Sialylation of IgG inhibits the formation of galactose-deficient IgA1-containing immune complexes and protects mesangial cells from injury in IgA nephropathy.

BACKGROUND: The addition of sialic acid alters IgG from a pro-inflammatory state to an anti-inflammatory state. However, there is a lack of research on the changes of IgG sialylation in IgA nephropathy (IgAN). METHODS: This study included a total of 184 IgAN patients. The sialylated IgG (SA-IgG), IgG-galactose-deficient IgA1 complex (IgG-Gd-IgA1-IC), IL-6, TNF-α, and TGF-ß were detected using commercial ELISA kits. SA-IgG, non-sialylated IgG (NSA-IgG), sialylated IgG-IgA1 complex (SA-IgG-IgA1), and non-sialylated IgG-IgA1 complex (NSA-IgG-IgA1) were purified from IgAN patients and healthy controls (HCs). RESULTS: The mean SA-IgG levels in plasma and B lymphocytes in IgAN patients were significantly higher than those of healthy controls. A positive correlation was found between SA-IgG levels in plasma and B lymphocytes. In vitro, the results showed that the release of IgG-Gd-IgA1-IC was significantly decreased in peripheral blood mononuclear cells (PBMCs) cultured with SA-IgG from both IgAN patients and healthy controls. The proliferation ability and the release of IL-6, TNF-α, and TGF-ß in human mesangial cells (HMCs) were measured after stimulating with SA-IgG-IgA1-IC and NSA-IgG-IgA1-IC. The mesangial cell proliferation levels induced by NSA-IgG-IgA1-IC derived from IgAN patients were significantly higher than those caused by SA-IgG-IgA1-IC derived from IgAN patients and healthy controls. Compared with NSA-IgG-IgA1 from healthy controls, IgAN-NSA-IgG-IgA1 could significantly upregulate the expression of IL-6 and TNF-α in mesangial cells. The data showed that there weren't any significant differences in the levels of IL-6, TNF-α, and TGF-ß when treated with IgAN-SA-IgG-IgA1 and HC-NSA-IgG-IgA1. CONCLUSIONS: The present study demonstrated that the sialylation of IgG increased in patients with IgA nephropathy. It exerted an inhibitory effect on the formation of Gd-IgA1-containing immune complexes in PBMCs and the proliferation and inflammation activation in mesangial cells.

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells.

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Immune complex-induced neutrophil functions: A focus on cell death.

Neutrophils are amongst the first cells to be recruited to sites of infection or trauma. Neutrophil functional responsiveness is tightly regulated by many agents including immune complexes. These immune cells can generate reactive oxygen species and degranulate to release abundant cytotoxic products, making them efficient at killing invading microorganisms. If neutrophil function is dysregulated, however, these cells have the potential to cause unwanted host tissue damage as exemplified by pathological and chronic inflammatory conditions. In physiological inflammation, once the initial insult has efficiently been dealt with, neutrophils are thought to leave the tissues or undergo programmed cells death, especially apoptosis. Apoptotic neutrophils are then rapidly removed by other phagocytes, primarily macrophages, by mechanisms that do not elicit a pro-inflammatory response. In this review, we discuss the interesting observations and consequences that immune complexes have on neutrophil cell death processes such as apoptosis.

Posible papel de Porphyromonas gingivalis en el desarrollo de la artritis reumatoide / Possible role of Porphyromonas gingivalis in the develpment of rheumatoid arthritis

Varios estudios han sugerido una asociación entre la periodontitissevera, la prevalencia de la bacteria Porphyromonas gingivalis y el desarrollo de artritis reumatoide. Como fundamento de esta relación, se ha observado que esta bacteria secreta una enzima, peptidil-arginina deiminasa, que es capaz de citrulinar proteínas del hospedero y así favorecer una respuesta autoinmune. Sin embargo, debido a la heterogeneidad de diseños experimentales, selección de pacientes y valoración de los desenlaces, los resultados no han mostrado la reproducibilidad deseada. Asimismo, observaciones recientes apuntan a que la actividad enzimática podría ser generada por otras especies bacterianas, lo que hace más compleja su relación. Sin embargo, por otro lado, algunos estudios sugieren que el tratamiento periodontal puede limitar el desarrollo de la artritis reumatoide.

Various studies have suggested a link between severe periodontitis,the prevalence of Porphyromonas gingivalis, and the development ofrheumatoid arthritis. As evidence of this relationship, P. gingivalis hasbeen found to secrete an enzyme, peptidyl arginine deiminase, which isable to citrullinate host proteins and thus help activate an autoimmuneresponse. However, due to the heterogeneity of experimental designs,patient selection, and assessment of clinical outcomes, the results havenot shown the desired reproducibility. Furthermore, recent fi ndingsindicate that the enzymatic activity may be produced by other species ofbacteria, which suggests the relationship is more complex. However, anumber of studies have shown that periodontal treatment could inhibitthe development of rheumatoid arthritis.

Microparticles That Form Immune Complexes as Modulatory Structures in Autoimmune Responses.

Microparticles (MPs) are induced during apoptosis, cell activation, and even "spontaneous" release. Initially MPs were considered to be inert cellular products with no biological function. However, an extensive research and functional characterization have shown that the molecular composition and the effects of MPs depend upon the cellular background and the mechanism inducing them. They possess a wide spectrum of biological effects on intercellular communication by transferring different molecules able to modulate other cells. MPs interact with their target cells through different mechanisms: membrane fusion, macropinocytosis, and receptor-mediated endocytosis. However, when MPs remain in the extracellular milieu, they undergo modifications such as citrullination, glycosylation, and partial proteolysis, among others, becoming a source of neoantigens. In rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), reports indicated elevated levels of MPs with different composition, content, and effects compared with those isolated from healthy individuals. MPs can also form immune complexes amplifying the proinflammatory response and tissue damage. Their early detection and characterization could facilitate an appropriate diagnosis optimizing the pharmacological strategies, in different diseases including cancer, infection, and autoimmunity. This review focuses on the current knowledge about MPs and their involvement in the immunopathogenesis of SLE and RA.

Optimization of a flow cytometric assay to evaluate the human neutrophil ability to phagocytose immune complexes via Fcγ and complement receptors.

INTRODUCTION: This study aims to optimize some experimental conditions of a flow cytometric assay to examine the human neutrophil ability to phagocytose immune complexes (ICs) via Fcγ and complement receptors (FcγR and CR, respectively). The parameters assessed were: number of cells, concentration of ICs, reaction time, pH and concentration of the Trypan Blue quenching solution. METHODS: Neutrophils were isolated from peripheral blood of healthy volunteers. Precipitated ICs composed of IgG and fluorescein isothiocyanate (FITC)-labeled ovalbumin, opsonized or not with serum complement, were used to trigger the neutrophil phagocytosis via FcγR, CR, and FcγR+CR. Fluorescence of the internalized ICs was measured by flow cytometry, after quenching the extracellular fluorescence with Trypan Blue. RESULTS: The optimal experimental conditions established for the phagocytosis assay were: 1 × 10(6) cells mL(-1) and 40 µg mL(-1) FITC-labeled ICs, incubated for 30 min, at 37°C, in 0.5 mL of reaction volume. Trypan Blue solution at 1.25 mg mL(-1) pH4.4 was the best fluorescence quencher of FITC-labeled ICs attached to the outer surface of neutrophils. DISCUSSION: The selected experimental conditions were viable to evaluate IC phagocytosis by neutrophils; they are also suitable to compare the efficiency of IC phagocytosis mediated by FcγR and CR classes of membrane receptors, alone or in combination. This method finds application in studies of (i) the receptor-specific phagocytic function of normal and pathogenic neutrophils as well as (ii) the impact of drugs and therapies on this essential effector function of neutrophils.

IgGs are made for walking on bacterial and viral surfaces.

Binding of antibodies to their cognate antigens is fundamental for adaptive immunity. Molecular engineering of antibodies for therapeutic and diagnostic purposes emerges to be one of the major technologies in combating many human diseases. Despite its importance, a detailed description of the nanomechanical process of antibody-antigen binding and dissociation on the molecular level is lacking. Here we utilize high-speed atomic force microscopy to examine the dynamics of antibody recognition and uncover a principle; antibodies do not remain stationary on surfaces of regularly spaced epitopes; they rather exhibit 'bipedal' stochastic walking. As monovalent Fab fragments do not move, steric strain is identified as the origin of short-lived bivalent binding. Walking antibodies gather in transient clusters that might serve as docking sites for the complement system and/or phagocytes. Our findings could inspire the rational design of antibodies and multivalent receptors to exploit/inhibit steric strain-induced dynamic effects.

Myristoylated Alanine Rich C Kinase Substrate (MARCKS) is essential to ß2-integrin dependent responses of equine neutrophils.

Neutrophil infiltration is a prominent feature in a number of pathologic conditions affecting horses including recurrent airway obstruction, ischemia-reperfusion injury, and laminitis. Cell signaling components involved in neutrophil migration represent targets for novel anti-inflammatory therapies. In order to migrate into tissue, neutrophils must respond to chemoattractant signals in their external environment through activation of adhesion receptors (i.e. integrins) and reorganization of the actin cytoskeleton. Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS), a highly conserved actin-binding protein, has a well demonstrated role in cytoskeletal dependent cellular functions (i.e. adhesion, spreading, and migration), but the details of MARCKS involvement in these processes remain vague. We hypothesized that MARCKS serves as a link between the actin cytoskeleton and integrin function in neutrophils. Using a MARCKS-specific inhibitor peptide known as MANS on equine neutrophils in vitro, we demonstrate that inhibition of MARCKS function significantly attenuates ß2-integrin-dependent neutrophil functions including migration, adhesion, and immune complex-mediated respiratory burst. The MANS peptide did not, however, inhibit the ß2-integrin-independent PMA mediated respiratory burst. These results attest to the essential role of MARCKS function in regulating neutrophil responses, and strongly implicate MARCKS as a potential regulator of ß2-integrins in neutrophils.

Neonatal Fc receptor promotes immune complex-mediated glomerular disease.

The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex-mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex-mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG.

IL-2/anti-IL-2 complex attenuates renal ischemia-reperfusion injury through expansion of regulatory T cells.

Regulatory T cells (Tregs) can suppress immunologic damage in renal ischemia-reperfusion injury (IRI), but the isolation and ex vivo expansion of these cells for clinical application remains challenging. Here, we investigated whether the IL-2/anti-IL-2 complex (IL-2C), a mediator of Treg expansion, can attenuate renal IRI in mice. IL-2C administered before bilateral renal IRI induced Treg expansion in both spleen and kidney, improved renal function, and attenuated histologic renal injury and apoptosis after IRI. Furthermore, IL-2C administration reduced the expression of inflammatory cytokines and attenuated the infiltration of neutrophils and macrophages in renal tissue. Depletion of Tregs with anti-CD25 antibodies abrogated the beneficial effects of IL-2C. However, IL-2C-mediated renal protection was not dependent on either IL-10 or TGF-ß. Notably, IL-2C administered after IRI also enhanced Treg expansion in spleen and kidney, increased tubular cell proliferation, improved renal function, and reduced renal fibrosis. In conclusion, these results indicate that IL-2C-induced Treg expansion attenuates acute renal damage and improves renal recovery in vivo, suggesting that IL-2C may be a therapeutic strategy for renal IRI.

Toll-like receptor 2 controls acute immune complex-driven arthritis in mice by regulating the inhibitory Fcγ receptor IIB.

OBJECTIVE: Previous studies have demonstrated a protective role of Toll-like receptor 2 (TLR-2) and a proinflammatory function of TLR-4 during chronic T cell-driven arthritis. The involvement of TLRs in T cell-independent arthritic processes, however, remains unclear. This study was undertaken to determine the functional significance of TLR-2 and TLR-4 in T cell-independent immune complex-driven arthritis. METHODS: Serum-transfer arthritis was induced in wild-type and TLR-deficient mice by intraperitoneal injections of arthritogenic K/BxN mouse serum. Arthritis was assessed macroscopically and by histologic analysis. The influence of TLR-2 on macrophage cytokine profile, Fcγ receptor (FcγR) expression, and response to immune complexes was determined. RESULTS: While TLR-4, unexpectedly, did not play any significant role, TLR-2 deficiency accelerated the onset and markedly increased the severity of acute immune complex-driven arthritis in mice. TLR-2 deficiency resulted in a substantial increase in joint inflammation, bone erosion, and cartilage pathology, indicating a protective function of TLR-2 in passive FcγR-driven disease. Ex vivo study of the macrophage inflammatory phenotype revealed increased production of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) despite similar levels of IL-10, along with a significant increase in FcγR-specific response, in TLR-2-/- mouse macrophages early in the disease. Although distinct FcγR messenger RNA expression was not affected, cell surface protein expression of the inhibitory FcγRIIB in TLR-2-/- naive primary macrophages was specifically diminished, resulting in a higher proinflammatory response. Accordingly, TLR-2 stimulation specifically up-regulated FcγRIIB, but not the activating FcγR, on macrophages. CONCLUSION: TLR-2 regulates acute immune complex-driven arthritis by controlling macrophage FcγR response. Our findings indicate that the protective role of TLR-2 is extended beyond its previously described role in promoting Treg cells during T cell-mediated arthritis.

Interleukin-33 primes mast cells for activation by IgG immune complexes.

Mast cells (MCs) are heterogeneous cells whose phenotype is modulated by signals received from the local microenvironment. Recent studies have identified the mesenchymal-derived cytokine IL-33 as a potent direct activator of MCs, as well as regulator of their effector phenotype, and have implicated this activity in the ability of mast cells to contribute to murine experimental arthritis. We explored the hypothesis that IL-33 enables participation of synovial MCs in murine K/BxN arthritis by promoting their activation by IgG immune complexes. Compared to wild-type (WT) control mice, transgenic animals lacking the IL-33 receptor ST2 exhibited impaired MC-dependent immune complex-induced vascular permeability (flare) and attenuated K/BxN arthritis. Whereas participation of MCs in this model is mediated by the activating IgG receptor FcγRIII, we pre-incubated bone marrow-derived MCs with IL-33 and found not only direct induction of cytokine release but also a marked increase in FcγRIII-driven production of critical arthritogenic mediators including IL-1ß and CXCL2. This "priming" effect was associated with mRNA accumulation rather than altered expression of Fcγ receptors, could be mimicked by co-culture of WT but not ST2(-/-) MCs with synovial fibroblasts, and was blocked by antibodies against IL-33. In turn, WT but not ST2(-/-) MCs augmented fibroblast expression of IL-33, forming a positive feedback circuit. Together, these findings confirm a novel role for IL-33 as an amplifier of IgG immune complex-mediated inflammation and identify a potential MC-fibroblast amplification loop dependent on IL-33 and ST2.

Noncovalent assembly of anti-dendritic cell antibodies and antigens for evoking immune responses in vitro and in vivo.

Targeting of Ags directly to dendritic cells (DCs) through anti-DC receptor Ab fused to Ag proteins is a promising approach to vaccine development. However, not all Ags can be expressed as a rAb directly fused to a protein Ag. In this study, we show that noncovalent assembly of Ab-Ag complexes, mediated by interaction between dockerin and cohesin domains from cellulose-degrading bacteria, can greatly expand the range of Ags for this DC-targeting vaccine technology. rAbs with a dockerin domain fused to the rAb H chain C terminus are efficiently secreted by mammalian cells, and many Ags not secreted as rAb fusion proteins are readily expressed as cohesin directly fused to Ag either via secretion from mammalian cells or as soluble cytoplasmic Escherichia coli products. These form very stable and homogeneous complexes with rAb fused to dockerin. In vitro, these complexes can efficiently bind to human DC receptors followed by presentation to Ag-specific CD4⁺ and CD8⁺ T cells. Low doses of the HA1 subunit of influenza hemagglutinin conjugated through this means to anti-Langerin rAbs elicited Flu HA1-specific Ab and T cell responses in mice. Thus, the noncovalent assembly of rAb and Ag through dockerin and cohesin interaction provides a useful modular strategy for development and testing of prototype vaccines for elicitation of Ag-specific T and B cell responses, particularly when direct rAb fusions to Ag cannot be expressed.

Inhibitory activity of liposomal flavonoids during oxidative metabolism of human neutrophils upon stimulation with immune complexes and phorbol ester.

CONTEXT AND OBJECTIVE: The massive production of reactive oxygen species by neutrophils during inflammation may cause damage to tissues. Flavonoids act as antioxidants and have anti-inflammatory effects. In this study, liposomes loaded with these compounds were evaluated as potential antioxidant carriers, in attempt to overcome their poor solubility and stability. MATERIALS AND METHODS: Liposomes containing quercetin, myricetin, kaempferol or galangin were prepared by the ethanol injection method and analyzed as inhibitors of immune complex (IC) and phorbol ester-stimulated neutrophil oxidative metabolism by luminol (CLlum) and lucigenin-enhanced (CLluc) chemiluminescence (CL) assays. The mechanisms involved this activity of liposomal flavonoids, such as cytotoxicity and superoxide anion scavenging capacity, and their effect on phagocytosis of ICs were also investigated. RESULTS AND DISCUSSION: The results showed that the inhibitory effect of liposomal flavonoids on CLlum and CLluc is inversely related to the number of hydroxyl groups in the flavonoid B ring. Moreover, phagocytosis of liposomes by neutrophils does not seem to necessarily promote such activity, as the liposomal flavonoids are also able to reduce CL when the cells are pretreated with cytochalasin B. Under assessed conditions, the antioxidant liposomes are not toxic to the human neutrophils and do not interfere with IC-induced phagocytosis. CONCLUSION: The studied liposomes can be suitable carriers of flavonoids and be an alternative for the treatment of diseases in which a massive oxidative metabolism of neutrophils is involved.

Immune complexes and late complement proteins trigger activation of Syk tyrosine kinase in human CD4(+) T cells.

In systemic lupus erythematosus (SLE), the autoantibodies that form immune complexes (ICs) trigger activation of the complement system. This results in the formation of membrane attack complex (MAC) on cell membrane and the soluble terminal complement complex (TCC). Hyperactive T cell responses are hallmark of SLE pathogenesis. How complement activation influences the T cell responses in SLE is not fully understood. We observed that aggregated human γ-globulin (AHG) bound to a subset of CD4(+) T cells in peripheral blood mononuclear cells and this population increased in the SLE patients. Human naive CD4(+) T cells, when treated with purified ICs and TCC, triggered recruitment of the FcRγ chain with the membrane receptor and co-localized with phosphorylated Syk. These events were also associated with aggregation of membrane rafts. Thus, results presented suggest a role for ICs and complement in the activation of Syk in CD4(+) T cells. Thus, we propose that the shift in signalling from ζ-chain-ZAP70 to FcRγ chain-Syk observed in T cells of SLE patients is triggered by ICs and complement. These results demonstrate a link among ICs, complement activation and phosphorylation of Syk in CD4(+) T cells.

Plasmacytoid dendritic cells and C1q differentially regulate inflammatory gene induction by lupus immune complexes.

Immune complexes (ICs) play a pivotal role in causing inflammation in systemic lupus erythematosus (SLE). Yet, it remains unclear what the dominant blood cell type(s) and inflammation-related gene programs stimulated by lupus ICs are. To address these questions, we exposed normal human PBMCs or CD14(+) isolated monocytes to SLE ICs in the presence or absence of C1q and performed microarray analysis and other tests for cell activation. By microarray analysis, we identified genes and pathways regulated by SLE ICs that are both type I IFN dependent and independent. We also found that C1q-containing ICs markedly reduced expression of the majority of IFN-response genes and also influenced the expression of multiple other genes induced by SLE ICs. Surprisingly, IC activation of isolated CD14(+) monocytes did not upregulate CD40 and CD86 and only modestly stimulated inflammatory gene expression. However, when monocyte subsets were purified and analyzed separately, the low-abundance CD14(dim) ("patrolling") subpopulation was more responsive to ICs. These observations demonstrate the importance of plasmacytoid dendritic cells, CD14(dim) monocytes, and C1q as key regulators of inflammatory properties of ICs and identify many pathways through which they act.

[The activation of TRIF-dependent signaling pathway in THP-1 cells induced by ß2 GPI/anti-ß2 GPI antibodies complex].

AIM: To observe whether the TIR-domain-containing adaptor inducing interferon-ß (TRIF) is activated in THP-1 cells treated with ß2 GPI/anti-ß2 GPI complex and investigate the roles of TRIF-dependent signaling pathway of Toll-like receptor 4 (TLR4) in antiphospholipid syndrome (APS). METHODS: The total RNA was extracted and the protein lysates were collected from THP-1 cells stimulated with ß2 GPI/anti-ß2 GPI complex. And the level of TRIF mRNA in THP-1 cells was detected by Real-time PCR (RT-PCR), TRIF protein expression was investigated by western blotting, respectively. Furthermore, whether TLR4 inhibitor, TAK-242, could interrupt the expression of TRIF as well as some inflammatory cytokines such as IL-6, IL-8 and TNF-α in THP-1 cells stimulated with ß2 GPI/anti-ß2 GPI complex was also investigated. RESULTS: Both mRNA and protein levels of TRIF could be significantly increased in THP-1 cells with treatment of ß2 GPI/anti-ß2 GPI complex (100 mg/L). The expression of TRIF was shown in a manner of time-dependence, with the maximal levels at 1 hour (mRNA) and 2 hour (protein) stimulation respectively. The ß2 GPI/anti-ß2 GPI complex-induced TRIF and inflammatory cytokines including IL-6, IL-8 and TNF-α expression in THP-1 cells could be inhibited by TAK-242 (5 µmol/L). CONCLUSION: TRIF-dependent signaling pathway of Toll-like receptor 4 is involved in the activation of THP-1 cells induced by ß2 GPI/anti-ß2GPI complex, suggesting that TRIF may play an important role in the pathogenesis of antiphospholipid syndrome.