The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways

Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained alpha-chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H(2)O(2)-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between alpha-globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.     

Intimal lining layer macrophages but not synovial sublining macrophages display an IL-10 polarized-like phenotype in chronic synovitis

Introduction

Synovial tissue macrophages play a key role in chronic inflammatory arthritis, but the contribution of different macrophage subsets in this process remains largely unknown. The main in vitro polarized macrophage subsets are classically (M1) and alternatively (M2) activated macrophages, the latter comprising interleukin (IL)-4 and IL-10 polarized cells. Here, we aimed to evaluate the polarization status of synovial macrophages in spondyloarthritis (SpA) and rheumatoid arthritis (RA).

Methods

Expression of polarization markers on synovial macrophages, peripheral blood monocytes, and in vitro polarized monocyte-derived macrophages from SpA versus RA patients was assessed by immunohistochemistry and flow cytometry, respectively. The polarization status of the intimal lining layer and the synovial sublining macrophages was assessed by double immunofluorescence staining.

Results

The expression of the IL-10 polarization marker cluster of differentiation 163 (CD163) was increased in SpA compared with RA intimal lining layer, but no differences were found in other M1 and M2 markers between the diseases. Furthermore, no significant phenotypic differences in monocytes and in vitro polarized monocyte-derived macrophages were seen between SpA, RA, and healthy controls, indicating that the differential CD163 expression does not reflect a preferential M2 polarization in SpA. More detailed analysis of intimal lining layer macrophages revealed a strong co-expression of the IL-10 polarization markers CD163 and cluster of differentiation 32 (CD32) but not any of the other markers in both SpA and RA. In contrast, synovial sublining macrophages had a more heterogeneous phenotype, with a majority of cells co-expressing M1 and M2 markers.

Conclusions

The intimal lining layer but not synovial sublining macrophages display an IL-10 polarized-like phenotype, with increased CD163 expression in SpA versus RA synovitis. These differences in the distribution of the polarized macrophage subset may contribute to the outcome of chronic synovitis.     

CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma

CD163 is a highly expressed macrophage membrane protein belonging to the scavenger receptor cysteine rich (SRCR) domain family. The CD163 expression is induced by interleukin-6, interleukin-10 and glucocorticoids. Its function has remained unknown until recently when CD163 was identified as the endocytic receptor binding hemoglobin (Hb) in complex with the plasma protein haptoglobin (Hp). This specific receptor-ligand interaction leading to removal from plasma of the Hp-Hb complex-but not free Hp or Hb-now explains the depletion of circulating Hp in individuals with increased intravascular hemolysis. Besides having a detoxificating effect by removing Hb from plasma, the CD163-mediated endocytosis of the Hp-Hb complex may represent a major pathway for uptake of iron in the tissue macrophages.The novel functional linkage of CD163 and Hp, which both are induced during inflammation, also reveal some interesting perspectives relating to the suggested anti-inflammatory properties of the receptor and the Hp phenotypes.     

Extracellular Mycobacterial DnaK Polarizes Macrophages to the M2-Like Phenotype

Macrophages are myeloid cells that play an essential role in inflammation and host defense, regulating immune responses and maintaining tissue homeostasis. Depending on the microenvironment, macrophages can polarize to two distinct phenotypes. The M1 phenotype is activated by IFN-γ and bacterial products, and displays an inflammatory profile, while M2 macrophages are activated by IL-4 and tend to be anti-inflammatory or immunosupressive. It was observed that DnaK from Mycobacterium tuberculosis has immunosuppressive properties, inducing a tolerogenic phenotype in dendritic cells and MDSCs, contributing to graft acceptance and tumor growth. However, its role in macrophage polarization remains to be elucidated. We asked whether DnaK was able to modulate macrophage phenotype. Murine macrophages, derived from bone marrow, or from the peritoneum, were incubated with DnaK and their phenotype compared to M1 or M2 polarized macrophages. Treatment with DnaK leads macrophages to present higher arginase I activity, IL-10 production and FIZZ1 and Ym1 expression. Furthermore, DnaK increased surface levels of CD206. Importantly, DnaK-treated macrophages were able to promote tumor growth in an allogeneic melanoma model. Our results suggest that DnaK polarizes macrophages to the M2-like phenotype and could constitute a virulence factor and is an important immunomodulator of macrophage responses.     

The Distribution of Macrophages with a M1 or M2 Phenotype in Relation to Prognosis and the Molecular Characteristics of Colorectal Cancer

High macrophage infiltration has been correlated to improved survival in colorectal cancer (CRC). Tumor associated macrophages (TAMs) play complex roles in tumorigenesis since they are believed to hold both tumor preventing (M1 macrophages) and tumor promoting (M2 macrophages) activities. Here we have applied an immunohistochemical approach to determine the degree of infiltrating macrophages with a M1 or M2 phenotype in clinical specimens of CRC in relation to prognosis, both in CRC in general but also in subgroups of CRC defined by microsatellite instability (MSI) screening status and the CpG island methylator phenotype (CIMP). A total of 485 consecutive CRC specimens were stained for nitric oxide synthase 2 (NOS2) (also denoted iNOS) as a marker for the M1 macrophage phenotype and the scavenger receptor CD163 as a marker for the M2 macrophage phenotype. The average infiltration of NOS2 and CD163 expressing macrophages along the invasive tumor front was semi-quantitatively evaluated using a four-graded scale. Two subtypes of macrophages, displaying M1 (NOS2⁺) or M2 (CD163⁺) phenotypes, were recognized. We observed a significant correlation between the amount of NOS2⁺ and CD163⁺ cells (P<0.0001). A strong inverse correlation to tumor stage was found for both NOS2 (P<0.0001) and CD163 (P<0.0001) infiltration. Furthermore, patients harbouring tumors highly infiltrated by NOS2⁺ cells had a significantly better prognosis than those infiltrated by few NOS2⁺ cells, and this was found to be independent of MSI screening status and CIMP status. No significant difference was found on cancer-specific survival in groups of CRC with different NOS2/CD163 ratios. In conclusion, an increased infiltration of macrophages with a M1 phenotype at the tumor front is accompanied by a concomitant increase in macrophages with a M2 phenotype, and in a stage dependent manner correlated to a better prognosis in patients with CRC.     

M2-Polarized Macrophages Determine Human Cutaneous Lesions in Lacaziosis

Lacaziosis is a cutaneous chronic mycosis caused by Lacazia loboi. Macrophages are important cells in the host immune response in fungal infections. The macrophage population exhibits strong plasticity that varies according to the stimuli in the microenvironment of lesions M1 profile promotes a Th1 pattern of cytokines and a microbicidal function and M2 is related to Th2 cytokines and immunomodulatory response. We investigated the population of M1 and M2 polarized macrophages in human cutaneous lesions. A total of 27 biopsies from human lesions were submitted to an immunohistochemistry protocol using antibodies to detect M1 and M2 macrophages (Arginase-1, CD163, iNOS, RBP-J and cMAF). We could observe high number of cells expressing Arginase1, CD163 and c-MAF that correspond to elements of the M2 profile of macrophage, over iNOS and RBP-J (elements of the M1 profile). The results suggest a predominant phenotype of M2 macrophages, which have an immunomodulatory role and probably contributing to chronicity of Lacaziosis.

     

Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury

The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.     

HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.     

Synthesis and characterization of a hemoglobin-ribavirin conjugate for targeted drug delivery

A novel conjugate of human hemoglobin (Hb) and the nucleoside analogue ribavirin (RBV) was synthesized to demonstrate the utility of Hb as a biocompatible drug carrier for improved drug delivery in the treatment of liver disease. RBV is used in combination with interferon for the treatment of hepatitis C, but its side effects can result in dose limitation or discontinuation of treatment. Targeted delivery of RBV may help to prevent or minimize its toxicity. The hemoglobin-ribavirin conjugate (Hb-RBV) was designed to release bioactive drug upon endocytosis by cells and tissues involved in extracellular Hb catabolism and clearance. Ribavirin-5'-monophosphate (RBV-P) was prepared from RBV and activated as the 5'-monophosphorimidazolide (RBV-P-Im) for reaction with carbonmonoxyhemoglobin to yield Hb-RBV consisting of multiple RBV drugs covalently attached as physiologically labile phosphoramidates via their 5'-hydroxyl groups. A molar drug ratio of six to eight RBV molecules per Hb tetramer was obtained with near complete haptoglobin (Hp) binding of the drug modified Hb maintained. The conjugate complex (Hp-Hb-RBV) was selectively taken up in vitro by cells that express the hemoglobin-haptoglobin receptor, CD163. Recovered ribavirin enzymatically cleaved from Hb-RBV showed equipotent antiproliferative activity compared to control unconjugated RBV against human HepG2 and mouse AML12 liver cell lines. Based upon the reported high level of Hb uptake in the liver, Hb-RBV may be useful in the treatment of certain liver diseases, as well as inflammatory disorders associated with CD163-positive macrophages.     

Induction of Murine Macrophage M2 Polarization by Cigarette Smoke Extract via the JAK2/STAT3 Pathway

Cigarette smoking is a major pathogenic factor in lung cancer. Macrophages play an important role in host defense and adaptive immunity. These cells display diverse phenotypes for performing different functions. M2 type macrophages usually exhibit immunosuppressive and tumor-promoting characteristics. Although macrophage polarization toward the M2 phenotype has been observed in the lungs of cigarette smokers, the molecular basis of the process remains unclear. In this study, we evaluated the possible mechanisms for the polarization of mouse macrophages that are induced by cigarette smoking (CS) or cigarette smoke extract (CSE). The results showed that exposure to CSE suppressed the production of reactive oxygen species (ROS) and nitric oxide (NO) and down-regulated the phagocytic ability of Ana-1 cells. The CD163 expressions on the surface of macrophages from different sources were significantly increased in in vivo and in vitro studies. The M1 macrophage cytokines TNF-α, IL-12p40 and enzyme iNOS decreased in the culture supernatant, and their mRNA levels decreased depending on the time and concentration of CSE. In contrast, the M2 phenotype macrophage cytokines IL-10, IL-6, TGF-β1 and TGF-β2 were up-regulated. Moreover, phosphorylation of JAK2 and STAT3 was observed after the Ana-1 cells were treated with CSE. In addition, pretreating the Ana-1 cells with the STAT3 phosphorylation inhibitor WP1066 inhibited the CSE-induced CD163 expression, increased the mRNA level of IL-10 and significantly decreased the mRNA level of IL-12. In conclusion, we demonstrated that the M2 polarization of macrophages induced by CS could be mediated through JAK2/STAT3 pathway activation.     

Macrophage Polarization Reflects T Cell Composition of Tumor Microenvironment in Pediatric Classical Hodgkin Lymphoma and Has Impact on Survival

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.     

M2 Macrophages Activate WNT Signaling Pathway in Epithelial Cells: Relevance in Ulcerative Colitis

Macrophages, which exhibit great plasticity, are important components of the inflamed tissue and constitute an essential element of regenerative responses. Epithelial Wnt signalling is involved in mechanisms of proliferation and differentiation and expression of Wnt ligands by macrophages has been reported. We aim to determine whether the macrophage phenotype determines the expression of Wnt ligands, the influence of the macrophage phenotype in epithelial activation of Wnt signalling and the relevance of this pathway in ulcerative colitis. Human monocyte-derived macrophages and U937-derived macrophages were polarized towards M1 or M2 phenotypes and the expression of Wnt1 and Wnt3a was analyzed by qPCR. The effects of macrophages and the role of Wnt1 were analyzed on the expression of β-catenin, Tcf-4, c-Myc and markers of cell differentiation in a co-culture system with Caco-2 cells. Immunohistochemical staining of CD68, CD206, CD86, Wnt1, β-catenin and c-Myc were evaluated in the damaged and non-damaged mucosa of patients with UC. We also determined the mRNA expression of Lgr5 and c-Myc by qPCR and protein levels of β-catenin by western blot. Results show that M2, and no M1, activated the Wnt signaling pathway in co-culture epithelial cells through Wnt1 which impaired enterocyte differentiation. A significant increase in the number of CD206+ macrophages was observed in the damaged mucosa of chronic vs newly diagnosed patients. CD206 immunostaining co-localized with Wnt1 in the mucosa and these cells were associated with activation of canonical Wnt signalling pathway in epithelial cells and diminution of alkaline phosphatase activity. Our results show that M2 macrophages, and not M1, activate Wnt signalling pathways and decrease enterocyte differentiation in co-cultured epithelial cells. In the mucosa of UC patients, M2 macrophages increase with chronicity and are associated with activation of epithelial Wnt signalling and diminution in enterocyte differentiation.     

S100A6通过招募和活化巨噬细胞促进血管形成*

目的:探讨S100A6经由巨噬细胞介导的促血管生成作用及机制。方法:(1)用重组蛋白 GST-hS100A6处理巨噬细胞后:①收集上清制备条件培养基(简称A6-Mφ-CM),并用之重悬人脐静脉内皮细胞(HUVEC),用体外血管形成试验检测各处理因素对血管形成的影响;②分别用实时荧光定量PCR和Western blot检测巨噬细胞的M2型标志物CD163及促血管形成因子CCL2、IL-6、VEGFA的mRNA和蛋白质水平,以及JAK2和STAT3的蛋白质及其磷酸化水平;③用Transwell迁移试验检测巨噬细胞迁移能力的变化。(2)使用JAK2抑制剂(XL019)预处理巨噬细胞后再加GST-hS100A6处理,检测S100A6促巨噬细胞迁移作用的变化。以重组蛋白GST为实验对照。 结果:(1)A6-Mφ-CM组的血管分支数和血管分支长度既明显高于GST-Mφ-CM组(P值均小于0.05),也明显高于GST-hS100A6直接处理的HUVEC组(P<0.001,P<0.01),提示S100A6处理后的巨噬细胞具有促进血管形成的作用;(2)GST-hS100A6处理后的巨噬细胞中,CD163、CCL2、IL-6、VEGFA的mRNA和蛋白质水平明显高于GST组(P值均小于0.05),提示S100A6诱导巨噬细胞向促血管表型(pro-angiogenic phenotype)转化;(3)GST-hS100A6处理后,巨噬细胞的迁移数是GST组的1.4倍(P<0.01),提示S100A6具有招募巨噬细胞的作用;(4)GST-hS100A6处理组巨噬细胞的JAK2和STAT3的蛋白质及其磷酸化水平都明显高于GST组(P值均小于0.05),而JAK2抑制剂XL019可部分抑制S100A6促进巨噬细胞迁移的作用(P<0.01),提示S100A6促进巨噬细胞迁移作用机制涉及JAK2/STAT3信号通路的激活。 结论:微环境中的S100A6可通过招募巨噬细胞并进一步诱导其向促血管表型转化,进而促进新生血管形成;其招募巨噬细胞的机制涉及JAK2/STAT3信号通路的激活。     

Depletion and Reconstitution of Macrophages in Mice

Macrophages are critical players in the innate immune response to infectious challenge or injury, initiating the innate immune response and directing the acquired immune response. Macrophage dysfunction can lead to an inability to mount an appropriate immune response and as such, has been implicated in many disease processes, including inflammatory bowel diseases. Macrophages display polarized phenotypes that are broadly divided into two categories. Classically activated macrophages, activated by stimulation with IFNγ or LPS, play an essential role in response to bacterial challenge whereas alternatively activated macrophages, activated by IL-4 or IL-13, participate in debris scavenging and tissue remodeling and have been implicated in the resolution phase of inflammation. During an inflammatory response in vivo, macrophages are found amid a complex mixture of infiltrating immune cells and may participate by exacerbating or resolving inflammation. To define the role of macrophages in situ in a whole animal model, it is necessary to examine the effect of depleting macrophages from the complex environment. To ask questions about the role of macrophage phenotype in situ, phenotypically defined polarized macrophages can be derived ex vivo, from bone marrow aspirates and added back to mice, with or without prior depletion of macrophages. In the protocol presented here clodronate-containing liposomes, versus PBS injected controls, were used to deplete colonic macrophages during dextran sodium sulfate (DSS)-induced colitis in mice. In addition, polarized macrophages were derived ex vivo and transferred to mice by intravenous injection. A caveat to this approach is that clodronate-containing liposomes deplete all professional phagocytes, including both dendritic cells and macrophages so to ensure the effect observed by depletion is macrophage-specific, reconstitution of phenotype by adoptive transfer of macrophages is necessary. Systemic macrophage depletion in mice can also be achieved by backcrossing mice onto a CD11b-DTR background, which is an excellent complementary approach. The advantage of clodronate-containing liposome-mediated depletion is that it does not require the time and expense involved in backcrossing mice and it can be used in mice regardless of the background of the mice (C57BL/6, BALB/c, or mixed background).     

Characterization of the effects of cross-linking of macrophage CD44 associated with increased phagocytosis of apoptotic PMN

Control of macrophage capacity for apoptotic cell clearance by soluble mediators such as cytokines, prostaglandins and lipoxins, serum proteins, and glucocorticoids may critically determine the rate at which inflammation resolves. Previous studies suggested that macrophage capacity for clearance of apoptotic neutrophils was profoundly altered following binding of CD44 antibodies. We have used a number of different approaches to further define the mechanism by which CD44 rapidly and specifically augment phagocytosis of apoptotic neutrophils. Use of Fab' fragments unequivocally demonstrated a requirement for cross-linking of macrophage surface CD44. The molecular mechanism of CD44-augmented phagocytosis was shown to be opsonin-independent and to be distinct from the Mer/protein S pathway induced by glucocorticoids and was not functional for clearance of apoptotic eosinophils. CD44-cross-linking also altered macrophage migration and induced cytoskeletal re-organisation together with phosphorylation of paxillin and activation of Rac2. Investigation of signal transduction pathways that might be critical for CD44 augmentation of phagocytosis revealed that Ca(2+) signalling, PI-3 kinase pathways and altered cAMP signalling were not involved, but did implicate a key role for tyrosine phosphorylation events. Finally, although CD44 antibodies were able to augment phagocytosis of apoptotic neutrophils by murine peritoneal and bone marrow-derived macrophages, we did not observe a difference in the clearance of neutrophils following induction of peritonitis with thioglycollate in CD44-deficient animals. Together, these data demonstrate that CD44 cross-linking induces a serum opsonin-independent mechanism of macrophage phagocytosis of apoptotic neutrophils that is associated with reduced macrophage migration and cytoskeletal reorganisation.     

Tumor cell apoptosis polarizes macrophages role of sphingosine-1-phosphate

Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAMs), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-alpha and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-kappaB signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization.     

Ratio of Intratumoral Macrophage Phenotypes Is a Prognostic Factor in Epithelioid Malignant Pleural Mesothelioma

Hypothesis

The tumor micro-environment and especially the different macrophage phenotypes appear to be of great influence on the behavior of multiple tumor types. M1 skewed macrophages possess anti-tumoral capacities, while the M2 polarized macrophages have pro-tumoral capacities. We analyzed if the macrophage count and the M2 to total macrophage ratio is a discriminative marker for outcome after surgery in malignant pleural mesothelioma (MPM) and studied the prognostic value of these immunological cells.

Methods

8 MPM patients who received induction chemotherapy and surgical treatment were matched on age, sex, tumor histology, TNM stage and EORTC score with 8 patients who received chemotherapy only. CD8 positive T-cells and the total macrophage count, using the CD68 pan-macrophage marker, and CD163 positive M2 macrophage count were determined in tumor specimens prior to treatment.

Results

The number of CD68 and CD163 cells was comparable between the surgery and the non-surgery group, and was not related to overall survival (OS) in both the surgery and non-surgery group. However, the CD163/CD68 ratio did correlate with OS in both in the total patient group (Pearson r −0.72, p<0.05). No correlation between the number of CD8 cells and prognosis was found.

Conclusions

The total number of macrophages in tumor tissue did not correlate with OS in both groups, however, the CD163/CD68 ratio correlates with OS in the total patient group. Our data revealed that the CD163/CD68 ratio is a potential prognostic marker in epithelioid mesothelioma patients independent of treatment but cannot be used as a predictive marker for outcome after surgery.     

巨噬细胞极化的研究进展

巨噬细胞是一群表型和功能均具有高度异质性的免疫细胞。巨噬细胞通过清除并修复受损的细胞和基质来维护组织完整性。巨噬细胞在不同的组织微环境、不同病理条件下,可极化成不同的表型即M1型巨噬细胞(经典活化的巨噬细胞)和M2型巨噬细胞(替代活化的巨噬细胞)。本文将对不同巨噬细胞亚群在抗细菌感染、抗寄生虫感染、哮喘、动脉粥样硬化和肿瘤产生中起到的的保护或致病作用,以及调控巨噬细胞极化的机制进行综述。掌握巨噬细胞极化在不同疾病中的作用以及调控巨噬细胞极化的具体机制,将为疾病的预防、诊断、治疗及药物研发提供新策略。     

Lymphangiogenesis in renal fibrosis arises from macrophages via VEGF-C/VEGFR3-dependent autophagy and polarization

Inflammation plays a crucial role in the occurrence and development of renal fibrosis, which ultimately results in end-stage renal disease (ESRD). There is new focus on lymphangiogenesis in the field of inflammation. Recent studies have revealed the association between lymphangiogenesis and renal fibrosis, but the source of lymphatic endothelial cells (LECs) is not clear. It has also been reported that macrophages are involved in lymphangiogenesis through direct and indirect mechanisms in other tissues. We hypothesized that there was a close relationship between macrophages and lymphatic endothelial progenitor cells in renal fibrosis. In this study, we demonstrated that lymphangiogenesis occurred in a renal fibrosis model and was positively correlated with the degree of fibrosis and macrophage infiltration. Compared to resting (M0) macrophages and alternatively activated (M2) macrophages, classically activated (M1) macrophages predominantly transdifferentiated into LECs in vivo and in vitro. VEGF-C further increased M1 macrophage polarization and transdifferentiation into LECs by activating VEGFR3. It was suggested that VEGF-C/VEGFR3 pathway activation downregulated macrophage autophagy and subsequently regulated macrophage phenotype. The induction of autophagy in macrophages by rapamycin decreased M1 macrophage polarization and differentiation into LECs. These results suggested that M1 macrophages promoted lymphangiogenesis and contributed to newly formed lymphatic vessels in the renal fibrosis microenvironment, and VEGF-C/VEGFR3 signaling promoted macrophage M1 polarization by suppressing macrophage autophagy and then increased the transdifferentiation of M1 macrophages into LECs.Subject terms: Lymphangiogenesis, End-stage renal disease