Alternative macrophage activation in periprosthetic osteolysis

Several disorders characterized by macrophages accumulating non-disposable (or hard to dispose of) material or formation of multinucleated giant cell containing granulomas have been linked to elicitation of an alternative macrophage activation phenotype. Gene profiling efforts have shown that alternative macrophage activation can exist in numerous forms, each specific for the particular biological niche in which the macrophage finds itself, accentuating the plasticity of this cell type. Periprosthetic osteolysis is characterized by macrophage phagocytosis of particles of wear debris and formation of foreign body granulomas, suggesting the hypothesis that it may represent a new member of this group of diseases characterized by alternative macrophage activation. Gene profiling has provided strong supportive evidence for this hypothesis, revealing that periprosthetic tissues of osteolysis patients show the presence of a pronounced alternative macrophage activation pathway, with the classical pro-inflammatory activation pathway being less evident. These findings have important implications for our understanding of periprosthetic osteolysis and how to approach future investigations into this disease.     

Adenosine receptor activation promotes macrophage class switching from LPS-induced acute inflammatory M1 to anti-inflammatory M2 phenotype

Lipopolysaccharide induced monocytes/macrophages exhibit a pro-inflammatory M1 phenotype. Elevated levels of the purine nucleoside adenosine play a major role in this response. The role of adenosine receptor modulation in directing the macrophage phenotype switch from proinflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype is investigated in this study. The mouse macrophage cell line RAW 264.7 was used as the experimental model and stimulated with Lipopolysaccharide (LPS) at a dose of 1 μg/ml. Adenosine receptors were activated by treating cells with the receptor agonist NECA (1 μM). Adenosine receptor stimulation in macrophages is found to suppress LPS-induced production of proinflammatory mediators (pro-inflammatory cytokines, Reactive Oxygen Species and nitrite levels). M1 marker CD38 (Cluster of Differentiation 38) and CD83 (Cluster of Differentiation 83) were significantly decreased while M2 markers Th2 cytokines, Arginase, TIMP (Tissue Inhibitor of Metalloproteinases) and CD206 (Cluster of Differentiation 206) exhibited an increase. Hence from our study we observed that activation of adenosine receptors can program the macrophages from a pro-inflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype. We report the significance and a time course profile of phenotype switching by receptor activation. Adenosine receptor targeting may be explored as a therapeutic intervention strategy in addressing acute inflammation.     

Torg osteolysis syndrome

We describe a 9-year-old girl who initially presented at age 4 with evidence of arthritis in her hands, feet, and large joints. Although she had a partial response to anti-inflammatory medications and had some laboratory results consistent with inflammatory disease, radiographs showed carpal and tarsal osteolysis associated with interphalangeal joint erosions. There was also widening of the shafts of the metacarpals and metatarsals with thinning of the cortices. Based on both the clinical progression of her illness and the radiologic characteristics, this child most likely has the Torg syndrome. Am. J. Med. Gen. 80:207–212, 1998. © 1998 Wiley-Liss, Inc.     

Effects of proteasome inhibitors on cytokines,metalloproteinases and their inhibitors and collagen type-I expression in periprosthetic tissues and fibroblasts from loose arthroplasty endoprostheses

ABSTRACT

Objective: Aseptic loosening is a major problem in total joint replacement. Implant wear debris provokes a foreign body host response and activates cells to produce a variety of mediators and ROS, leading to periprosthetic osteolysis. Elevated ROS levels can harm proteasome function. Proteasome inhibitors have been reported to alter the secretory profile of cells involved in inflammation and also to induce ROS production. In this work, we aimed to document the effects of proteasome inhibitors MG-132 and Epoxomicin, on the production of factors involved in aseptic loosening, in periprosthetic tissues and fibroblasts, and investigate the role of proteasome impairment in periprosthetic osteolysis.

Materials and methods: IL-6 levels in tissue cultures were determined by sandwich ELISA. MMP-1, -3, -13, -14 and TIMP-1 levels in tissue or cell cultures were determined by indirect ELISA. Results for MMP-1 and TIMP-1 in tissue cultures were confirmed by Western blotting. MMP-2 and MMP-9 levels were determined by gelatin zymography. Gene expression of IL-6, MMP-1,-3,-14, TIMP-1 and collagen type-I was determined by RT-PCR.

Results: Results show that proteasome inhibition induces the expression of ΜΜΡ-1, -2, -3, -9 and suppresses that of IL-6, MMP-14, -13, TIMP-1 and collagen type I, enhancing the collagenolytic and gelatinolytic activity already present in periprosthetic tissues, as documented in various studies.

Conclusions: These findings suggest that proteasome impairment could be a contributing factor to aseptic loosening. Protection and enhancement of proteasome efficacy could thus be considered as an alternative strategy toward disease treatment.     

Regulators of macrophage activation

Macrophages are ubiquitous phagocytes that can constitute up to 15% of the cellular content of tissues. These heterogeneous cells of the innate immune system perform important functions during health and disease. Equipped with receptors for the T helper cell cytokines INF-γ and IL-4, macrophages undergo specific activation programs during Th1 or Th2 immune responses. These activation profiles, termed classical (M1) or alternative (M2) activation respectively, are further tuned by the presence and recognition of microbial-associated molecular patterns, other cytokines, lipids, and even adhesion to the substratum. The activation of macrophages also relies on the maturation background of the cells, elicitation of complicated intracellular signalling cascades, and the crosstalk between the different signalling elements. Of interest, not all genes participating in the activation-related signalling cascades are equally important for the elicitation of functional profiles and a regulator gene hierarchy is emerging for the different types of activation. In this issue of the European Journal of Immunology, two papers add to our understanding of how cellular kinases and phosphatases, related to the PI3K pathway, regulate M1 or M2 activation programmes in macrophages.     

Comparison of VEGF-producing cells in periprosthetic osteolysis

The pro-angiogenic cytokine vascular endothelial growth factor (VEGF) has been implicated in periprosthetic osteolysis and subsequent aseptic loosening of implants following total hip arthroplasty (THA). The goal of this study was to investigate whether increased VEGF at the bone-implant interface is secondary to a greater number of VEGF-producing cells or to increased VEGF production by individual cells. Real time polymerase chain reaction (RT-PCR) techniques were used to assess the expression of VEGF mRNA (isoforms 121, 165, 189) in periprosthetic tissues from revision THAs. Immunofluorescence was used to determine both differences in overall cellularity and in VEGF-producing cell type (macrophages, fibroblasts, endothelial cells) between patients with periprosthetic osteolysis (OL) and a control group undergoing primary THA for osteoarthritis (OA). Quantitative analysis of VEGF release in periprosthetic membranes via RT-PCR demonstrated no significant difference in the per-cell mRNA production of VEGF isoforms 121 165, or 189 between OL and OA patient groups. Immunofluorescence showed both higher cellularity and higher overall VEGF expression in the OL group. Immunofluorescence also showed a significant increase in macrophages in the OL group, but no significant difference in the proportion of fibroblasts or endothelial cells between the OL and OA groups. Co-localization of CD68+ and CD11b+ macrophage fluorescent signals with VEGF signal was greater in the OL group than in the OA group. Our results demonstrate that increased VEGF in OL periprosthetic tissue compared to OA synovium is correlated to increased numbers of VEGF-producing CD68+ and CD11b+ macrophages. Impact statement: Aseptic loosening, caused in large part by OL, remains the major cause of failed THAs leading to revision surgery. At the bone-implant interface, we found increased numbers of macrophages-cellular mediators of OL-and increased VEGF expression. VEGF may be a possible target for therapeutic intervention in mitigating OL.     

Chemokine receptor Ccr5 deficiency induces alternative macrophage activation and improves long‐term renal allograft outcome

The chemokine (C‐C motif) receptor 5 (CCR5) has been implicated in experimental and clinical allograft rejection. To dissect the function of CCR5 in acute and chronic renal allograft rejection, bilaterally nephrectomized WT and Ccr5^?/? C57BL/6 mice were used as recipients of WT BALB/c renal allografts and analyzed 7 and 42 days after transplantation. Lesion scores (glomerular damage, vascular rejection, tubulointerstitial inflammation) and numbers of CD4⁺, CD8⁺, CD11c⁺ and alpha smooth muscle actin (αSMA)⁺ cells were reduced in allografts from Ccr5^?/? recipients during the chronic phase. Increasing creatinine levels indicated deterioration of allograft function over time. While mRNA expression of Th1‐associated markers decreased between 7 and 42 days, Th2‐associated markers increased. Markers for alternatively activated macrophages (arginase 1, chitinase 3‐like 3, resistin‐like α, mannose receptor, C type 1), were strongly upregulated (mRNA and/or protein level) only in allografts from Ccr5^?/? recipients at 42 days. Ccr5 deficiency shifted intragraft immune responses during the chronic phase towards the Th2 type and led to accumulation of alternatively activated macrophages. Additionally, splenocytes from unchallenged Ccr5^?/? mice showed significantly increased arginase 1 and mannose receptor 1 mRNA levels, suggesting constitutive alternative activation of splenic macrophages. We conclude that Ccr5 deficiency favors alternative macrophage activation. This finding may be relevant for other inflammatory diseases that involve macrophage activation and may also influence future therapeutic strategies targeting CCR5.     

cAMP modulates macrophage development by suppressing M-CSF-induced MAPKs activation

M-CSF is a key cytokine in macrophage development by inducing MAPKs activation, and cAMP can inhibit MAPKs activation induced by inflammatory stimuli. To explore the effects of cAMP on M-CSF-induced MAPKs activation and on macrophage development, the model of bone marrow-derived murine macrophages （BMMs） was used. The effects of cAMP on M-CSF-induced MAPKs activation were analyzed by Western blotting assay, and the effects of cAMP on CD14 and F4/80 expression during macrophage development were examined by FACS analysis. Macrophage morphology showed the successful establishment of the model of macrophage development. Western blotting assay revealed that M-CSF activated ERK, JNK and p38 in both mature and immature macrophages, and cAMP inhibited M-CSF-induced ERK, JNK and p38 activation in a time-dependent manner. FACS analysis revealed that macrophage development was impaired with cAMP pretreatment. In conclusion, cAMP modulates macrophage development by suppressing M-CSF-induced MAPKs activation. Cellular ＆ Molecular Immunology.     

Platelet-derived growth factor-BB attenuates titanium-particle-induced osteolysis in vivo

Inflammation and osteoclastogenesis play critical roles in wear-particle-induced periprosthetic osteolysis (WPO). Platelet-derived growth factor-BB (PDGF-BB) could promote osteogenesis and inhibit inflammatory response. The aim of this study was to investigate the impact of PDGF-BB on WPO. Mice were divided into four groups, namely, sham, vehicle, low-, and high-dose PDGF-BB groups. Mice in the rhPDGF-BB groups were treated with PDGF-BB at 0.25 or 1?mg/ml/kg/day. Mice in the sham and vehicle groups received PBS daily. Two weeks after surgery, calvariae were harvested. Immunohistochemical analysis and μ-CT showed that PDGF-BB significantly reduced osteoclast formation and bone resorption. ELISA showed that rhPDGF-BB decreased the secretion of TNF-α, IL-1β, and IL-6. Western blotting revealed that rhPDGF-BB stimulated the expression of osteocalcin and osteoprotegerin. Furthermore, more VEGF and CD31 proteins were observed due to PDGF-BB by immunofluorescence. In conclusion, these findings suggest that rhPDGF-BB represents a potential treatment for WPO.     

Alternative activation of macrophage by IL-10.

Macrophage activation by proinflammatory stimuli is suppressed by IL-10. We tested the hypothesis that IL-10 induces an alternative state of macrophage activation rather than solely mediating suppression. We therefore searched for genes the expression of which might be up-rather than downregulated in response to IL-10. Total RNA was obtained from mouse macrophages J774 A.1 before or after stimulation with IL-10 (20 ng/ml). Poly(A)+RNA was isolated in both cases in order to obtain driver and tester mRNA. Subtraction suppression hybridization was performed using the PCR-select cDNA subtraction procedure. After evaluation of the subtraction efficiency the subtracted cDNA library was cloned into pCRII.1. A total of 1,300 clones were obtained. Southern blot hybridization analysis was performed as the first screening step of this total number of clones. 140 (10.7%) were identified as upregulated in response to IL-10. Sequence analyses so far showed perfect or near perfect matches with already known genes for the majority of clones. Our results clearly indicate that IL-10 stimulates the expression of a large number of genes in macrophages. We conclude that IL-10 induces in macrophages a noninflammatory state of reactivity which may serve to contain proinflammatory conditions.     

Innate immunity, macrophage activation, and atherosclerosis

Summary:  Inflammation underpins the development of atherosclerosis. Initiation and progression of vascular inflammation involves a complex cellular network, with macrophages as major contributors. Activated macrophages produce proinflammatory mediators, bridge innate and adaptive immunity, regulate lipid retention, and participate directly in vascular repair and remodeling. Recent efforts to elucidate molecular mechanisms involved in the regulation of vascular inflammation in atherosclerosis have implicated several families of innate immune recognition receptors in inflammatory activation during the course of this disease. This article reviews our current understanding of innate immune recognition receptors, signaling pathways, and putative ligands implicated in activation of macrophages in the disease. In its final section, we propose a model for the role of macrophages in bridging inflammation and atherosclerosis from the perspective of innate immune recognition and activation.     

The role of hydrogen sulphide signalling in macrophage activation

Hydrogen sulphide (H₂S) is the latest identified small gaseous mediator enabled by its lipophilic nature to freely permeate the biological membranes. Initially, H₂S was recognized by its roles in neuronal activity and vascular relaxation, which makes it an important molecule involved in paracrine signalling pathways. Recently, the immune regulatory function of gasotransmitters, H₂S in particular, is increasingly being appreciated. Endogenous H₂S level has been linked to macrophage activation, polarization and inflammasome formation. Mechanistically, H₂S‐induced protein S‐sulphydration suppresses several inflammatory pathways including NF‐κB and JNK signalling. Moreover, H₂S serves as a potent cellular redox regulator to modulate epigenetic alterations and to promote mitochondrial biogenesis in macrophages. Here in this review, we intend to summarize the recent advancements of H₂S studies in macrophages, and to discuss with focus on the therapeutic potential of H₂S donors by targeting macrophages. The feasibility of H₂S signalling component as a macrophage biomarker under disease conditions would be also discussed.     

Vessel-associated myogenic precursors control macrophage activation and clearance of apoptotic cells

Swift and regulated clearance of apoptotic cells prevents the accumulation of cell remnants in injured tissues and contributes to the shift of macrophages towards alternatively activated reparatory cells that sustain wound healing. Environmental signals, most of which are unknown, in turn control the efficiency of the clearance of apoptotic cells and as such determine whether tissues eventually heal. In this study we show that vessel-associated stem cells (mesoangioblasts) specifically modulate the expression of genes involved in the clearance of apoptotic cells and in macrophage alternative activation, including those of scavenger receptors and of molecules that bridge dying cells and phagocytes. Mesoangioblasts, but not immortalized myoblasts or neural precursor cells, enhance CD163 membrane expression in vitro as assessed by flow cytometry, indicating that the effect is specific. Mesoangioblasts transplanted in acutely or chronically injured skeletal muscles determine the expansion of the population of CD163⁺ infiltrating macrophages and increase the extent of CD163 expression. Conversely, macrophages challenged with mesoangioblasts engulf significantly better apoptotic cells in vitro. Collectively, the data reveal a feed-forward loop between macrophages and vessel-associated stem cells, which has implications for the skeletal muscle homeostatic response to sterile injury and for diseases in which homeostasis is jeopardized, including muscle dystrophies and inflammatory myopathies.     

Alternative versus classical macrophage activation during experimental African trypanosomosis

The type I/type II cytokine balance may influence the development of different subsets of suppressive macrophages, i.e., classically activated macrophages (caMphi, type I) versus alternatively activated macrophages (aaMphi, type II). Recently, we showed that although mice infected with phospholipase C-deficient (PLC-/-) Trypanosoma brucei brucei exhibit a clear shift from type I to the type II cytokine production, wild type (WT)-infected mice remain locked in a type I cytokine response. In the present study, phenotype and accessory cell function of macrophages elicited during WT and PLC-/- T. b. brucei infection were compared. Results indicate that caMphi develop in a type I cytokine environment in the early phase of WT and PLC-/- trypanosome infection, correlating with inhibition of T cell activation triggered by a mitogen, a superantigen, or an antigen. In the late stage of infection, only PLC(-/-)-infected mice resisting the infection develop type II cytokine-associated aaMphi correlating with impaired antigen- but not mitogen- or superantigen-induced T cell activation.     

Temporal radiographic texture analysis in the detection of periprosthetic osteolysis

Periprosthetic osteolysis is one of the most serious long-term problems in total hip arthroplasty. It has been primarily attributed to the body's inflammatory response to submicron polyethylene particles worn from the hip implant, and it leads to bone loss and structural deterioration in the surrounding bone. It was previously demonstrated that radiographic texture analysis (RTA) has the ability to distinguish between osteolysis and normal cases at the time of clinical detection of the disease; however, that analysis did not take into account the changes in texture over time. The goal of this preliminary analysis, however, is to assess the ability of temporal radiographic texture analysis (tRTA) to distinguish between patients who develop osteolysis and normal cases. Two tRTA methods were used in the study: the RTA feature change from baseline at various follow-up intervals and the slope of the best-fit line to the RTA data series. These tRTA methods included Fourier-based and fractal-based features calculated from digitized images of 202 total hip replacement cases, including 70 that developed osteolysis. Results show that separation between the osteolysis and normal groups increased over time for the feature difference method, as the disease progressed, with area under the curve (AUC) values from receiver operating characteristic analysis of 0.65 to 0.72 at 15 years postsurgery. Separation for the slope method was also evident, with AUC values ranging from 0.65 to 0.76 for the task of distinguishing between osteolysis and normal cases. The results suggest that tRTA methods have the ability to measure changes in trabecular structure, and may be useful in the early detection of periprosthetic osteolysis.     

Class A scavenger receptor activation inhibits endoplasmic reticulum stress-induced autophagy in macrophage

Macrophage death in advanced atherosclerosis promotes plaque necrosis and destabilization. Involvement of autophagy in bulk degradation of cellular components has been recognized recently as an important mechanism for cell survival under endoplasmic reticulum (ER) stress. We previously found that the engagement of class A scavenger receptor (SR-A) triggered JNK-dependent apoptosis in ER-stressed macrophages. However, pro-apoptotic mechanisms mediated by SR-A are not fully understood. Therefore, we sought to see if SR-A mediated apoptosis was associated with autophagy in macrophages. Here, we showed that fucoidan inhibited microtubule-associated protein light chain 3-phospholipid conjugates (LC3-II) formation as well as the number of autophagosomes under ER stress. The inhibition of LC3-II formation was paralleled by the activation of the mTOR pathway, and the inhibition of mTOR allowed LC3-II induction in macrophages treated with thapsigargin plus fucoidan. Furthermore, apoptosis induced by fucoidan was prevented under ER stress by the mTOR inhibitor. We propose that fucoidan, a SR-A agonist, may contribute to macrophage apoptosis during ER stress by inhibiting autophagy.