present study, we investigated radiation-induced differences in release of damage associated molecular patterns (DAMPs), cytokines and activation of dendritic cells (DCs) in HPV-positive and negative HNSCC cancer cell lines. Material and methodsCalreticulin (CRT) exposure was detected on cancer cell surface. ATP, HMGB1 and cytokines were measured in culture supernatants. Maturation marker CD83 surface exposure was determined on DCs after co-incubation with irradiated tumor cells. ResultsThere was no increase in DAMPs and cytokine profiles after radiation treatment and no difference between HPV+ and HPV− cell lines. The HPV/p16-positive SCC90 cells showed a trend for increased total CRT, HMGB1, and number of cytokines compared to all other cell lines. None of the irradiated cancer cell lines could affect DC maturation. ConclusionsRadiation treatment did not increase immunogenicity of HNSCC cell lines assessed by membrane CRT, ATP, HMGB1, cytokines production, and by activation of immature DCs. There was no difference between HPV-positive and HPV-negative cell lines.

ФУНКЦІОНАЛЬНИЙ СТАН АНТИГЕНПРЕЗЕНТУЮЧИХ КЛІТИН ПРИ ЗОВНІШНЬОМУ ГЕНІТАЛЬНОМУ ЕНДОМЕТРІОЗІ.

<p>У статті наведені результати спостереження 120 безплідних хворих з зовнішнім генітальним ендометріозом. Вік обстежених хворих був від 21 до 35 років. Серед клінічних проявів захворювання найбільш часто зустрічалися<br />хронічні тазові болі у 83 (69,2 %) пацієнток, альгодисменорея - у 59 (49,2 %), диспареунія - у 46 (38,3 %). При діагностичній лапароскопії ендометріоз І та ІІ стадій зустрічався частіше - у 37 (30,8 %) і 41 (34,2 %) випадків<br />відповідно. У черевній порожнині у більшості хворих (61 %) був виявлений злуковий процес різного ступеня тяжкості. При імуногістохімічному дослідженні було виявлено зниження активності зрілих дендритних клітин (до<br />5,2 ± 0,5 кл./мм2 CD23 у функціональному шарі ендометрія і до 6,0±0,5 кл./мм2 у базальному шарі), а також підвищення активності незрілих дендритних клітин у базальному шарі ендометрія (CD1a до 34,2±1,2 кл./мм2).<br />Все вищезазначене свідчить про функціональну супресію місцевого імунітету і може бути чинником, що знижує репродуктивну функцію, що, у свою чергу, вимагає проведення імунокорекції.<br /><br /></p>

Pecularities of functional state of antigen-presenting cells of the endometrium in patients with deep infiltrating endometriosis

Проведено дослідження, метою якого була оцінка функціонального стану антигенпрезентуючих клітин ендометрія у хворих із тяжкими формами зовнішнього генітального ендометріозу. Показано, що у здорових жінок, які увійшли до контрольної групи, перитонеальна рідина у 3–15 мл містила від 7 до 12 млн лейкоцитів, представлених переважно макрофагами (90%), лімфоцитами (5–10%) і поліморфноядерними лейкоцитами (до 5%), тоді як у жінок, хворих на зовнішній генітальний ендометріоз, кількість макрофагів зростала до 95% за рахунок відносного збіднення популяції поліморфноядерних лейкоцитів. При цьому загальна кількість клітин у перитонеальній рідині зростала до 18–20 млн в 1 мл. Характерним для пацієнток із зовнішнім генітальним ендометріозом є зниження активності зрілих дендритних клітин (до 5,2 ± 0,5 клітин/мм2 СD23 у функціональному шарі та до 6,0 ± 0,5 клітин/мм2 у базальному шарі ендометрія) та підвищення активності незрілих дендритних клітин у базальному шарі (CD1a до 34,2 ± 1,2 клітин/мм2).

Biologic activity of immature dendritic cells modified by FasL and TGF-β1 gene

Objective To investigate the biological activities of immature dendritic cells (imDCs)modified by hFasL and hTGF-β1 gene,from bone marrow stem cells of rats.Methods The experiment was divided into six groups:mDC group,imDC group,empty vector group,hFasL gene modification group (FasL group),hTGF-β1 gene modification group (TGF group) and hFasL-hTGF-β1 co-transfection group (namely F-T group).The plasmids of pIRES2-EGFP-hFasL or(and) pIRES2-EGFP-hTGF-β1 were transfected into imDCs which were cultivated for six days.The expression of hFasL and hTGF-β1 gene was detected by using fluorescence microscopy,ELISA,Western blotting,flow cytometry and MLR after imDCs were transfected for 24-48 h.Results After transfection,green fluorescence was observed in the transfected imDCs under the fluorescence microscopy.ELISA revealed that the expression of hTGF-β1 in F-T group and TGF group was significantly higher than that in other groups (P ＜0.01 ) ; the expression of hTGF-β1 in imDC group,empty vector group and FasL group was significantly higher than that in mDC group (P ＜0.05) ; and the hFasL gene expression among groups had no significant difference ( P ＞ 0.05 ).Western blotting showed the positive expression of hFasL and hTGF-β1 protein in hFasL and hTGF-β1 gene modification groups.After the plasmids of pIRES2-EGFP-hFasL or(and) pIRES2-EGFP-hTGF-β1 were transfected into imDCs,CD86 and CD80 expression on the imDCs surface were lower than in mDC group by flow cytometry.The result of MLR demonstrated that the imDCs induced lower T-cells in F-T group than in TGF group (P ＜ 0.05 ) and FasL group ( P ＜ 0.05 ).Conclusion The ability of imDCs with hFasL and hTGF-β1 co-transfection was stronger than that transfected by single hFasL or hTGF-β1 in inducing allogeneic immunology tolerance. Key words: Dendritic cells; FasL; TGF-β1 ; Gene modification; Liver transplantation

IL-32γ Induces the Maturation of Dendritic Cells with Th1- and Th17-Polarizing Ability through Enhanced IL-12 and IL-6 Production

IL-32, a newly described multifunctional cytokine, has been associated with a variety of inflammatory diseases, including rheumatoid arthritis, vasculitis, and Crohn's disease. In this study, we investigated the immunomodulatory effects of IL-32γ on bone marrow-derived dendritic cell (DC)-driven Th responses and analyzed the underlying signaling events. IL-32γ-treated DCs exhibited upregulated expression of cell-surface molecules and proinflammatory cytokines associated with DC maturation and activation. In particular, IL-32γ treatment significantly increased production of IL-12 and IL-6 in DCs, which are known as Th1- and Th17-polarizing cytokines, respectively. This increased production was inhibited by the addition of specific inhibitors of the activities of phospholipase C (PLC), JNK, and NF-κB. IL-32γ treatment increased the phosphorylation of JNK and the degradation of both IκBα and IκBβ in DCs, as well as NF-κB binding activity to the κB site. The PLC inhibitor suppressed NF-κB DNA binding activity and JNK phosphorylation increased by IL-32γ treatment, thereby indicating that IL-32γ induced IL-12 and IL-6 production in DCs via a PLC/JNK/NF-κB signaling pathway. Importantly, IL-32γ-stimulated DCs significantly induced both Th1 and Th17 responses when cocultured with CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb abolished the secretion of IFN-γ in a dose-dependent manner; additionally, the blockage of IL-1β and IL-6, but not of IL-21 or IL-23p19, profoundly inhibited IL-32γ-induced IL-17 production. These results demonstrated that IL-32γ could effectively induce the maturation and activation of immature DCs, leading to enhanced Th1 and Th17 responses as the result of increased IL-12 and IL-6 production in DCs.

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.

Intratumoral Expression of Mature Human Neutrophil Peptide-1 Mediates Antitumor Immunity in Mice

Human neutrophil peptides (HNP1-3), small molecular antimicrobial peptides, are expressed within tumors and associated with tumor necrosis and inhibition of angiogenesis. Recent investigations have suggested that HNP1-3 are likely to be involved in the host immune responses to tumors. We used recombinant pSec-HNP1, which expresses a secretable form of HNP1, to obtain expression of HNP1 in the tumor milieu in immunocompetent mice to explore the possible roles of HNP1 in tumor immunity. The antitumor effects were investigated in established CT26 colon cancer and 4T1 breast cancer models. HNP1-mediated chemotactic and activating effects on immature dendritic cells were detected both in vitro and in vivo. Intratumoral expression of HNP1 resulted in not only significant tumor growth inhibition but also increased CTL infiltration within tumors. Adoptive transfer of splenocytes and a (51)Cr release assay revealed specific cellular immune responses. Furthermore, increased antibodies were also found in sera from pSec-HNP1-treated mice supporting specific humoral immune responses. Increased apoptosis and decreased angiogenesis were also shown in treated tumors. These findings indicate that HNP1 can exert multiple antitumor effects through different mechanisms; more importantly, HNP1 mediates host immune responses to tumors in situ through the recruitment and subsequent activation of immature dendritic cells and thus shows promising potential in cancer therapy.

Polyamine compound deoxyspergualin inhibits heat shock protein-induced activation of immature dendritic cells

Polyamine compound deoxyspergualin (DSG) is a potent immunosuppressive agent that has been applied clinically for protecting graft rejection and treatment of Wegener's granulomatosis. Though DSG can bind to heat-shock proteins (HSPs) in cells, its mechanism of immunosuppressive action remains unknown. It is widely accepted that extracellular HSPs are capable of stimulating dendritic cells (DC) through cell surface receptors, leading to DC activation and cytokine release. In this study, we examined if DSG analogs could inhibit HSP70-induced DC activation. Bone marrow derived immature mouse DCs and peripheral blood mononuclear cell-derived immature human DCs were generated and incubated with Alexa 488-labeled Hsp70 in the presence of methoxyDSG (Gus-1) that had comparable HSP70-binding affinity to DSG or DSG analog GUS-7, which had much more reduced binding affinity for HSP70. The binding of HSP70 to immature DCs was analyzed by laser microscopy and flow cytometry. HSP70-induced DC activation was assessed by TNF-alpha release by enzyme-linked immunosorbent assay. Binding of Hsp70 to the cell surface of immature DCs was inhibited under the presence of Gus-1, but not under the presence of Gus-7. Immature DCs were activated and released TNF-alpha by the stimulation with HSP70 for 12 hours; however, the HSP70-induced TNF-alpha release was suppressed under the presence of Gus-1, and partially suppressed under the presence of Gus-7. Similar results were observed when immature human DCs were stimulated under the same conditions. Immunosuppressive mechanism of DSG may be explained, at least in part, by the inhibition of extracellular HSP70-DC interaction and HSP70-induced activation of immature DCs.

The Solution Structure and Dynamics of Human Pancreatic Ribonuclease Determined by NMR Spectroscopy Provide Insight into Its Remarkable Biological Activities and Inhibition

Human pancreatic ribonuclease (RNase 1) is expressed in many tissues; has several important enzymatic and biological activities, including efficient cleavage of single-stranded RNA, double-stranded RNA and double-stranded RNA–DNA hybrids, digestion of dietary RNA, regulation of vascular homeostasis, inactivation of the HIV, activation of immature dendritic cells and induction of cytokine production; and furthermore shows potential as an anti-tumor agent. The solution structure and dynamics of uncomplexed, wild-type RNase 1 have been determined by NMR spectroscopy methods to better understand these activities. The family of 20 structures determined on the basis of 6115 unambiguous nuclear Overhauser enhancements is well resolved (pairwise backbone RMSD = 1.07 Å) and has the classic RNase A type of tertiary structure. Important structural differences compared with previously determined crystal structures of RNase 1 variants or inhibitor-bound complexes are observed in the conformation of loop regions and side chains implicated in the enzymatic as well as biological activities and binding to the cytoplasmic RNase inhibitor. Multiple side chain conformations observed for key surface residues are proposed to be crucial for membrane binding as well as translocation and efficient RNA hydrolysis. 15N– 1H relaxation measurements interpreted with the standard and our extended Lipari–Szabo formalism reveal rigid regions and identify more dynamic loop regions. Some of the most dynamic areas are key for binding to the cytoplasmic RNase inhibitor. This finding and the important differences observed between the structure in solution and that bound to the inhibitor are indications that RNase 1 to inhibitor binding can be better described by the “induced fit” model rather than the rigid “lock-into-key” mechanism. Translational diffusion measurements reveal that RNase 1 is predominantly dimeric above 1 mM concentration; the possible implications of this dimeric state for the remarkable biological properties of RNase 1 are discussed.

Estrogen Modulates Bone Marrow-Derived DCs in SLE Murine Model-(NZB × NZW) F1 Female Mice

Dendritic cells (DCs) in the patient and animal models of systemic lupus erythematosus (SLE) are abnormal, but the detailed mechanism is unclear. Estrogen can modulate DCs in biological condition and estrogen consentration is related to the onset and development of SLE. So the control of estrogen on DCs might lead to the disorder of DCs. To prove the hypothesis, we detected the effects of 17β-estradiol (E2) on bone marrow (BM)-derived DCs in SLE murine model-(NZB × NZW) F1 (NZB/w F1) female mice before and after the disease onset. We found that E2 mainly enhanced the expression of surface molecule CD40, MHCII and the stimulation activity of immature DCs, but weakened the activity of mature DCs. E2 decreased the production of cytokines IL-6, IL-10, IL-12 and TNFα of DCs in young mice, but increased them in old mice. Tamoxifen could antagonize the E2 effect. E2 changed the expression of estrogen receptor-α (ERα) in DCs. The level of ERα in DCs of various old mice and the differentiation states varied. The results suggest that E2 can modulate the functions of BM-derived DCs in SLE pathology. The modulation is achieved by binding ER. The effects of E2 on DCs are different depending on the progression of SLE and cell differentiation status. This might be due to the difference of ER expression.

Neutrophils induce the maturation and activation of immature dendritic cells during adaptive immune responses

To investigate whether polymorphonuclear neutrophilic leucocyte (PMN) can induce the maturation and activation of immature dendritic cells, thus promoting the Th1 immunity to microbial pathogens in vitro. PMN isolated from mouse bone marrow were stimulated by lipopolysaccharide (LPS), and were cultured with immature DC (imDCs) isolated from mouse bone marrow for 18 hours. Then the analysis of DC surface markers CD40 and CD86 was done by flow cytometry, and ELISA was used to detect the levels of TNF-alpha and IL-12. Purified DC cocultured with LPS-PMN (PMN-DC) and imDC were incubated with fluorescein isothiocyanate conjugated OVA (FITC-OVA), and the phagocytic capacity of PMN-DC and imDC was assessed by flow cytometry. Splenic CD4(+) T cells from DO11.10 x C57BL/6 F1 hybrid mice were obtained, and then cultured with PMN-DC or imDC in the presence of OVA (323 - 339). The cells were double stained with anti-CD4-PE and 7-amino-actinomycin D. The cellular data were acquired for 56s with a flow cytometer and the assay of IFN-gamma and IL-4 was done by intracellular staining. LPS-PMN induced strong up-regulation of CD40 and CD86, and this capacity was remarkably inhibited after being neutralized by anti-TNF-alpha monoclonal antibody. LPS-PMN also stimulated imDCs to produce IL-12 and TNF-alpha. PMN-DCs demonstrated decreased phagocytic capacity compared with that of imDCs. Furthermore, the PMN-DC induced considerable DO11.10 T cell proliferation, and stimulated DO11.10 T cell to produce a large amount of IFN-gamma, but a relatively low amount of IL-4. LPS-PMN can induce the maturation and activation of imDCs, including the cytokine secretion, specific antigen presentation and Th1 differentiation, thus promoting the type I immunity to microbial pathogens.

Prolongation of skin graft survival by modulation of the alloimmune response with alternatively activated dendritic cells.

Activation of immature dendritic cells (DC) in the presence of the glucocorticoid hormone dexamethasone (DEX) results in alternatively matured DC that present antigen in the absence of a proper co-stimulatory context. This maturation process is irreversible, making these cells an attractive potential tool for the induction of antigen-specific T-cell tolerance in vivo. The authors explored the possibility of using these DC for the induction of transplantation tolerance in a fully allogeneic setting in mice. Immature dendritic cells (D1, an immature splenic DC line derived from B6 mice) were pretreated with DEX for 24 hr, after which lipopolysaccharide or nothing was added to the culture for another 48 hr. These cells were analyzed for their in vitro and in vivo stimulating or tolerizing capacities. In line with their phenotype, including decreased interleukin (IL)-12 production, in vitro co-culture of alternatively matured D1 (B6 origin; H-2b) with completely allogeneic T cells of BALB/c origin led to a significant decrease in the alloreactive T-cell response. A single injection of 1 x 10(6) alternatively matured H-2b DC into BALB/c mice induced a different alloimmune response compared with mature DC. The responding T cells showed a lower proliferation rate and a lower interferon-gamma production, whereas a significantly higher proportion of the cells produced IL-10 as measured ex vivo by enzyme-linked immunospot assay. Furthermore, injection with alternatively matured DC, followed by transplantation of fully mismatched skin grafts (C57BL/6), led to a significantly prolonged survival compared with that of mature DC-pretreated mice or untreated mice. The immunomodulatory effect was antigen specific, as third-party reactive alloresponses were not affected. The authors' data constitute the first direct demonstration that DC alternatively matured in the presence of glucocorticoid hormones can be exploited for the specific suppression of the alloreactive Th1 response, resulting in a delayed skin graft rejection in a complete major histocompatibility complex-incompatible strain combination.

Silent infection of bone marrow-derived dendritic cells by Leishmania mexicana amastigotes.

Resolution of infection by Leishmania sp. is critically dependent on activation of CD4(+) T helper cells. Naive CD4(+) T helper cells are primed by dendritic cells which have responded to an activation signal in the periphery. However, the role of Leishmania-infected dendritic cells in the activation of an anti-Leishmania immune response has not been comprehensively addressed. Using the highly controlled model system of bone marrow-derived dendritic cell infection by Leishmania mexicana cultured in vitro, we show that uptake of L. mexicana parasites does not result in activation of immature dendritic cells or secretion of IL-12. Incubation with L. mexicana promastigotes results in the activation of a small percentage of dendritic cells which do not appear to contain whole parasites. Activation of dendritic cells is not suppressed by infection, since infected cells can be fully activated on addition of activating stimuli. Therefore, uptake of intact Leishmania mexicana parasites is not sufficient to activate dendritic cells in vitro. We propose that these data provide a basis for interpreting the interactions between dendritic cells and all Leishmania sp.

A putative chemoattractant receptor, C5L2, is expressed in granulocyte and immature dendritic cells, but not in mature dendritic cells

Leukocyte chemoattractants are involved in the inflammatory response act via G protein-coupled receptors. We report the cloning of a novel human gene encoding the putative orphan receptor, C5L2, belonging to a subfamily of C3a, C5a and formyl Met-Leu-Ph receptors that are related to the chemokine receptor family. C5L2 transcript levels were abundant in granulocytes and immature dendritic cells but not in mature dendritic cells. We speculate that this receptor may regulate the activation of immature dendritic cells and play a role in the chemoattraction of leukocytes to inflammatory regions.