<Emphasis Type="Italic">Glycine tomentella</Emphasis> Hayata inhibits IL-1β and IL-6 production,inhibits MMP-9 activity,and enhances RAW264.7 macrophage clearance of apoptotic cells

Background  

To assess the effects of Glycine tomentella Hayata (GTH), a traditional herbal medicine for treatment of rheumatic diseases on the expression of the proinflammatory cytokines and on the clearance of apoptotic cells by macrophages.     

Accelerated Fas-mediated apoptosis of monocytes and maturing macrophages from patients with systemic lupus erythematosus: relevance to in vitro impairment of interaction with iC3b-opsonized apoptotic cells.

Impaired handling of apoptotic cells has been suggested as an important factor in the development of systemic lupus erythematosus (SLE), and a role for complement in the removal of apoptotic cells was shown recently. We studied the in vitro function of macrophages from 40 patients with SLE and their matched controls in the removal of heterologous apoptotic cells opsonized by iC3b. Interaction index of apoptotic cells opsonized by iC3b was significantly lower in patients with SLE and averaged 71% +/- 37 of that of healthy individuals (p < 0.002) and 69% +/- 35 of patients with rheumatoid arthritis (p < 0.007). SLE patients had increased apoptosis of both freshly isolated monocytes (p < 0.001) and maturing macrophages (p < 0.04) that led to decreased density of monocyte-derived macrophages. Apoptosis was inhibited by adding soluble Fas receptor indicating Fas-mediated apoptosis. As demonstrated in both healthy controls and patients with SLE, decreased macrophage density by itself caused significant decreased uptake of apoptotic cells by the remaining macrophages. Maintaining normal density in SLE patients either by an increased initial density or by using soluble Fas restored the interaction capacity of the individual macrophages in the majority of patients. We concluded that impaired in vitro interaction of iC3b-opsonized apoptotic cells with macrophages from patients with SLE was mainly associated with Fas-dependent accelerated apoptosis of the monocytes/macrophages. Accelerated apoptosis of phagocytes may represent a novel in vitro mechanism of impairment of interaction with apoptotic cells that, apart from reducing the number of professional phagocytes, alters the function of the remaining macrophages.     

High mobility group box 1 (HMGB1) and anti-HMGB1 antibodies and their relation to disease characteristics in systemic lupus erythematosus

Introduction  

High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein. HMGB1, which is secreted by inflammatory cells and passively released from apoptotic and necrotic cells, may act as a pro-inflammatory mediator. As apoptotic cells accumulate in systemic lupus erythematosus (SLE), HMGB1 levels might be increased in SLE. HMGB1 may also serve as an autoantigen, leading to the production of anti-HMGB1 antibodies. In this study we determined levels of HMGB1 and anti-HMGB1 in SLE patients in comparison to healthy controls (HC) and analysed their relation with disease activity.     

Is disturbed clearance of apoptotic keratinocytes responsible for UVB-induced inflammatory skin lesions in systemic lupus erythematosus?

Apoptotic cells are thought to play an essential role in the pathogenesis of systemic lupus erythematosus (SLE). We hypothesise that delayed or altered clearance of apoptotic cells after UV irradiation will lead to inflammation in the skin of SLE patients. Fifteen SLE patients and 13 controls were irradiated with two minimal erythemal doses (MEDs) of ultraviolet B light (UVB). Subsequently, skin biopsies were analysed (immuno)histologically, over 10 days, for numbers of apoptotic cells, T cells, macrophages, and deposition of immunoglobulin and complement. Additionally, to compare results with cutaneous lesions of SLE patients, 20 biopsies of lupus erythematosus (LE) skin lesions were analysed morphologically for apoptotic cells and infiltrate. Clearance rate of apoptotic cells after irradiation did not differ between patients and controls. Influx of macrophages in dermal and epidermal layers was significantly increased in patients compared with controls. Five out of 15 patients developed a dermal infiltrate that was associated with increased epidermal influx of T cells and macrophages but not with numbers of apoptotic cells or epidermal deposition of immunoglobulins. Macrophages were ingesting multiple apoptotic bodies. Inflammatory lesions in these patients were localised near accumulations of apoptotic keratinocytes similar as was seen in the majority of LE skin lesions. In vivo clearance rate of apoptotic cells is comparable between SLE patients and controls. However, the presence of inflammatory lesions in the vicinity of apoptotic cells, as observed both in UVB-induced and in LE skin lesions in SLE patients, suggests that these lesions result from an inflammatory clearance of apoptotic cells.     

Impaired Clearance of Early Apoptotic Cells Mediated by Inhibitory IgG Antibodies in Patients with Primary Sj?gren's Syndrome

Objectives

Deficient efferocytosis (i.e. phagocytic clearance of apoptotic cells) has been frequently reported in systemic lupus erythematosus (SLE). Todate, patients with primary Sjögren''s syndrome (SS) have not been assessed for phagocytosis of apoptotic cells (ApoCell-phagocytosis) and of particulate targets (microbeads, MB-phagocytosis).

Design

ApoCell-phagocytosis and MB-phagocytosis were comparatively assessed by flow cytometry in peripheral blood specimens and monocyte-derived macrophage (MDM) preparations from healthy blood donors (HBD) and consecutive SS, SLE and rheumatoid arthritis (RA) patients. Cross-admixture ApoCell-phagocytosis experiments were also performed using phagocytes from HBD or patients, and apoptotic cells pretreated with whole sera or purified serum IgG derived from patients or HBD.

Results

Compared to HBD, approximately half of SS and SLE patients studied (but not RA) manifested significantly reduced ApoCell-phagocytosis (p<0.001) and MB-phagocytosis (p<0.003) by blood-borne phagocytes that correlated inversely with disease activity (p≤0.004). In cross-admixture assays, healthy monocytes showed significantly reduced ApoCell-phagocytosis when fed with apoptotic cells that were pretreated with sera or purified serum IgG preparations from SS and SLE patients (p<0.0001, compared to those from HBD or RA). Such aberrant effect of the SS and SLE sera and IgG preparations correlated linearly with their content of IgG antibodies against apoptotic cells (p≤0.0001). Phagocytic dysfunction maybe also present in certain SS and SLE patients, as supported by deficient capacity of MDM for ApoCell-phagocytosis and MB-phagocytosis under patients'' serum-free conditions.

Conclusion

Similarly to SLE, efferocytosis is frequently impaired in SS and is primarily due to the presence of inhibitory IgG anti-ApoCell antibodies and secondarily to phagocytes'' dysfunction.     

The phosphatidylserine receptor from <Emphasis Type="Italic">Hydra</Emphasis> is a nuclear protein with potential Fe(II) dependent oxygenase activity

Background  

Apoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR) and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts.     

Epithelial cell-directed efferocytosis in the post-partum mammary gland is necessary for tissue homeostasis and future lactation

Background  

Mammary glands harbor a profound burden of apoptotic cells (ACs) during post-lactational involution, but little is known regarding mechanisms by which ACs are cleared from the mammary gland, or consequences if this process is interrupted. We investigated AC clearance, also termed efferocytosis, during post-lactational remodeling, using mice deficient for MerTK, Axl, and Tyro3, three related receptor tyrosine kinases (RTKs) regulating macrophage-mediated efferocytosis in monocytes. MerTK expression, apoptosis and the accumulation of apoptotic debris were examined in histological sections of MerTK-deficient, Axl/Tyro3-deficient, and wild-type mammary glands harvested at specific time points during lactation and synchronized involution. The ability of primary mammary epithelial cells (MECs) to engulf ACs was assessed in culture. Transplant of MerTK-deficient mammary epithelium into cleared WT mammary fat pads was used to assess the contribution of WT mammary macrophages to post-lactational efferocytosis.     

Increased plasma levels of the soluble Mer tyrosine kinase receptor in systemic lupus erythematosus relate to disease activity and nephritis

Introduction  

Mer and Tyro3 are receptor tyrosine kinases important for the phagocytosis of apoptotic cells. Together with Axl, they constitute the TAM receptor family. These receptors can be shed from the cell membrane and their soluble extracellular regions can be found in plasma. The objective of this study was to elucidate whether the plasma levels of soluble Mer (sMer) and Tyro3 (sTyro3) were increased in systemic lupus erythematosis (SLE), rheumatoid arthritis (RA), or critical limb ischemia (CLI).     

IgM promotes the clearance of small particles and apoptotic microparticles by macrophages

Background

Antibodies are often involved in enhancing particle clearance by macrophages. Although the mechanisms of antibody-dependent phagocytosis have been studied for IgG in greater detail, very little is known about IgM-mediated clearance. It has been generally considered that IgM does not support phagocytosis. Recent studies indicate that natural IgM is important to clear microbes and other bioparticles, and that shape is critical to particle uptake by macrophages; however, the relevance of IgM and particle size in their clearance remains unclear. Here we show that IgM has a size-dependent effect on clearance.

Methodology/Principal Findings

We used antibody-opsonized sheep red blood cells, different size beads and apoptotic cells to determine the effect of human and mouse IgM on phagocytosis by mouse alveolar macrophages. Our microscopy (light, epifluorescence, confocal) and flow cytometry data show that IgM greatly enhances the clearance of small particles (about 1–2 micron) by these macrophages. There is an inverse relationship between IgM-mediated clearance by macrophages and the particle size; however, macrophages bind and internalize many different size particles coated with IgG. We also show that IgM avidly binds to small size late apoptotic cells or bodies (2–5 micron) and apoptotic microparticles (<2 µm) released from dying cells. IgM also promotes the binding and uptake of microparticle-coated beads.

Conclusions/Significance

Therefore, while the shape of the particles is important for non-opsonized particle uptake, the particle size matters for antibody-mediated clearance by macrophages. IgM particularly promotes the clearance of small size particles. This finding may have wider implications in IgM-mediated clearing of antigens, microbial pathogens and dying cells by the host.     

Mannose-Binding Lectin Blunts Macrophage Polarization and Ameliorates Lupus Nephritis

Background

Deficiency in clearance of self nuclear antigens, including DNA, is the hallmark of systemic lupus erythematosus (SLE), a chronic autoimmnue disease characterized by the production of various autoantibodies, immune complex deposition and severe organ damage. Our previous studies revealed that administration of syngeneic BALB/c mice with activated lymphocyte-derived DNA (ALD-DNA) could induce SLE disease. Mannose-binding lectin (MBL), a secreted pattern recognition receptor with binding activity to DNA, has been proved to be a modulator of inflammation, but whether MBL takes responsibility for DNA clearance, modulates the DNA-mediated immune responses, and is involved in the development of DNA-induced SLE disease remain poorly understood.

Methodology/Principal Findings

The levels of serum MBL significantly decreased in lupus mice induced by ALD-DNA and were negatively correlated with SLE disease. MBL blunted macrophage M2b polarization by inhibiting the MAPK and NF-κB signaling while enhancing the activation of CREB. Furthermore, MBL suppressed the ability of ALD-DNA–stimulated macrophages to polarize T cells toward Th1 cells and Th17 cells. Importantly, MBL supplement in vivo could ameliorate lupus nephritis.

Conclusion/Significance

These results suggest MBL supplement could alleviate SLE disease and might imply a potential therapeutic strategy for DNA-induced SLE, which would further our understanding of the protective role of MBL in SLE disease.     

Is disturbed clearance of apoptotic keratinocytes responsible for UVB-induced inflammatory skin lesions in systemic lupus erythematosus?

Apoptotic cells are thought to play an essential role in the pathogenesis of systemic lupus erythematosus (SLE). We hypothesise that delayed or altered clearance of apoptotic cells after UV irradiation will lead to inflammation in the skin of SLE patients. Fifteen SLE patients and 13 controls were irradiated with two minimal erythemal doses (MEDs) of ultraviolet B light (UVB). Subsequently, skin biopsies were analysed (immuno)histologically, over 10 days, for numbers of apoptotic cells, T cells, macrophages, and deposition of immunoglobulin and complement. Additionally, to compare results with cutaneous lesions of SLE patients, 20 biopsies of lupus erythematosus (LE) skin lesions were analysed morphologically for apoptotic cells and infiltrate. Clearance rate of apoptotic cells after irradiation did not differ between patients and controls. Influx of macrophages in dermal and epidermal layers was significantly increased in patients compared with controls. Five out of 15 patients developed a dermal infiltrate that was associated with increased epidermal influx of T cells and macrophages but not with numbers of apoptotic cells or epidermal deposition of immunoglobulins. Macrophages were ingesting multiple apoptotic bodies. Inflammatory lesions in these patients were localised near accumulations of apoptotic keratinocytes similar as was seen in the majority of LE skin lesions. In vivo clearance rate of apoptotic cells is comparable between SLE patients and controls. However, the presence of inflammatory lesions in the vicinity of apoptotic cells, as observed both in UVB-induced and in LE skin lesions in SLE patients, suggests that these lesions result from an inflammatory clearance of apoptotic cells.     

Association between monocyte Fcγ subclass expression and acute coronary syndrome

Background  

Atherosclerosis lesions contain abundant immunoglobulins complexed with oxidized LDL (OxLDL) that are endocytosed by macrophages to form foam cells. While recent evidence supports a role for the macrophage scavenger receptor pathway in 75–90% of OxLDL uptake, in vitro evidence suggests another potential uptake pathway could involve autoantibody binding to IgG subclass-specific Fc receptors.     

Poractant alfa (Curosurf?) increases phagocytosis of apoptotic neutrophils by alveolar macrophages in vivo

Background

Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e.g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf^®) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages.

Methods

Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages.

Results

Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage.

Conclusions

We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.     

A novel receptor – ligand pathway for entry of <Emphasis Type="Italic">Francisella tularensis</Emphasis> in monocyte-like THP-1 cells: interaction between surface nucleolin and bacterial elongation factor Tu

Background  

Francisella tularensis, the causative agent of tularemia, is one of the most infectious human bacterial pathogens. It is phagocytosed by immune cells, such as monocytes and macrophages. The precise mechanisms that initiate bacterial uptake have not yet been elucidated. Participation of C3, CR3, class A scavenger receptors and mannose receptor in bacterial uptake have been already reported. However, contribution of an additional, as-yet-unidentified receptor for F. tularensis internalization has been suggested.     

Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity

Introduction

CD200 is a type I transmembrane glycoprotein that can regulate the activation threshold of inflammatory immune responses, polarize cytokine production, and maintain immune homeostasis. We therefore evaluated the functional status of CD200/CD200 receptor 1 (CD200R1) interactions in subjects with systemic lupus erythematosus (SLE).

Methods

Serum CD200 level was detected by ELISA. The expression of CD200/CD200R1 by CD4⁺ T cells and dendritic cells (DCs) was examined by flow cytometry, and then compared between SLE patients and healthy controls. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidyl ester and annexin V/propidium iodide for evaluation of the effect of CD200 on cell proliferation and apoptosis. In addition, the effect of CD200 on DC function was determined by transwell migration assay as well as by measurement of binding and phagocytosis of apoptotic cells.

Results

In SLE patients, the number of CD200⁺ cells and the level of soluble CD200 were significantly higher than in healthy controls, whereas the expression of CD200R1 by CD4⁺ T cells and DCs was decreased. Furthermore, the increased CD200 expression by early apoptotic cells contributed to their diminished binding and phagocytosis by DCs in SLE. Importantly, the engagement of CD200 receptor on CD4⁺ T cells with CD200-Fc fusion protein in vitro reduced the differentiation of T-helper type 17 cells and reversed the defective induction of CD4⁺CD25^highFoxP3⁺ T cells by transforming growth factor beta in SLE patients. Conversely, blockade of CD200-CD200R1 interaction with anti-CD200R1 antibody promoted CD4⁺ T-cell proliferation.

Conclusion

CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in SLE.     

Increased expression of Mer tyrosine kinase in circulating dendritic cells and monocytes of lupus patients: correlations with plasma interferon activity and steroid therapy

Introduction

The requirement for the immunoregulatory Mer tyrosine kinase (Mer) for optimal removal of apoptotic cells prompted us to look at its expression in systemic lupus erythematosus (SLE), in which apoptotic cell clearance is abnormal. We compared the levels of expression of Mer in normal human subjects and in patients with SLE.

Methods

We used flow cytometry of isolated peripheral blood mononuclear cells to compare the levels of Mer on leukocyte subsets. We used a Mer-specific enzyme-linked immunosorbent assay (ELISA) to quantify soluble Mer (sMer) in plasmas.

Results

Monocytes, CD1c⁺ myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs) from both normal individuals and from SLE patients expressed Mer. In both normal and SLE patients, the CD14⁺⁺CD16⁺ subpopulation of monocytes expressed the highest levels of Mer, with somewhat lower levels on the CD14^intCD16⁺ population. Mer levels on CD1c⁺ mDCs and pDCs, and sMer levels in blood were increased in SLE patients compared with controls. In patients, Mer levels on CD14^intCD16⁺, CD14⁺⁺CD16^- monocytes, and CD1c⁺ dendritic cells correlated positively with type I interferon (IFN-I) activity detected in blood. In SLE patients treated with corticosteroids, Mer expression on monocytes correlated with prednisone dose, CD1c⁺ myeloid dendritic cells in patients treated with prednisone had higher levels of Mer expression than those in patients not receiving prednisone.

Conclusions

We found no global defect in Mer expression in lupus blood. In contrast, we observed increased levels of Mer expression in DC populations, which could represent a response to increased IFN-I in SLE patients. Enhanced Mer expression induced by corticosteroids may contribute to its beneficial effects in SLE.     

Vitamin K-dependent proteins GAS6 and Protein S and TAM receptors in patients of systemic lupus erythematosus: correlation with common genetic variants and disease activity

Introduction

Growth arrest-specific gene 6 protein (GAS6) and protein S (ProS) are vitamin K-dependent proteins present in plasma with important regulatory functions in systems of response and repair to damage. They interact with receptor tyrosine kinases of the Tyro3, Axl and MerTK receptor tyrosine kinase (TAM) family, involved in apoptotic cell clearance (efferocytosis) and regulation of the innate immunity. TAM-deficient mice show spontaneous lupus-like symptoms. Here we tested the genetic profile and plasma levels of components of the system in patients with systemic lupus erythematosus (SLE), and compare them with a control healthy population.

Methods

Fifty SLE patients and 50 healthy controls with matched age, gender and from the same geographic area were compared. Genetic analysis was performed in GAS6 and the TAM receptor genes on SNPs previously identified. The concentrations of GAS6, total and free ProS, and the soluble forms of the three TAM receptors (sAxl, sMerTK and sTyro3) were measured in plasma from these samples.

Results

Plasma concentrations of GAS6 were higher and, total and free ProS were lower in the SLE patients compared to controls, even when patients on oral anticoagulant treatment were discarded. Those parameters correlated with SLE disease activity index (SLEDAI) score, GAS6 being higher in the most severe cases, while free and total ProS were lower. All 3 soluble receptors increased its concentration in plasma of lupus patients.

Conclusions

The present study highlights that the GAS6/ProS-TAM system correlates in several ways with disease activity in SLE. We show here that this correlation is affected by common polymorphisms in the genes of the system. These findings underscore the importance of mechanism of regulatory control of innate immunity in the pathology of SLE.     

Non‐erythropoietic erythropoietin‐derived peptide protects mice from systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease, which results in various organ pathologies. However, current treatment towards SLE is suboptimal. Erythropoietin (EPO) has been shown to promote SLE recovery, but clinical application can be limited by its haematopoiesis‐stimulating effects. EPO‐derived helix‐B peptide (ARA290) is non‐erythrogenic but has been reported to retain the anti‐inflammatory and tissue‐protective functions of EPO. Therefore, here we investigated the effects and potential mechanisms of ARA290 on SLE. The administration of ARA290 to pristane‐induced SLE and MRL/lpr mice significantly suppressed the level of serum antinuclear autoantibodies (ANAs) and anti‐dsDNA autoantibodies, reduced the deposition of IgG and C3, and ameliorated the nephritis symptoms. Moreover, the serum concentrations of inflammatory cytokine IL‐6, MCP‐1 and TNF‐α in SLE mice were reduced by ARA290. Further, ARA290 decreased the number of apoptotic cells in kidney. In vitro experiment revealed that ARA290 inhibited the inflammatory activation of macrophages and promoted the phagocytotic function of macrophages to apoptotic cells. Finally, ARA290 did not induce haematopoiesis during treatment. In conclusion, ARA290 ameliorated SLE, which at least could be partly due to its anti‐inflammatory and apoptotic cell clearance promoting effects, without stimulating haematopoiesis, suggesting that ARA290 could be a hopeful candidate for SLE treatment.     

Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

Background/Objective

Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.

Methodology/Principal Findings

In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.

Conclusion/Significance

Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.     

Dysfunctional interferon-α production by peripheral plasmacytoid dendritic cells upon Toll-like receptor-9 stimulation in patients with systemic lupus erythematosus

Background  

It is well known that interferon (IFN)-α is important to the pathogenesis of systemic lupus erythematosus (SLE). However, several reports have indicated that the number of IFN-α producing cells are decreased or that their function is defective in patients with SLE. We studied the function of plasmacytoid dendritic cells (pDCs) under persistent stimulation of Toll-like receptor (TLR)9 via a TLR9 ligand (CpG ODN2216) or SLE serum.