Regulated expression of FcgammaR in human dendritic cells controls cross-presentation of antigen-antibody complexes

Receptors for IgG (FcgammaR) expressed in dendritic cells (DCs) influence the initiation of Ab-mediated immunity. Dynamic variations in FcgammaR expression allow DCs to adjust their capacity to capture Ab-opsonized Ag. The current paradigm predicts a progressive decline in FcgammaR-mediated phagocytic function upon DC maturation. Surprisingly, we find that expression of the phagocytic receptor FcgammaRIIa is preserved in immature and mature DCs at comparable levels with macrophages. Moreover, phagocytosis of antigenic peptides directed to FcgammaRIIa on DCs leads to dramatic increases in Ag cross-presentation and T cell activation. In immature DCs, high expression of inhibitory FcgammaRIIb correlates with decreased uptake and cross-presentation of Ab-Ag complexes. In contrast, engagement of FcgammaRIIb is not associated with changes in cross-presentation in mature DCs. We provide evidence that FcgammaRIIb expression is patently reduced in mature DCs, an effect that is modulated by treatment with cytokines. The regulated expression of activating and inhibitory FcgammaRs in DCs emerges as a critical checkpoint in the process of Ag uptake and cross-presentation.     

A promoter haplotype of the immunoreceptor tyrosine-based inhibitory motif-bearing FcgammaRIIb alters receptor expression and associates with autoimmunity. I. Regulatory FCGR2B polymorphisms and their association with systemic lupus erythematosus

FcgammaRIIb, the immunoreceptor tyrosine-based inhibitory motif-containing receptor for IgG (Mendelian Inheritance in Man no. 604590), plays an important role in maintaining the homeostasis of immune responses. We have identified 10 novel single-nucleotide polymorphisms in the promoter region of human FCGR2B gene and characterized two functionally distinct haplotypes in its proximal promoter. In luciferase reporter assays, the less frequent promoter haplotype leads to increased expression of the reporter gene in both B lymphoid and myeloid cell lines under constitutive and stimulated conditions. Four independent genome-wide scans support linkage of the human FcgammaR region to the systemic lupus erythematosus (SLE; Online Mendelian Inheritance in Man no. 152700) phenotype. Our case-control study in 600 Caucasians indicates a significant association of the less frequent FCGR2B promoter haplotype with the SLE phenotype (odds ratio = 1.65; p = 0.0054). The FCGR2B haplotype has no linkage disequilibrium with previously identified FCGR2A and FCGR3A polymorphisms, and after adjustment for FCGR2A and FCGR3A, FCGR2B showed a persistent association with SLE (odds ratio = 1.72; p = 0.0083). These results suggest that an expression variant of FCGR2B is a risk factor for human lupus and implicate FCGR2B in disease pathogenesis.     

Regulated expression and inhibitory function of Fcgamma RIIb in human monocytic cells.

Human monocytes/macrophages express three classes of receptors for IgG: FcgammaRI, FcgammaRII, and FcgammaRIII. The expression and function of these receptors has been extensively studied with the exception of one, FcgammaRIIb. While the mRNA for FcgammaRIIb has been detected in human monocytes, the protein has remained elusive. Studies in mouse models indicated that the macrophage FcgammaRIIb serves to down-regulate FcgammaR-mediated phagocytosis and immune complex-induced inflammation. FcgammaRIIb has also been shown to modulate the action of cytotoxic antibodies against tumors in mouse models. Hence, an understanding of how FcgammaRIIb expression is regulated is of great importance. Here we demonstrate for the first time FcgammaRIIb protein expression and function in human monocytes. We also report that the expression of FcgammaRIIb is highly up-regulated by interleukin-4, a Th2 cytokine, and that the up-regulation of FcgammaRIIb results in a decrease in the phagocytic efficiency of interleukin-4-treated THP-1 cells. Furthermore co-clustering FcgammaRIIb with FcgammaRIIa resulted in enhanced phosphorylation of the inositol phosphatase SHIP, association of SHIP with Shc, and phosphorylation of additional proteins around 120 and 60-65 kDa, with a concomitant attenuation of Akt activation. We, therefore, propose that FcgammaRIIb serves to inhibit FcgammaRI/IIa-mediated macrophage activation using SHIP as its effector.     

In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo.     

TGF-beta 1 suppresses [correction of supresses] myeloid Fc gamma receptor function by regulating the expression and function of the common gamma-subunit

We have previously reported that FcgammaR-mediated function in myeloid cells is a tightly regulated event that is influenced by the cytokines present in the milieu. TGF-beta1 is an immunosuppressive cytokine with pleiotropic effects on immune responses; however, the molecular mechanism by which TGF-beta suppresses immune responses is poorly understood. In this study, we have analyzed the effect of TGF-beta on FcgammaR-mediated activation of myeloid cells. We report that TGF-beta1-treated THP-1 human myeloid cells displayed reduced ability to phagocytose IgG-coated particles. Because FcgammaR expression is modulated by cytokines, we analyzed expression levels of FcgammaRI, FcgammaRIIa, FcgammaRIIb, and FcgammaRIIIa in cells cultured with or without TGF-beta1 and found while total protein levels of the FcgammaR were not reduced, surface expression of FcgammaRI and FcgammaRIII was lower in cells cultured with TGF-beta1. Concomitantly, there was a dose-dependent reduction in the expression of the FcgammaR-associated gamma-subunit. This suppressive effect of TGF-beta was likewise observed in bone marrow-derived murine myeloid cells and human monocytes. Importantly, TGF-beta1 also significantly reduced the production of monocyte chemoattractant protein-1 induced by immobilized IgG, which would further reduce monocyte recruitment to the site of inflammation. In contrast, human alveolar macrophages were refractory to this effect, expressing low levels of TGF-beta type II receptors compared with peripheral blood monocytes from the same donor. These data provide insight into the regulation of immune responses by TGF-beta1 and demonstrate the selectivity of these effects.     

Defective expression and function of the leukocyte associated Ig-like receptor 1 in B lymphocytes from systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is characterized by the production of a wide array of autoantibodies and dysregulation of B cell function. The leukocyte associated Immunoglobulin (Ig)-like receptor (LAIR)1 is a transmembrane molecule belonging to Ig superfamily which binds to different types of collagen. Herein, we have determined the expression and function of LAIR1 on B lymphocyte from SLE patients. LAIR1 expression in peripheral blood B lymphocytes from 54 SLE, 24 mixed connective tissue disease (MCTD), 20 systemic sclerosis (SSc) patients, 14 rheumatoid arthritis (RA) and 40 sex and age matched healthy donors (HD) have been analyzed by immunofluorescence. The effect of LAIR1 ligation by specific monoclonal antibodies, collagen or collagen producing mesenchymal stromal cells from reactive lymph nodes or bone marrow on Ig production by pokeweed mitogen and B cell receptor (BCR)-mediated NF-kB activation was assessed by ELISA and TransAM assay. The percentage of CD20(+) B lymphocytes lacking or showing reduced expression of LAIR1 was markedly increased in SLE and MCTD but not in SSc or RA patients compared to HD. The downregulation of LAIR1 expression was not dependent on corticosteroid therapy. Interestingly, LAIR1 engagement by collagen or collagen-producing mesenchymal stromal cells in SLE patients with low LAIR1 expression on B cells delivered a lower inhibiting signal on Ig production. In addition, NF-kB p65 subunit activation upon BCR and LAIR1 co-engagement was less inhibited in SLE patients than in HD. Our findings indicate defective LAIR1 expression and function in SLE B lymphocytes, possible contributing to an altered control of B lymphocytes behavior.     

Fc gamma RIIa is expressed on natural IFN-alpha-producing cells (plasmacytoid dendritic cells) and is required for the IFN-alpha production induced by apoptotic cells combined with lupus IgG

An ongoing production of IFN-alpha may be of etiopathogenic significance in systemic lupus erythematosus (SLE). It may be due to the natural IFN-producing cells (NIPC), also termed plasmacytoid dendritic cells (PDC), activated by immune complexes that contain nucleic acids derived from apoptotic cells. We here examined the role of FcgammaR in the IFN-alpha production in vitro by PBMC induced by the combination of apoptotic U937 cells and autoantibody-containing IgG from SLE patients (SLE-IgG). The Fc portion of the SLE-IgG was essential to induce IFN-alpha production, because Fab fragments or F(ab')(2) were ineffective. Normal, especially heat-aggregated, IgG inhibited the IFN-alpha production, suggesting a role for FcgammaR on PBMC. Using blocking anti-FcgammaR Abs, the FcgammaRIIa,c (CD32) but not FcgammaRI or FcgammaRIII were shown to be involved in the IFN-alpha induction by apoptotic cells combined with SLE-IgG, but not by HSV or CpG DNA. In contrast, the action of all of these inducers was inhibited by the anti-FcgammaRIIa,b,c mAb AT10 or heat-aggregated IgG. Flow cytometric analysis revealed that approximately 50% of the BDCA-2-positive PBMC, i.e., NIPC/PDC, expressed low but significant levels of FcgammaRII, as did most of the actual IFN-alpha producers activated by HSV. RT-PCR applied to NIPC/PDC purified by FACS demonstrated expression of FcgammaRIIa, but not of FcgammaRIIb or FcgammaRIIc. We conclude that FcgammaRIIa on NIPC/PDC is involved in the activation of IFN-alpha production by interferogenic immune complexes, but may also mediate inhibitory signals. The FcgammaRIIa could therefore have a key function in NIPC/PDC and be a potential therapeutic target in SLE.     

The intracellular granzyme B inhibitor,proteinase inhibitor 9, is up-regulated during accessory cell maturation and effector cell degranulation,and its overexpression enhances CTL potency

Granzyme B (grB) is a serine proteinase released by cytotoxic lymphocytes (CLs) to kill abnormal cells. GrB-mediated apoptotic pathways are conserved in nucleated cells; hence, CLs require mechanisms to protect against ectopic or misdirected grB. The nucleocytoplasmic serpin, proteinase inhibitor 9 (PI-9), is a potent inhibitor of grB that protects cells from grB-mediated apoptosis in model systems. Here we show that PI-9 is present in CD4(+) cells, CD8(+) T cells, NK cells, and at lower levels in B cells and myeloid cells. PI-9 is up-regulated in response to grB production and degranulation, and associates with grB-containing granules in activated CTLs and NK cells. Intracellular complexes of PI-9 and grB are evident in NK cells, and overexpression of PI-9 enhances CTL potency, suggesting that cytoplasmic grB, which may threaten CL viability, is rapidly inactivated by PI-9. Because dendritic cells (DCs) acquire characteristics similar to those of target cells to activate naive CD8(+) T cells and therefore may also require protection against grB, we investigated the expression of PI-9 in DCs. PI-9 is evident in thymic DCs (CD3(-), CD4(+), CD8(-), CD45(+)), tonsillar DCs, and DC subsets purified from peripheral blood (CD16(+) monocytes and CD123(+) plasmacytoid DCs). Furthermore, PI-9 is expressed in monocyte-derived DCs and is up-regulated upon TNF-alpha-induced maturation of monocyte-derived DCs. In conclusion, the presence and subcellular localization of PI-9 in leukocytes and DCs are consistent with a protective role against ectopic or misdirected grB during an immune response.     

Change in expression of MGMT during maturation of human monocytes into dendritic cells

Dendritic cells (DCs) maturated from monocytes play an important role in the immune system, not only in defense against conventional infections but also in cancer rejection. Because of the central role of DCs in tumor host defense it is highly important that DCs as well as the progenitor cell population are protected during cancer therapy. Since most anticancer drugs target DNA, the DNA repair capacity is most importance for the response of DCs and their precursor cells. Here, we studied the expression of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) in monocytes obtained from peripheral blood of healthy donors and DCs maturated from monocytes (moDCs). We show that MGMT is expressed at high level in monocytes, comparable to peripheral lymphocytes. The MGMT expression level declines, however, during DC maturation reaching the low level of CD34+ haematopoetic stem cells. Decline of MGMT was observed on activity, protein and RNA level. It is not related to MGMT promoter methylation, suggesting silencing of the MGMT gene in moDCs occurs by other means. Since maturation of monocytes into DCs is provoked by IL-4 and GM-CSF, the data indicate that MGMT is subject to cytokine-mediated regulation. Despite of the high MGMT level, monocytes were more sensitive to methylating agents (MNNG, temozolomide) and equally sensitive to the chloroethylating agent fotemustine than moDCs, undergoing apoptosis upon treatment. The data provide an example that high MGMT expression level does not necessarily implicate a higher level of resistance against O6-alkylating agents.     

Flt3 ligand preferentially increases the number of functionally active myeloid dendritic cells in the lungs of mice

In the present study, we investigated the effects of in vivo Flt3L administration on the generation, phenotype, and function of lung dendritic cells (DCs) to evaluate whether Flt3L favors the expansion and maturation of a particular DC subset. Injection of Flt3L into mice resulted in an increased number of CD11c-expressing lung DCs, preferentially in the alveolar septa. FACS analysis allowed us to quantify a 19-fold increase in the absolute numbers of CD11c-positive, CD45R/B220 negative DCs in the lungs of Flt3L-treated mice over vehicle-treated mice. Further analysis revealed a 90-fold increase in the absolute number of myeloid DCs (CD11c positive, CD45R/B220 negative, and CD11b positive) and only a 3-fold increase of lymphoid DCs (CD11c positive, CD45R/B220 negative, and CD11b negative) from the lungs of Flt3L-treated mice over vehicle-treated mice. Flt3L-treated lung DCs were more mature than vehicle-treated lung DCs as demonstrated by a significantly higher percentage of cells expressing MHC class II, CD86, and CD40. Freshly isolated Flt3L lung DCs were not fully mature, because after an overnight culture they continued to increase accessory molecule expression. Functionally, Flt3L-treated lung DCs were more efficient than vehicle-treated DCs at stimulating naive T cell proliferation. Our data show that administration of Flt3L favors the expansion of myeloid lung DCs over lymphoid DCs and enhanced their ability to stimulate naive lymphocytes.     

Neutrophils and B cells express XCR1 receptor and chemotactically respond to lymphotactin

The C chemokine lymphotactin (Lptn) has been reported to act specifically on CD4(+) and CD8(+) T lymphocytes and natural killer (NK) cells, but not monocytes. However, the chemotactic effect of Lptn on other types of hematopoietic cells has not been well studied. In this study we investigated (i) the chemotactic influences of Lptn on T and B lymphocytes, neutrophils, monocytes, and dendritic cells, and (ii) the expression of the Lptn receptor (XCR1) by these cells, using RT-PCR. Our data showed that Lptn is chemotactic for B lymphocytes and neutrophils as well as T lymphocytes, but not for monocytes or dendritic cells, and that XCR1 expression is found only in association with T and B lymphocytes and neutrophils, but not monocytes or dendritic cells. Thus, this study is the first demonstration of a chemotactic effect of Lptn on neutrophils and confirms the association of this effect with expression of the XCR1 receptor on these cells. These data suggest that Lptn could potentially be an important protein in the regulation of T and B lymphocytes and neutrophil trafficking, and thereby also their roles in inflammatory and immunological responses.     

Down-regulated NOD2 by immunosuppressants in peripheral blood cells in patients with SLE reduces the muramyl dipeptide-induced IL-10 production

Background

Pattern recognition receptors (PRRs) such as Toll-like receptors are aberrantly expressed of peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients, for playing immunopathological roles.

Methodology/Principal Findings

We investigated the expression and function of the PRR nucleotide-binding oligomerization domain (NOD2) in SLE. NOD2 expression in T, B lymphocytes, monocytes, myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) was assessed in SLE patients and healthy controls (HCs) using flow cytometric analysis. Ex vivo production of cytokines from PBMCs upon NOD2 agonist muramyl dipeptide (MDP) stimulation was assessed using Cytometric Bead Array. Over-expression of NOD2 in monocytes was observed in immunosuppressant naïve SLE patients, and was positively associated with longer disease duration. Immunosuppressive therapy was an independent explanatory variable for downregulating NOD2 expression in CD8+ T, monocytes, mDCs and pDCs. Ex vivo basal productions of cytokines (IL-6, IL-8 and IL-10) were significantly increased in immunosuppressant naïve patients and patients with active disease despite immunosuppressants compared with HCs. Upon MDP stimulaiton, relative induction (%) of cytokines (IL-1β) from PBMC was significantly increased in immunosuppressant naïve patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with HCs. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naïve patients and HCs.

Conclusions/Significance

Bacterial exposure may increase the NOD2 expression in monocytes in immunosuppressant naïve SLE patients which can subsequently lead to aberrant activation of PBMCs to produce proinflammatory cytokines, implicating the innate immune response for extracellular pathogens in the immunopathological mechanisms in SLE. Immunosuppressant therapy may downregulate NOD2 expression in CD8+ T lymphocytes, monocytes, and DCs in SLE patients which subsequently IL-10 reduction, contributing towards the regulation of immunopathological mechanisms of SLE, at the expense of increasing risk of bacterial infection.     

Increased lymphocytic aminopeptidase N/CD13 promoter activity after cell-cell contact

Aminopeptidase N (APN)/CD13 is a transmembrane ectoenzyme expressed on a wide variety of cells. With respect to haematopoietic cells, APN/CD13 has been considered specific for the myeloid lineage, because granulocytes and monocytes/macrophages, but not lymphocytes of peripheral blood, show a surface expression of CD13 antigen. However, we could recently show that cell-cell contact of lymphocytes with endothelial cells, monocytes, and fibroblast-like synoviocytes (SFCs) results in an increase of steady-state APN/CD13 mRNA and a rapid expression of cell-surface protein on the lymphocytes. In this study using the Dual-Luciferase reporter assay, we demonstrate that interaction of the T-cell line Jurkat with SFCs results in a higher activity of the APN/CD13 myeloid promoter in T cells. An enhancer located between the myeloid and epithelial APN/CD13 promoter increases the response of the promoter to the cell-cell contact-induced expression of APN/CD13 in lymphocytes. Adhesion of lymphocytes to extracellular matrix did not result in increased promoter activity. The lymphocytic promoter response induced by direct cell-cell contact with SFCs is not affected by mutations of a proximal promoter element (nucleotides -48 to -35), which has a possible functional role in the basal APN/CD13 gene expression in lymphocytes. Upregulated peptidase-promoter activity via cell-cell contact shown in this study for the first time is discussed as a general mechanism in peptidase induction.     

Cutting edge: the role of IFN-α receptor and MyD88 signaling in induction of IL-15 expression in vivo

IL-15 plays a multifaceted role in immune homeostasis, but the unreliability of IL-15 detection has stymied exploration of IL-15 regulation in vivo. To visualize IL-15 expression, we created a transgenic mouse expressing emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP/IL-15 was prevalent in innate cells including dendritic cells (DCs), macrophages, and monocytes. However, DC subsets expressed varying levels of EmGFP/IL-15 with CD8(+) DCs constitutively expressing EmGFP/IL-15 and CD8(-) DCs expressing low EmGFP/IL-15 levels. Virus infection resulted in IL-15 upregulation in both subsets. By crossing the transgenic mice to mice deficient in specific elements of innate signaling, we found a cell-intrinsic dependency of DCs and Ly6C(+) monocytes on IFN-α receptor expression for EmGFP/IL-15 upregulation after vesicular stomatitis virus infection. In contrast, myeloid cells did not require the expression of MyD88 to upregulate EmGFP/IL-15 expression. These findings provide evidence of previously unappreciated regulation of IL-15 expression in myeloid lineages during homeostasis and following infection.     

Redox remodeling by dendritic cells protects antigen-specific T cells against oxidative stress

Microorganisms and microbial products induce the release of reactive oxygen species (ROS) from monocytes and other myeloid cells, which may trigger dysfunction and apoptosis of adjacent lymphocytes. Therefore, T cell-mediated immunity is likely to comprise mechanisms of T cell protection against ROS-inflicted toxicity. The present study aimed to clarify the dynamics of reduced sulfhydryl groups (thiols) in human T cells after presentation of viral and bacterial Ags by dendritic cells (DCs) or B cells. DCs, but not B cells, efficiently triggered intra- and extracellular thiol expression in T cells with corresponding Ag specificity. After interaction with DCs, the Ag-specific T cells acquired the capacity to neutralize exogenous oxygen radicals and resisted ROS-induced apoptosis. Our results imply that DCs provide Ag-specific T cells with antioxidative thiols during Ag presentation, which suggests a novel aspect of DC/T cell cross-talk of relevance to the maintenance of specific immunity in inflamed or infected tissue.