Cytokine response of human macrophage-like cells after contact with polyethylene and pure titanium particles.

The aim of this study was to establish a human macrophage cell culture system to examine the effect of polyethylene (PE) and titanium particles on cytokine release by macrophage-like cells (MLC) and to quantify this response with respect to the nature and concentration of particles. Human monocytic leukemia cells were differentiated under standard conditions with vitamin D3 and granulocyte macrophage-colony-stimulating factor. Cells were characterized by fluorescence-activated cell-sorter Scan of CD 14 expression analysis as well as a phagocytosis test exploiting fluorescence-labeled particles of bacteria] walls. To achieve a relevant contact between the floating PE particles (approximately 1 microm in size) and MLC, a rotation device was used (15 rotations/min) during incubation. The same was done with the titanium particles. Cell culture supernatants were then analyzed for interleukin (IL)-1beta, IL-8, and tumor necrosis factor (TNF)-alpha using the enzyme-linked immunosorbent assay technique in the absence or presence of particles. Rotation of incubated MLC alone did not influence the secretion of TNF-alpha, but it enhanced secretion of IL-1beta and IL-8 about 30-fold compared to background levels. Both PE and titanium particles significantly enhanced MLC cytokine release, the amount of which depended on the concentration of particles. Using 40 X 10(8) PE particles (0.7 x 10(8) titanium particles) and 10(6) MLC, the maximal release of IL-1beta was about 20-fold (7-fold titanium particles) higher than that of the rotating control sample. The stimulation of IL-8 release was 4-fold (3-fold titanium particles) and of TNF-alpha. 300-fold (170-fold titanium particles) compared to controls. MLC were viable (>90% cell survival) at concentrations less than 108 x 10(8) polyethylene particles per 10(6) MLC and 16 x 10(8) titanium particles per 10(6) MLC. Rotation per se as well as exposure to increasing concentrations of PE and titanium particles stimulates cytokine release (TNF-alpha, IL-1beta, IL-8) by macrophages in vitro. This in vitro model resembles the in vivo situation near arthroplasties, where implant particles make contact with inflammatory cells, such as macrophages. Cytokine release by macrophages may impair osteoblast function as well as stimulate bone resorption by osteoclasts and macrophages, thereby causing aseptic loosening of arthroplasties. Our in vitro model provides a reproducible human cell system that might shed light on the pathogenesis of particle disease and might serve as a reproducible in vitro test system for the biocompatibility of foreign materials.     

Pathogenesis of beta(2)-microglobulin amyloidosis: role of monocytes/macrophages

beta(2)-microglobulin (beta(2)M) amyloidosis (A beta(2)M) is a serious, often incapacitating complication for patients undergoing long-term hemodialysis. Amyloid deposits composed of beta(2)M fibrils as the major constituent protein are mainly localized in joints and periarticular bone and lead to chronic arthralgias, carpal tunnel syndrome, and eventually destructive arthropathy. Although recent histologic studies have shown the accumulation of monocytes/macrophages around amyloid deposits, the factor(s) causing their infiltration and pathologic involvement have yet to be fully elucidated. Immunohistochemical staining reveals that macrophages in tenosynovial tissues express CD13, CD14, CD33, HLA-DR, and CD68 antigens on their surfaces and express interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and IL-6. Many of these cells also express LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and VLA-4 (CD49d/CD29) on their surfaces. AGE-modified beta(2)M enhances chemotaxis of monocytes and stimulates macrophages to release bone-resorbing cytokines, such as IL-1 beta, TNF-alpha and IL-6. Via a RAGE-mediated pathway, AGE-modified, but not unmodified beta(2)M, significantly delays constitutive apoptosis of human peripheral blood monocytes. Monocytes survival in an advanced glycation end product (AGE) beta(2)M-containing microenvironment is associated with their phenotypic alteration into macrophage-like cells that generate more reactive oxygen species and elaborate greater quantities of IL-1 beta and TNF-alpha. Thus through regulation of their survival and differentiation, AGE beta(2)M in amyloid deposits may be able to influence the presence and quantity of infiltrated monocytes, and hence their biologic effects.     

Synergistic effect of particles and cyclic pressure on cytokine production in human monocyte/macrophages: proposed role in periprosthetic osteolysis.

Macrophages, activated by particulate wear debris, are important in the process of osteolysis, which occurs during joint implant loosening. We previously found increased levels of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor-alpha in cultured macrophages subjected to cyclical pressure of 0.138 MPa, suggesting that cyclic pressure may be another relevant cause of macrophage activation. The current study first investigated the effects of a range of cyclic pressures on cultured macrophages, including an investigation of the time course of cytokine expression. At 0.138 MPa, supernatant levels of TNF-alpha were maximal at 12 h, whereas IL-6 and IL-1beta were maximal at 24 h. All four cyclic pressure levels tested (without particles) resulted in increased production of all three cytokines relative to control. These increases were most marked at 0.069 and 0.035 MPa, and the increase in cytokine production at 0.017 MPa was not statistically significant. Further studies demonstrated that conditioned media from cyclically pressurized macrophages stimulated bone resorption in a neonatal mouse calvarial assay system. There were increased levels of calcium released from calvaria cultured in conditioned media from pressurised monocytes, and an increase in tartate-resistant acid phosphatase-positive osteoclasts was observed microscopically. As particulate wear debris is important in implant loosening, ultra high molecular weight polyethylene particles were also added to the pressurized cell cultures. The experiments compared the effect of atmospheric pressure, cyclic pressure alone, particles alone, and particles and cyclic pressure combined. A combination of ultra high molecular weight polyethylene particles and cyclic pressure at 0.017 MPa resulted in a dramatic synergistic elevation of levels of all three cytokines compared with the levels found with either pressure or particles alone. We propose that monocyte/macrophage activation by cyclic pressure plays a major role in the osteolysis seen in aseptic loosening of implants. The synergistic effect observed between particles and pressure could accelerate implant loosening, and implies that reduction in either cyclic pressure (by improving implant fixation) or wear debris load would reduce osteolysis.     

Cytokine receptor profile of arthroplasty macrophages, foreign body giant cells and mature osteoclasts

In the arthroplasty pseudomembrane surrounding a loose prosthesis there is a marked macrophage and foreign body giant cell (FBGC) response to implant-derived wear particles. These cells contribute to the osteolysis of loosening by releasing cytokines and growth factors which influence the formation and activity of osteoclasts. Using a panel of monoclonal antibodies directed against known cytokine/growth factor receptors, we have determined by immunohis-tochemistry whether arthroplasty macrophages, FB-GCs and osteoclasts express receptors for cytokines and growth factors that are known to modulate osteolysis.

All these cell types reacted with antibodies directed against the following cytokine/growth factor receptors: gp130, IL-1R type 1, IL-2R, IL-4R, IL-6R, TNFR, M-CSFR, GM-CSFR and SCFR but not with antibodies directed against IL-3R and IL-8R. Arthroplasty macrophages, FBGCs and osteoclasts thus show a similar pattern of cytokine/growth factor receptor expression. This reflects the fact that arthroplasty macrophages are capable of osteoclast differentiation and that these cell types form part of the mononuclear phagocyte system. As regards the osteolysis of aseptic loosening, it also indicates that these cells are targets for numerous cytokines and growth factors which influence osteoclast formation and bone resorption.     

Cytokine receptor profile of arthroplasty macrophages, foreign body giant cells and mature osteoclasts

In the arthroplasty pseudomembrane surrounding a loose prosthesis there is a marked macrophage and foreign body giant cell (FBGC) response to implant-derived wear particles. These cells contribute to the osteolysis of loosening by releasing cytokines and growth factors which influence the formation and activity of osteoclasts. Using a panel of monoclonal antibodies directed against known cytokine/growth factor receptors, we have determined by immunohistochemistry whether arthroplasty macrophages, FB-GCs and osteoclasts express receptors for cytokines and growth factors that are known to modulate osteolysis. All these cell types reacted with antibodies directed against the following cytokine/growth factor receptors: gp130, IL-1R type 1, IL-2R, IL-4R, IL-6R, TNFR, M-CSFR, GM-CSFR and SCFR but not with antibodies directed against IL-3R and IL-8R. Arthroplasty macrophages, FBGCs and osteoclasts thus show a similar pattern of cytokine/growth factor receptor expression. This reflects the fact that arthroplasty macrophages are capable of osteoclast differentiation and that these cell types form part of the mononuclear phagocyte system. As regards the osteolysis of aseptic loosening, it also indicates that these cells are targets for numerous cytokines and growth factors which influence osteoclast formation and bone resorption.     

The effect of cyclic pressure on human monocyte-derived macrophages in vitro

Aseptic loosening and osteolysis around prosthetic joints are the principal causes of failure and consequent revision. During this process activated macrophages produce cytokines which are thought to promote osteolysis by osteoclasts. Changes in pressure within the space around implants have been proposed as a cause of loosening and osteolysis. We therefore studied the effect of two different regimes of cyclic pressure on the production of interleukin-1beta (IL-1beta), IL-6 and tumour necrosis factor-alpha (TNF-alpha) by cultured human monocyte-derived (M-D) macrophages. There was a wide variation in the expression of cytokines in non-stimulated M-D macrophages from different donors and therefore cells from the same donor were compared under control and pressurised conditions. Both regimes of cyclic pressure were found to increase expression of IL-6 and TNF-alpha. Expression of IL-1beta was increased by a higher-frequency regime only. Our findings suggest that M-D macrophages are activated by cyclic pressure. Further work will be required to understand the relative roles of frequency, amplitude and duration of applied pressure in the cellular effects of cyclic pressure in this system.     

The biological reaction to polyethylene wear debris can be related with oxidation of the UHMWPE cups

BACKGROUND: Oxidised UHMWPE due to gamma irradiation in air has a greater susceptibility to wear than non-oxidised UHMWPE (ethylene oxide, EtO). AIM: To evaluate, the biological reaction of loose implants with oxidised and non-oxidised PE components. MATERIALS AND METHODS: Ten loose PE cups sterilised by EtO (group 1) and 13 sterilised by gamma irradiation in air (group 2) were studied. PE wear and oxidation were related to biological reaction. RESULTS: Group 1: PE cups had low wear score and no oxidation. In the oldest implants, a few PE particles and macrophages were observed in the interface membrane. Group 2: PE inserts had medium-to-high PE wear score and variable oxidation. In the interface membrane, the number and total area of PE particles were high and variable; giant cells were more numerous than macrophages. CONCLUSION: Junctional tissues around loose oxidised PE components contain more PE debris and giant cells than membranes around PE components that are not oxidised.     

Exposure to particles stimulates superoxide production by human THP-1 macrophages and avian HD-11EM osteoclasts activated by tumor necrosis factor-alpha and PMA

Wear of orthopaedic implants generates particles capable of inducing bone resorption and aseptic loosening of the implant. The present study shows the combined effect of particles and cell activation on macrophage (THP-1) and osteoclast (HD-11EM) release of reactive oxygen and nitrogen species, providing insight into mechanisms that can lead to osteolysis. In the absence of cell activation, exposure of either cell type to submicron zirconia or latex particles did not elicit an increase in reactive oxygen and nitrogen species production. Suboptimal stimulation with 4 beta-phorbol-12-myristate-13-acetate (PMA) plus particles resulted in a synergistic release of superoxide (O2-), however, and a low-level production of nitric oxide small middle dot by THP-1 macrophages. Similarly, particle stimulation of tumor necrosis factor-alpha-activated THP-1 cells increased O2- release. Our findings show the synergistic effect of cell activation and wear particles on O2- production by activated macrophages and osteoclasts, suggesting O2- involvement in mediating osteolysis.     

The osteoclastogenic molecules RANKL and RANK are associated with periprosthetic osteolysis

Extensive osteolysis adjacent to implants is often associated with wear particles of prosthetic material. We have investigated if RANKL, also known as osteoprotegerin ligand, osteoclast differentiation factor or TRANCE, and its natural inhibitor, osteoprotegerin (OPG), may be important in controlling this bone loss. Cells isolated from periprosthetic tissues containing wear particles expressed mRNA encoding for the pro-osteoclastogenic molecules, RANKL, its receptor RANK, monocyte colony-stimulating factor (M-CSF), interleukin (IL)-1beta, tumour necrosis factor (TNF)alpha, IL-6, and soluble IL-6 receptor, as well as OPG. Osteoclasts formed from cells isolated from periprosthetic tissues in the presence and absence of human osteoblastic cells. When osteoclasts formed in the absence of osteoblastic cells, markedly higher levels of RANKL mRNA relative to OPG mRNA were expressed. Particles of prosthetic materials also stimulated human monocytes to express osteoclastogenic molecules in vitro. Our results suggest that ingestion of prosthetic wear particles by macrophages results in expression of osteoclast-differentiating molecules and the stimulation of macrophage differentiation into osteoclasts.     

Comparison of the roles of IL-1, IL-6, and TNFalpha in cell culture and murine models of aseptic loosening

Pro-inflammatory cytokines, such as IL-1, IL-6, and TNF, are considered to be major mediators of osteolysis and ultimately aseptic loosening. This study demonstrated that synergistic interactions among these cytokines are required for the in vitro stimulation of osteoclast differentiation by titanium particles. In contrast, genetic knock out of these cytokines or their receptors does not protect murine calvaria from osteolysis induced by titanium particles. Thus, the extent of osteolysis was not substantially altered in single knock out mice lacking either the IL-1 receptor or IL-6. Osteolysis also was not substantially altered in double knock out mice lacking both the IL-1 receptor and IL-6 or in double knock out mice lacking both TNF receptor-1 and TNF receptor-2. The differences between the in vivo and the cell culture results make it difficult to conclude whether the pro-inflammatory cytokines contribute to aseptic loosening. One alternative is that in vivo experiments are more physiological and that therefore the current results do not support a role for the pro-inflammatory cytokines in aseptic loosening. We however favor the alternative that, in this case, the cell culture experiments can be more informative. We favor this alternative because the role of the pro-inflammatory cytokines may be obscured in vivo by compensation by other cytokines or by the low signal to noise ratio found in measurements of particle-induced osteolysis.     

Orthopedic wear debris mediated inflammatory osteolysis is mediated in part by NALP3 inflammasome activation

Activation of myeloid cells by orthopedic particulate debris is a key event in the pathogenesis of periprosthetic osteolysis and implant loosening after total joint replacement (TJR). Several lines of evidence implicate NACHT, LRR, and PYD domains‐containing protein 3 (NALP3) inflammasome‐mediated production of interleukin 1 beta (IL‐1β) in the pathogenesis of clinical disorders ascribable to foreign particulate materials, including asbestos, silica, and urate crystals. Recent reports indicate that orthopedic polymer products and metallic particulates and ions may activate the same pathway. Here, we investigated the contribution of the NALP3 inflammasome to the pathogenesis of peri‐implant osteolysis. Pharmaceutical and genetic perturbations of caspase‐1 and inflammasome components were used to assess the role of the NALP3 inflammasome in IL‐1β production and osteoclast formation by human monocytes and mouse macrophages in response to polymethylmethacrylate (PMMA) particle phagocytosis. The role of caspase‐1 in a mouse calvarial model of particle‐mediated osteolysis was assessed using µCT. Phagocytosis of PMMA particles induces caspase‐1 dependent release of IL‐1β from human monocytes and mouse macrophages. Importantly, using macrophages from mice deficient in components of the NALP3 inflammasome, we show PMMA‐induced IL‐1β production is strictly dependent on these components. Mice lacking caspase‐1, the sole effector of the NALP3 inflammasome, show reduced orthopedic wear particle‐induced calvarial osteolysis compared to wild‐type controls. Absence of NALP3 inflammasome components fails to alter osteoclast formation in vitro. Our findings identify the NALP3 inflammasome as a critical mediator of orthopedic wear‐induced osteolysis and as a viable therapeutic target for the treatment of periprosthetic osteolysis. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:73–80, 2012     

The importance of cytokines in the posttraumatic inflammatory reaction

Alterations in the immune response after multiple trauma, posttraumatic sepsis and surgery are recognized as physiological reactions of the organism to restore homeostasis. The level of these immunological changes correlates with the degree of tissue damage as well as with the severity of haemorrhage and ischaemia. Cytokines are known to be integral components of this immune response. The local release of pro- and antiinflammatory cytokines after severe trauma indicates their potential to induce systemic immunological alterations. It appears that the balance or imbalance of these different cytokines partly controls the clinical course in these patients. Overproduction of either proinflammatory cytokines or antiinflammatory mediators may result in organ dysfunction. Whereas predominance of the proinflammatory response leads to the systemic inflammatory response syndrome (SIRS), the antiinflammatory reaction may result in immune suppression with an enhanced risk of infectious complications. Systemic inflammation, as well as immune suppression, are thought to play a decisive role in the development of multiple organ dysfunction syndrome (MODS).The major proinflammatory cytokines involved in the response to trauma and surgery include tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6 and IL-8. These cytokines, which are predominantly produced by monocytes and macrophages, mediate a variety of frequently overlapping effects, and their actions can be additive. TNF-alpha and IL-1beta are early regulators of the immune response and both induce the release of secondary cytokines, such as IL-6 and IL-8. IL-10 is an antiinflammatory cytokine which reduces the synthesis of proinflammatory mediators. Other important antiinflammatory mediators are soluble TNF receptors and the IL-1 receptor antagonist, which interfere with the effects of TNF-alpha and IL-1beta.Early evaluation of the prognosis of polytraumatized patients and assessment of their clinical status is known to be difficult. Therefore, in several clinical studies, cytokine levels during the posttraumatic course have been determined with the aim of finding predictive markers of patient outcome. The purpose of this review was to highlight our current knowledge on the interaction of posttraumatic immune reactivity and the development of complications. A better understanding of these mechanisms might lead to the introduction of preventive and therapeutic strategies into clinical practice.     

Modulation of IL-1beta,IL-6, TNF-alpha and PGE(2) by pharmacological agents in explants of membranes from failed total hip replacement

Introduction and goal Proinflammatory cytokines and prostaglandin E(2) (PGE(2)) play an important role in the pathophysiology of osteolysis and implant loosening. The aim of this study was to evaluate the role of pharmacological agents in the inhibition of Interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) and PGE(2) in explants of interface membranes from failed total hip replacements (fTHR).Material and methods Membranes from fTHR were retrieved (N=20) and explants were incubated for 72h in the absence or presence of tenidap at three different concentrations (5, 20 or 50 microg/ml) or diclofenac (125 microg/l). IL-1beta, IL-6, TNF-alpha, and PGE(2) levels were measured in the culture medium using ELISA Capture or EIA kits. Statistical analysis was done using the Mann-Whitney U-test.Results A statistically significant inhibition in IL-1beta synthesis was found at tenidap concentrations of 20 microg/ml (71.3%, P< 0.05) and 50 microg/ml (79.3%,P< 0.02). Tenidap reduced IL-6 levels by 90.4% at 20 microg/ml (P< 0.005) and 96.0% (P< 0.05) at 50 microg/ml. Tenidap also reduced the synthesis of TNF-alpha by 66.9% (P< 0.05) and 77.4% at concentrations of 20 microg/ml and 50 microg/ml. Tenidap had a marked suppressive effect of over 90% (P< 0.0001) on PGE(2) synthesis in all three concentrations. Diclofenac (125 microg/l) decreased PGE(2) production by 95% (P< 0.0001), but had no significant effect in IL-1beta, IL-6, and TNF-alpha levels in the culture medium.Conclusion The ability to simultaneously suppress the release of proinflammatory cytokines and PGE(2) may help control osteolysis and prevent aseptic loosening of THR. This effect could increase implant longevity and lead the way to the pharmacological treatment of this pathology.     

阿伦膦酸钠防治人工关节松动的实验研究

目的：评价二膦酸盐阿伦膦酸钠在防治人工关节松动中的作用。方法：36只SD大鼠右膝置入自制人工关节假体,建立人工关节松动的动物模型,分成3组：阴性对照组,阳性对照组和实验组。阴性对照组关节腔内注射磷酸缓冲液与鼠血清混合液,阳性对照组关节腔内注射10^10/ml关节磨屑（超高分子聚乙烯颗粒）,实验组关节腔内注射10^10/ml关节磨屑同时用阿伦膦酸钠灌胃（每日1mg/kg）。术后12周,处死各组动物行组织切片对比观察假体周围骨溶解情况。体外分离培养人外周血单个核细胞并分成5组,A组为单核细胞组,B组为单核细胞和关节磨屑混合培养组,C组为单核细胞和关节磨屑混合培养加入10^-4mol/L阿伦膦酸钠,D组单核细胞和关节磨屑混合培养加入10^-5mol/L阿伦膦酸钠,E组为单核细胞和关节磨屑混合培养加入10^-6mol/L阿伦膦酸钠,检测各组单个核细胞分泌溶骨因子的情况。结果：关节磨屑可引起假体周围骨溶解,刺激单个核细胞分泌溶骨因子,阿伦膦酸钠可阻止这种作用。结论：阿伦膦酸钠可有效防止关节磨屑诱导的人工关节松动,有望用于临床。     

IL-1 beta induces COX2, MMP-1, -3 and -13, ADAMTS-4, IL-1 beta and IL-6 in human tendon cells.

Overuse injuries and trauma in tendon often involve acute or chronic pain and eventual matrix destruction. Anti-inflammatory drugs have been used as a treatment, however, the cellular and molecular mechanisms of the destructive processes in tendon are not clearly understood. It is thought that an inflammatory event may be involved as an initiating factor. Mediators of the inflammatory response include cytokines released from macrophages and monocytes. Interleukin-1 beta (IL-1 beta) is a candidate proinflammatory cytokine that is active in connective tissues such as bone and cartilage. We hypothesized that tendon cells would express receptors and respond to IL-1 beta in an initial "molecular inflammation" cascade, that is, connective tissue cell expression of cytokines that induce matrix destructive enzymes. This cascade results in expression of matrix metalloproteinases (MMPs) and aggrecanases that may lead to matrix destruction. Normal human tendon cells from six patients were isolated, grown to quiescence and treated with human recombinant IL-1 beta in serum-free medium for 16 h. Total RNA was isolated and mRNA expression assessed by semiquantitative RT-PCR. IL-1 beta (1 nM) induced mRNAs for cyclooxygenase 2 (COX2), MMP-1, -3, -13 and aggrecanase-1 as well as IL-1 beta and IL-6, whereas mRNAs for COX1 and MMP-2 were expressed constitutively. The IL-1 beta-treated tendon cells released prostaglandin E(2) (PGE(2)) in the medium, suggesting that the inducible COX2 catalyzed this synthesis. Induction of PGE(2) was detectable at 10 pM IL-1 beta. IL-1 beta also stimulated MMP-1 and -3 protein secretion. Induction of MMP-1 and -3 was detectable at 10 pM IL-1 beta. Post-injury or after some other inciting events, exogenous IL-1 beta released upon bleeding or as leakage of local capillaries may drive a proinflammatory response at the connective tissue cell level. The resulting induction of COX2, MMP-1 and -3 may underscore a potential for nonlymphocyte-mediated cytokine production of MMPs that causes matrix destruction and a loss of tendon biomechanical properties. Endogenous IL-1 beta might contribute to the process through a positive feedback loop by stimulating expression and accumulation of MMPs in the tendon matrix.     

Role of polyethylene particles in peri-prosthetic osteolysis: A review

There is convincing evidence that particles produced by the wear of joint prostheses are causal in the peri-prosthetic loss of bone, or osteolysis, which, if it progresses, leads to the phenomenon of aseptic loosening. It is important to fully understand the biology of this bone loss because it threatens prosthesis survival, and loosened implants can result in peri-prosthetic fracture, which is disastrous for the patient and presents a difficult surgical scenario. The focus of this review is the bioactivity of polyethylene (PE) particles, since there is evidence that these are major players in the development and progression of osteolysis around prostheses which use PE as the bearing surface. The review describes the biological consequences of interaction of PE particles with macrophages, osteoclasts and cells of the osteoblast lineage, including osteocytes. It explores the possible cellular mechanisms of action of PE and seeks to use the findings to date to propose potential non-surgical treatments for osteolysis. In particular, a non-surgical approach is likely to be applicable to implants containing newer, highly cross-linked PEs (HXLPEs), for which osteolysis seems to occur with much reduced PE wear compared with conventional PEs. The caveat here is that we know little as yet about the bioactivity of HXLPE particles and addressing this constitutes our next challenge.     

Transforming growth factor-beta is involved in the pathogenesis of dialysis-related amyloidosis

BACKGROUND: Advanced glycation end product-modified beta2-microglobulin (AGE-beta2m) is an important component of dialysis-related amyloidosis (DRA). Its presence induces monocyte chemotaxis and the release of the proinflammatory cytokines through macrophage activation. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that also has chemotactic activity for monocytes at very low (0.1 to 10 pg/mL) concentrations and inhibits proinflammatory cytokine production of macrophages. In this study, we investigated the role of TGF-beta in the pathogenesis of DRA. METHODS: We performed an immunohistochemical study of DRA tissues (8 cases) to confirm the existence of TGF-betas and their receptors; we also performed a chemotaxis assay of human monocytes as well as enzyme-linked immunosorbent assay (ELISA) of TGF-beta1, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-1 receptor antagonist (IL-1Ra) in the supernatant of human monocyte-derived macrophage cell culture under varying conditions of incubation with TGF-beta1, AGE-beta2m, and TGF-beta1 antibody additions. RESULTS: There was positive staining for TGF-betas (types 1, 2, and 3) and their receptors (types I, II, and III) in infiltrated macrophages (CD68+), synovial lining cell, as well as vascular walls around amyloid deposition. AGE-beta2m also induced TGF-beta1 production by macrophages in a dose-dependent manner (410 +/- 80 pg/mL at 12.5 microg/mL, 621 +/- 62 pg/mL at 25 microg/mL, and 776 +/- 62 pg/mL at 50 microg/mL of AGE-beta2m). AGE-beta2m induced significant TNF-alpha and IL-1Ra production by macrophage. The addition of exogenous TGF-beta1 (0.1 to 10 ng/mL) decreased AGE-beta2m-induced TNF-alpha production and increased IL-1Ra production in a dose-dependent fashion. IL-1beta production was not effected by any experimental conditions. In chemotaxis assay, anti-TGF-beta1 antibody (0.1 to 10 microg/mL) attenuated AGE-beta2m-induced monocyte chemotaxis. CONCLUSIONS: These results provide the first evidence to our knowledge for the presence of TGF-beta in DRA tissue, as well as the stimulatory action of AGE-beta2m on tissue macrophages. In turn, TGF-beta suppresses the proinflammatory activation of macrophages, suggesting a dual role for TGF-beta in the inflammatory process of DRA. These observations may provide a pathophysiologic link between TGF-beta and DRA.