C1q inhibits immune complex–induced interferon‐α production in plasmacytoid dendritic cells: A novel link between C1q deficiency and systemic lupus erythematosus pathogenesis

Objective

C1q deficiency is the strongest risk factor known for the development of systemic lupus erythematosus (SLE), since almost all humans with a genetic deficiency of C1q develop this disease. Low C1q serum concentration is also a typical finding in SLE during flares, emphasizing the involvement of C1q in SLE pathogenesis. Recent studies have revealed that C1q has a regulatory effect on Toll‐like receptor–induced cytokine production. Therefore, we undertook this study to investigate whether C1q could regulate production of interferon‐α (IFNα).

Methods

Peripheral blood mononuclear cells (PBMCs) and plasmacytoid dendritic cells (PDCs) were stimulated with 3 known interferogenic stimuli and cultured with physiologic concentrations of C1q. IFNα production was determined by an immunoassay.

Results

C1q significantly inhibited PBMC IFNα production induced by RNA‐containing immune complexes (ICs), herpes simplex virus (HSV), and CpG DNA. C1q also inhibited PDC IFNα production induced by ICs and CpG DNA but increased PDC IFNα production induced by HSV. The regulatory role of C1q was not specific for IFNα but was also seen for interleukin‐6 (IL‐6), IL‐8, and tumor necrosis factor α. We demonstrated binding of C1q to PDCs both by surface plasmon resonance interaction analysis and by flow cytometry, and we also demonstrated intracellular detection of 2 C1q binding proteins.

Conclusion

Our findings contribute to the understanding of why C1q deficiency is such a strong risk factor for SLE and suggest an explanation for the up‐regulation of the type I IFN system seen in SLE patients.

     

C1q limits dendritic cell differentiation and activation by engaging LAIR-1

C1q, the first component of complement, and leukocyte-associated Ig-like receptor 1 (LAIR-1; CD305), an inhibitory receptor expressed on hematopoietic cells, have both been associated with arrest of monocyte-derived dendritic cell (DC) differentiation and inhibition of Toll-like receptor activity in plasmacytoid DCs. Defects in both molecules have been implicated in susceptibility to, and progression of, systemic lupus erythematosus. Inhibitory signaling partners for C1q on monocytes and DCs remain undefined. Because C1q contains collagen-like motifs and LAIR-1 is a universal collagen receptor, we hypothesized that C1q is a functional ligand for LAIR-1. Binding analyses in cell-free systems and on the cell membrane demonstrate that C1q and its collagen tail associate with LAIR-1 and LAIR-2 (CD306), a soluble inhibitor of LAIR-1. Both C1q and its collagen tail trigger phosphorylation of LAIR-1 immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in monocytes. Functional analyses show that C1q-mediated inhibition of monocyte-DC differentiation and C1q-mediated inhibition of IFN-α production by plasmacytoid DCs were both reversed by LAIR-2. Moreover, C1q-mediated inhibition of DC differentiation was reversed by LAIR-1 siRNA. Thus, C1q is a functional ligand for LAIR-1 restricting immune cell differentiation and activation. The discovery of C1q interactions with LAIR-1 and LAIR-2 lends much needed insight into molecular mechanisms operating to prevent the loss of tolerance, particularly in systemic lupus erythematosus.     

Levo- but not dextro-1-methyl tryptophan abrogates the IDO activity of human dendritic cells

Clinical trials have been started with the aim of inducing tumor immunity by blocking the immunosuppressive action of indoleamine-2,3-dioxygenase (IDO) with the IDO2-inhibitor dextro-1-methyl-tryptophan (D-1MT). Here we show that human dendritic cells (DCs) express both IDO-1 and IDO-2, but that only IDO1 mediates tryptophan catabolism; furthermore, its activity is blocked by levo-1MT, whereas D-1MT is inefficient. Consequently, in humans any possible antitumor effects of D-1MT cannot be attributed to abrogation of IDO activity in DCs as described in this study.     

Alternatively activated dendritic cells regulate CD4+ T-cell polarization in vitro and in vivo

Interleukin-4 is a cytokine widely known for its role in CD4(+) T cell polarization and its ability to alternatively activate macrophage populations. In contrast, the impact of IL-4 on the activation and function of dendritic cells (DCs) is poorly understood. We report here that DCs respond to IL-4 both in vitro and in vivo by expression of multiple alternative activation markers with a different expression pattern to that of macrophages. We further demonstrate a central role for DC IL-4Rα expression in the optimal induction of IFNγ responses in vivo in both Th1 and Th2 settings, through a feedback loop in which IL-4 promotes DC secretion of IL-12. Finally, we reveal a central role for RELMα during T-cell priming, establishing that its expression by DCs is critical for optimal IL-10 and IL-13 promotion in vitro and in vivo. Together, these data highlight the significant impact that IL-4 and RELMα can have on DC activation and function in the context of either bacterial or helminth pathogens.     

Differentiation of myeloid dendritic cells into CD8alpha-positive dendritic cells in vivo

Bone marrow-derived dendritic cells (DC) represent a family of antigen-presenting cells (APC) with varying phenotypes. For example, in mice, CD8alpha(+) and CD8alpha(-) DC are thought to represent cells of lymphoid and myeloid origin, respectively. Langerhans cells (LC) of the epidermis are typical myeloid DC; they do not express CD8alpha, but they do express high levels of myeloid antigens such as CD11b and FcgammaR. By contrast, thymic DC, which derive from a lymphoid-related progenitor, express CD8alpha but only low levels of myeloid antigens. CD8alpha(+) DC are also found in the spleen and lymph nodes (LN), but the origin of these cells has not been determined. By activating and labeling CD8alpha(-) epidermal LC in vivo, it was found that these cells expressed CD8alpha on migration to the draining LN. Similarly, CD8alpha(-) LC generated in vitro from a CD8 wild-type mouse and injected into the skin of a CD8alphaKO mouse expressed CD8alpha when they reached the draining LN. The results also show that CD8alpha(+) LC are potent APC. After migration from skin, they localized in the T-cell areas of LN, secreted high levels of interleukin-12, interferon-gamma, and chemokine-attracting T cells, and they induced antigen-specific T-cell activation. These results demonstrate that myeloid DC in the periphery can express CD8alpha when they migrate to the draining LN. CD8alpha expression on these DC appears to reflect a state of activation, mobilization, or both, rather than lineage. (Blood. 2000;96:1865-1872)     

DC-SIGN, C1q, and gC1qR form a trimolecular receptor complex on the surface of monocyte-derived immature dendritic cells

C1q modulates the differentiation and function of cells committed to the monocyte-derived dendritic cell (DC) lineage. Because the 2 C1q receptors found on the DC surface-gC1qR and cC1qR-lack a direct conduit into intracellular elements, we postulated that the receptors must form complexes with transmembrane partners. In the present study, we show that DC-SIGN, a C-type lectin expressed on DCs, binds directly to C1q, as assessed by ELISA, flow cytometry, and immunoprecipitation experiments. Surface plasmon resonance analysis revealed that the interaction was specific, and both intact C1q and the globular portion of C1q bound to DC-SIGN. Whereas IgG reduced this binding significantly, the Arg residues (162-163) of the C1q-A chain, which are thought to contribute to the C1q-IgG interaction, were not required for C1q binding to DC-SIGN. Binding was reduced significantly in the absence of Ca(2+) and by preincubation of DC-SIGN with mannan, suggesting that C1q binds to DC-SIGN at its principal Ca(2+)-binding pocket, which has increased affinity for mannose residues. Antigen-capture ELISA and immunofluorescence microscopy revealed that C1q and gC1qR associate with DC-SIGN on blood DC precursors and immature DCs. The results of the present study suggest that C1q/gC1qR may regulate DC differentiation and function through the DC-SIGN-mediated induction of cell-signaling pathways.     

树突状细胞体外分离扩增技术研究进展

树突状细胞（DC）是目前所知的体内功能最强的专职抗原呈递细胞,是机体免疫反应的始动者,被称为机体防御病原微生物侵袭的重要“哨兵”,在移植排斥、抗肿瘤、抗病毒感染和自身免疫疾病中发挥着重要作用。获得大量高纯度且表型、功能典型的DC是上述基础和临床研究的前提。从直接分离提取到添加细胞因子诱导扩增DC研究获得很大进展,成为当今免疫学及肿瘤治疗学等相关学科的研究热点。本文对DC体外分离、纯化及扩增方法作一简要综述。     

Functional comparison of thymic B cells and dendritic cells in vivo

In this report we present a transgenic mouse model in which we targeted gene expression specifically to B-lymphocytes. Using the human CD19 promoter, we expressed major histocompatibility complex class II I-E molecules specifically on B cells of all tissues, but not on other cell types. If only B cells expressed I-E in a class II-deficient background, positive selection of CD4(+) T cells could not be observed. A comparison of the frequencies of I-E reactive Vbeta5(+) and Vbeta11(+) T cells shows that I-E expression on thymic B cells is sufficient to negatively select I-E reactive CD4(+) T cells partially, but not CD8(+) T cells. Thus partial negative but no positive selection events can be induced by B-lymphocytes in vivo. (Blood. 2000;95:2610-2616)     

Maturation and function of human dendritic cells are regulated by B lymphocytes

Mature dendritic cells (DCs) are stimulators of T-cell immune response, whereas immature DCs support T-cell tolerance. Murine B cells can inhibit the production of IL-12 by DCs and thereby hinder the inflammatory response. Notwithstanding the importance of this modulation, only a few studies are available in humans. Here, we have developed an in vitro model of cocultures to assess its significance. We establish that human activated B cells restrained the development of monocytes into immature DCs and their differentiation into mature DCs. In addition, they decreased the density of HLA-DR from mature DCs, the expression of CD80 and CD86 coactivation molecules, the production of IL-12p70 required for antigen presentation and Th1 differentiation, and inhibited the DC-induced T-cell proliferation. These modulations were mediated by CD19(+)IgD(low)CD38(+)CD24(low)CD27(-) B cells and needed direct cell-to-cell contacts that involved CD62L for the control of CD80 and CD86 expression and a soluble factor for the control of IL-12 production. Moreover, mature DCs from patients with systemic lupus erythematosus displayed insensitivity to the regulation of IL-12. Overall, it appears that human B cells can regulate DC maturation and function and that inefficient B-cell regulation may influence an improper balance between an effector inflammatory response and tolerance induction.     

In vivo and in vitro observations on laminin production by Schwann cells.

Recent reports on the dystrophic mouse mutant suggest that the prominent extracellular matrix component of peripheral nerve tissues plays an important role in peripheral nerve development. We have examined the disposition of two prominent extracellular matrix components, fibronectin and laminin, both in mature peripheral nerve in vivo and in an in vitro system that allows study of Schwann cells in various functional states. In frozen sections of whole nerve, staining with antibodies to fibronectin and laminin shows that fibronectin stains throughout the endoneurium while laminin staining is restricted to regions known to contain basal lamina, particularly the basal lamina of each ensheathing Schwann cell. Tissue culture studies indicate that fibronectin staining at the light microscopic level is a reliable marker for fibroblasts (and not Schwann cells) in culture; conversely, antibodies to laminin stain components related to the Schwann cell surface but not components related to fibroblasts. Unexpectedly, Schwann cells in culture produce laminin at all stages in development, whether in contact with axons or not. As Schwann cells in culture begin to ensheathe axons, punctate regions of laminin on their surfaces become confluent. After ensheathment is completed, a continuous line of staining is found in the region of the Schwann cell basal lamina. It has been established that Schwann cells produce a basal lamina only when in contact with axons. Therefore, the production of laminin appears to be necessary but not sufficient for basal lamina formation. The constancy of laminin production by Schwann cells provides a reliable basis for distinguishing between Schwann cells and fibroblasts in tissue culture.     

Interferon gamma increases in vitro and in vivo expression of C1 inhibitor

C1 inhibitor (C1 INH) is the major protease inhibitor of the first components of the classic complement system and of the proteases of the Hageman factor pathways. Since C1 INH may modulate inflammatory reactions associated with complement and contact system activation, we sought to determine if the cytokine gamma interferon (IFN-gamma) could modulate C1 INH production. Initial studies investigated the effect of IFN-gamma on the molecular and protein expression of C1 INH in human erythroleukemia (HEL) cells. HEL cells constitutively expressed the 2.1 kb mRNA for C1 INH. IFN-gamma (50 to 1,000 U/mL), but not interferon alpha or beta, increased twofold the amount of C1 INH mRNA expressed within HEL cells. Similarly, this cytokine increased HEL cell C1 INH synthesis of a 105 Kd protein 10-fold, from 1.9 +/- 0.5 microgram C1 INH antigen per 10(8) cells (mean +/- SEM) to 19 +/- 8 micrograms/10(8) cells in 8 days. C1 INH produced by HEL cells after IFN-gamma stimulation had fully intact kallikrein neutralizing activity. Moreover, conditioned media of IFN-gamma-treated HEL cells accumulated more secreted C1 INH in 8 days (6.7 micrograms/mL/10(8) cells) than untreated cells (0.6 microgram/mL/10(8) cells). Additional studies were done on plasma specimens from 22 patients with metastatic colorectal carcinoma who received IFN-gamma daily for 4 days by intravenous infusion. Before treatment, the mean +/- SEM C1 INH levels in these patients was 438 +/- 16 micrograms/mL. At day 10 from the start of the infusion, the plasma C1 INH in these patients increased to 586 +/- 32 micrograms/mL (P less than .0001). The extent of rise of plasma C1 INH after IFN-gamma treatment was independent of dose from 0.01 to 40 U/m2. After 30 days, the mean plasma C1 INH levels decreased to 502 +/- 27 micrograms/mL. These combined studies indicate that IFN-gamma can increase C1 INH protein expression in vitro and in vivo.     

Functional comparison of spleen dendritic cells and dendritic cells cultured in vitro from bone marrow precursors

We have compared dendritic cells (DC) isolated from mouse spleen, or generated in vitro from bone marrow (BM) precursors cultured in granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), for the ability to process and present soluble antigen and stimulate major histocompatibility complex (MHC) Class II- restricted T cells. DC from spleen or BM cultures were equally able to stimulate the in vitro proliferation of allogeneic T cells or of antigen-specific T-cell receptor (TCR)-transgenic T cells. Both DC populations also induced comparable levels of IL-2 secretion by a T- cell hybridoma. Therefore, splenic and BM-derived DC express comparable levels of (Antigen + MHC Class II) ligands and/or costimulatory molecules and have comparable ability to stimulate T-cell responses. When presentation of a native protein antigen, rather than peptide, was evaluated, BM-derived DC were at least 50 times better than splenic DC at stimulating the proliferation of TCR-transgenic T cells. The antigen processing ability of the two populations was similar only when splenic DC were used immediately ex vivo. Therefore, unlike spleen DC, BM- derived DC maintain the capacity to process protein antigen for MHC Class II presentation during in vitro culture. Due to these characteristics, BM-derived DC may represent a useful tool in immunotherapy studies, as they combine high T-cell stimulatory properties with the capacity to process and present native antigen.     

Selective Rac1 inhibition in dendritic cells diminishes apoptotic cell uptake and cross-presentation in vivo

To better understand the influence of cytoskeletal regulation on dendritic cell (DC) function in vivo, the Rho guanosine triphosphatase (GTPase) Rac1 was selectively inhibited in DCs in transgenic (Tg) mice. Although transgene expression did not interfere with the migratory capacities of DC in vivo, a decreased uptake of fluorescent probes was observed. Interestingly, the absence of full Rac1 function most strongly affected the development and function of CD8+ DCs. Apoptotic cell uptake was severely reduced in Tg mice, impairing subsequent DC-mediated cross-presentation and priming of bacteria-specific T-cell responses. These findings highlight a special role for Rac1 in the capacity of CD8+ DCs to endocytose apoptotic cells and prime T cells via cross-presentation.     

树突状细胞负载肝癌相关抗原后成熟调控的研究

目的 研究树突状细胞（DC）负载肝癌相关抗原后的成熟调控。方法 用SDS－PAGE制备电泳纯化牛结核分枝杆菌热休克蛋白70，用其诱导DC的分化与成熟。结果 DC负载肝癌可溶性抗原后，10％2细胞失去了DC特征，同时其表面CD54（90．0％），CD83（78．0％），CD86（85．0％）分子表达下降；牛分枝杆菌卡介苗－热休克蛋白70（BCG HSP70）的活化有利于负载肝癌可溶性抗原后的DC维持其特异性标志，同时DC表面CD54（92．0％），CD83（90．0％），CD86（91．0％）分子表达增加，DC诱导同种异体淋巴细胞转化的能力增加，淋巴细胞增殖加快，从而促进DC成熟，增加其抗原呈递能力。结论 预示BCG HSP70有可能成为促进DC活化和成熟的另一重要分子。     

Murine dendritic cells pulsed with whole tumor lysates mediate potent antitumor immune responses in vitro and in vivo

The highly efficient nature of dendritic cells (DC) as antigen-presenting cells raises the possibility of uncovering in tumor-bearing hosts very low levels of T cell reactivity to poorly immunogenic tumors that are virtually undetectable by other means. Here, we demonstrate the in vitro and in vivo capacities of murine bone marrow-derived, cytokine-driven DC to elicit potent and specific anti-tumor responses when pulsed with whole tumor lysates. Stimulation of naive spleen-derived T cells by tumor lysate-pulsed DC generated tumor-specific proliferative cytokine release and cytolytic reactivities in vitro. In addition, in two separate strains of mice with histologically distinct tumors, s.c. injections of DC pulsed with whole tumor lysates effectively primed these animals to reject subsequent lethal challenges with viable parental tumor cells and, important to note, also mediated significant reductions in the number of metastases established in the lungs. Tumor rejection depended on host-derived CD8⁺ T cells and, to a lesser extent, CD4⁺ T cells. Spleens from mice that had rejected their tumors contained specific precursor cytotoxic T lymphocytes. The use of whole tumor lysates as a source of tumor-associated antigen(s) for pulsing of DC circumvents several limitations encountered with other methods as well as provides certain distinct advantages, which are discussed. These data serve as rationale for our recent initiation of a phase I clinical trial of immunization with autologous tumor lysate-pulsed DC in adult and pediatric cancer patients.     

Conditioned medium from HeLa cells enhances motility of human monocyte-derived dendritic cells but abrogates their maturation and endocytic activity

Progressive tumor proliferation may be associated with suppression of the immune response. Several different mechanisms can contribute to immune evasion. It is generally proposed that inhibition of dendritic cell functions would be a key mechanism by which tumors could escape immune surveillance. Therefore, the purpose of this study was to evaluate the capacity of HeLa cells conditioned medium (HeLa-CM) to modulate phenotypic and functional parameters of human peripheral blood monocyte-derived dendritic cells (DCs). Two types of reference DCs population were generated in vitro, the first cultured in the presence of IL-4 and GM-CSF which represented immature DCs (iDCs) and the second, representing mature DCs (mDCs), was raised from the iDCs by additional stimulation with a maturation cocktail - TNF-alpha, IL-1beta, IL-6, PGE2. In parallel, the iDCs were treated with HeLa-CM collected from the tumor cells. The analysis of DC populations demonstrated that the HeLa-CM prevented maturation of these cells and also impaired their capacity to uptake an antigen and stimulate proliferation of allogeneic T cells. In contrast, HeLa-CM modulated DCs exhibited a 3-fold increase mobility over iDCs. The latter functional capacity did not correlate with the levels of matrix metalloproteinase expression in the analysed cells. Altogether, our results provide evidence that HeLa cells produce soluble factors that might dramatically alter basic phenotypic and functional characteristics of DCs.     

Distinct contributions of TNF and LT cytokines to the development of dendritic cells in vitro and their recruitment in vivo

TNF/LTalpha/LTbeta (tumor necrosis factor/lymphotoxin-alpha/lymphotoxin-beta) triple knockout (KO) mice show a significant reduction of dendritic cell (DC) number in the spleen, presumably due to defective recruitment and/or production. To distinguish between these possibilities, DCs were generated from bone marrow (BM) cultures prepared from wild-type (wt) and mutant mice in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The yield of CD11c(+) major histocompatibility complex (MHC) class II(+) DCs generated from TNF/LTalpha/LTbeta(-/-) BM culture was significantly reduced compared with wt BM culture. In order to further dissect the individual pathways responsible for defective DC properties observed in TNF/LTalpha/LTbeta(-/-) mice, the panel of TNF/LT ligand and receptor single KO mice were used. The production of DCs from BM culture was significantly reduced in TNF(-/-) and TNF receptor (TNFR) p55(-/-) mice, but normal in LTalpha(-/-), LTbeta(-/-), LTbetaR(-/-) mice. Recombinant TNF (rTNF) exogenously added to TNF/LTalpha/LTbeta(-/-) BM cultures could reverse this defect, and blocking antibodies showed partial effect on BM cultures of wt mice. Conversely, numbers of mature DCs in spleen were significantly decreased in LTalpha(-/-), LTbeta(-/-), LTbetaR(-/-) mice, but not in TNF(-/-) and TNFRp55(-/-) mice. These results reveal 2 distinct contributions of TNF/LT cytokines. First, TNF acting through TNF receptor is involved in the development/maturation of DCs in BM progenitor cultures, but this function appears to be redundant in vivo. Second, the microenvironment in peripheral lymphoid organs associated with LTalpha/LTbeta-LTbetaR signaling and chemokine production is critical for recruitment efficiency of DCs, and this pathway is indispensable.