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SAA induces reactive oxygen species production

Clinical and Experimental Immunology



Serum amyloid A induces reactive oxygen species (ROS) production and proliferation of ?broblast
cei_4300 362..367

E. Hatanaka,*1 A. Dermargos,?1 H. A. Armelin,? R. Curi? and A. Campa§
*Instituto de Ciências da Atividade Física e Esportes, Universidade Cruzeiro do Sul, ? Departamento de Bioquímica, Instituto de

Summary Serum amyloid A (SAA) levels are elevated highly in acute phase response and elevated slightly and persistently in chronic diseases such as rheumatoid arthritis and diabetes. Given that ?broblasts exert profound effects on progression of in?ammatory chronic diseases, the aim of this study was to investigate the response of ?broblasts to SAA. A dose-dependent increase in O2levels was observed by treatment of ?broblasts with SAA (r = 0·99 and P 0·001). In addition, the expression of p47-phox was up-regulated by SAA (P < 0·001) and diphenyliodonium (DPI), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, reduced the release of O2- by 50%. Also, SAA raised ?broblast proliferation (P < 0·001) and this effect was completely abolished by the addition of anti-oxidants (P < 0·001). These ?ndings support the notion that, in chronic in?ammatory sites, SAA activated ?broblast proliferation and ROS production. Keywords: in?ammation, proliferation, reactive oxygen species, SAA

Química, Universidade de S?o Paulo, Departamento de Fisiologia e Biofísica,

Universidade de S?o Paulo, and §Departamento de Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, S?o Paulo, Brazil Accepted for publication 9 November 2010 Correspondence: E. Hatanaka, Universidade Cruzeiro do Sul, Rua Galv?o Bueno, 868, 13° andar, Liberdade, 01506-000, S?o Paulo, SP, Brazil. E-mail: ehata@usp.br; elaine.hatanaka@cruzeirodosul.edu.br

These authors contributed equally to the study.

Serum amyloid A (SAA), a classical acute phase protein, plays a role in the in?ammatory process by increasing production of cytokines [1–4] nitric oxide [5] and reactive oxygen species (ROS) by leucocytes [6]. These SAA functions are particularly relevant in conditions of persistent elevated plasma SAA levels, as observed in chronic diseases such as rheumatic arthritis [7], cancer [8] and diabetes [9]. Several SAA receptors have been described, including CD36 and LIMPII analogous-1 (CLA-1), [10] lipoxin A1 receptor/formyl peptide receptor 1 (FPRL1) [11], tanis, a hepatic receptor activated by glucose [12] and Toll-like receptor-4 (TLR-4) [5] and TLR-2 [13]. It has been reported recently that SAA activates rheumatoid synovial ?broblasts by binding to receptors of advanced glycation end products (RAGE) [14]. Also, a high-density lipoprotein receptor, the scavenger receptor class B type 1 (SR-B1), is expressed in RA synovial tissue and is apparently involved in SAA-induced in?ammation in arthritis [15]. Persistent in?ammation, ?broblast de?cient/excessive proliferation and endothelial cell hypertrophy have been implicated in various aspects of chronic diseases [16]. Under

these conditions the role of ROS in cellular dysfunction, in particular in signal transduction, is also increasingly recognized [17]. Identi?cation of SAA cellular targets is very important for the full understanding of the SAA effects. Persistently elevated SAA levels induce a reactive form of amyloidosis in peripheral tissues, leading to progressive organ failure associated with amyloidal accumulation [18]. Amyloidosis is associated commonly with ROS and the formation/ development of excess ?brous connective tissue in an organ or tissue by ?broblast. As a result, ?broblasts exert profound effects on progression of chronic degenerative diseases. Thus, the purpose of this study was to investigate the effect of SAA on Swiss 3T3 ?broblasts ROS production and proliferation.

Materials and methods
Phenol red, hydroethidine and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Lucigenin was supplied by Sigma Chemical Co. (St Louis, MO, USA). Recombinant human Apo-SAA 1 was obtained from Peprotech (Rocky Hill, NJ, USA).

? 2010 The Authors Clinical and Experimental Immunology ? 2010 British Society for Immunology, Clinical and Experimental Immunology, 163: 362–367

SAA effects on ?broblasts

Cell line
Swiss 3T3 ?broblasts, obtained from the American Type Culture Collection, were maintained at 37°C in Dulbecco’s modi?ed Eagle’s medium (DMEM) supplemented with 10% (v : v) fetal bovine serum (FBS), ampicilin (25 mg/ml) and streptomycin (100 mg/ml). Adherent ?broblasts were treated with trypsin, washed gently, and resuspended in phosphatebuffered saline (PBS). Cell viability was always greater than 98%, as indicated by ?ow cytometry.


[H]-Thymidine incorporation

Fibroblasts were plated in DMEM with 10% fetal calf serum (FCS). Six hours later, the medium was removed and replaced by DMEM with 0·5% FCS. Thirty-six hours afterwards, the medium was removed and cells were stimulated with SAA to trigger DNA synthesis. Twelve hours later, [3H]-methyl-thymidine (1 mCi/ml) was added and incorporation measurement was detected 12 h later. Cells then were lysed, DNA was precipitated and collected on ?lter paper and counted in a scintillation counter.

Superoxide anion determination
Lucigenin (1 mM) was added to ?broblast (2·5 ? 106 cells/ ml) incubation medium. Immediately afterwards, cells were treated with SAA (0, 5, 10 and 15 mg/ml). ROS release was monitored for 30 min in a Microplate Luminometer (EG&G Berthold LB96V, New Haven, CT, USA). The assays were run in PBS buffer supplemented with CaCl2 (1 mM), MgCl2 (1·5 mM) and glucose (10 mM) at 37°C [19].

Growth curve
Fibroblasts (2 ? 104 cells/cm2) were plated in DMEM with 0·5% FCS and SAA (1 mg/ml), changing the medium every third day, and the total number of cells was counted in a Fuchs–Rosenthal camera.

Statistical analysis
Comparisons were performed by one-way analysis of variance (anova) and Dunnett test. Results were obtained from three to ?ve separate experiments and are expressed as mean standard error of the mean (s.e.m.).

Hydrogen peroxide determination
Fibroblasts (2·5 ? 106 cells/ml) were treated with 15 mg/ml of SAA in the presence of phenol red (0·28 mM) and 1 U/ml horseradish peroxidase (HRP) type II. H2O2 release was monitored for 30 min by measurement at 610 nm. The reaction was stopped by addition of 10 ml 1 N NaOH [20].

Results and discussion
To test if SAA was able to trigger ROS production in 3T3 cells we used three assays: hydroethidine reduction, phenol red reduction and lucigenin-ampli?ed chemiluminescence. The ROS measurements were taken under conditions in which the interference of the other effects was minimized; appropriate controls were carried out using SAA in the assays without cells. The SAA did not directly affect the luminol, lucigenin and phenol red assays. The possible interference of SAA in the reaction of ROS with the reagents was tested in a previous study conducted by our group by adding xanthine and xanthine oxidase to lucigenin and luminol assays, without cells. These tests did not indicate a direct effect of SAA on the lucigenin, luminol and phenol red ROS detecting systems. Furthermore, the lucigenin-ampli?ed chemiluminescence probe is the most sensitive test to measure the rise in O2- levels and causes less ROS cycling interference than other probes for measuring ROS. Due probably to the higher sensitivity of the lucigenin-ampli?ed chemiluminescence probe, we were able to measure the rise in O2- levels only by the lucigenin-ampli?ed chemiluminescence assay (Fig. 1a). No detectable ROS production was observed with assays using hydroethidine reduction and phenol red reduction. There was a positive correlation between SAA concentration and O2- production (Fig. 1b), and Pearson’s correlation found r = 0·99 and P = 0·001 (Fig. 1a).

Flow cytometric measurement of reactive oxygen metabolites using hydroethidine
Fibroblasts (2·5 ? 106 cells/ml) were treated with 15 mg/ml of SAA in the presence of hydroethidine (1 mM). Fluorescence was measured using the FL3 (480 nm) channel in a ?uorescence activated cell sorter (FACSCalibur) ?ow cytometer (Becton Dickinson, San Juan, CA, USA). Ten thousand events were analysed per experiment [21].

RNA extraction and quantitative reverse transcriptase–polymerase chain reaction (qRT–PCR)
After incubation at 37°C for 1 h in the absence or presence of SAA, RNA of ?broblast was extracted by using Trizol reagent (Invitrogen Life Technologies). Total RNA was then reversetranscribed into cDNA by using Superscript II RT (Invitrogen) with oligo random hexamers. Prepared cDNA was subjected to qPCR analysis by using ROTOR GENE 3000 equipment (Corbett Research, Mortlake, Australia) with SYBR Green Master Mix (Applied Biosystems, Warrington, UK). Quanti?cation of gene expression was based on cycle threshold (Ct) values, using b2 myosin genes as inner control [22].

? 2010 The Authors 363 Clinical and Experimental Immunology ? 2010 British Society for Immunology, Clinical and Experimental Immunology, 163: 362–367

E. Hatanaka et al.
(a) 160 Chemiluminescence (RLU)
Table 1. Effect of serum amyloid A (SAA) (15 mg/ml) on mRNA expression of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase component p47-phox in Swiss 3T3 ?broblasts. p47-phox mRNA (relative units) (mean standard deviation) Control SAA 1 791 0·04 186***

Control + SAA



***P < 0·001 for comparison between control and SAA treatment.

40 0 3 6 9 12 Time (min) 15 18 21

(b) 7500 Chemiluminescence (RLU) 6000 4500 3000 1500
Chemiluminescence (RLU)
r = 0?99 P < 0?004


5 10 SAA (μg/mL)






SAA (μg/ml) (c) 5000 Chemiluminescence (URL) 4000 3000 2000 1000 ##




Fig. 1. (a) Representative kinetic of O2- production measured by lucigenin-enhanced chemiluminescence in 3T3 ?broblasts (2·5 106 cells/ml) in the presence of serum amyloid A (SAA) (15 mg/ml). (b) Dose-dependent effect of SAA (5, 10 and 15 mg/ml) on O2- anion production measured by lucigenin-enhanced chemiluminescence in 3T3 ?broblasts (2·5 ? 106 cells/ml). (c) Effect of diphenyliodonium (DPI) (20 mM on the lucigenin-enhanced chemiluminescence of SAA (10 mg/ml)-treated 3T3 ?broblasts. ***P < 0·001 for comparison between control and SAA treatment. ##P < 0·01 for comparison between SAA and SAA plus DPI treatments.

ROS production seems to occur, at least in part, due to activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes, as diphenyleneiodonium (DPI), a speci?c inhibitor of NADPH oxidase, reduced O2production induced by SAA by 50% (Fig. 1c). Also, the expression of p47-phox was up-regulated by SAA, as evaluated by qPCR (Table 1). The mechanism of O2- production by a membrane-bound NADPH oxidase involves translocation of the cytosolic subunits to the membrane. Nonphagocytic cells express a speci?c pattern of NADPH oxidase subunits. In ?broblasts, p47phox is one of the cytosolic subunits that have a regulatory function. In these cells the active enzyme migrates from the cytosol to the plasma membrane during the activation process [17]. In neutrophils, we have already shown that SAA primes cells, increasing the amount of ROS produced in response to opsonized stimulus [6]. Recently it has been described that SAA directly induces activation of the neutrophil NADPH oxidase [23]. Activation of the NADPH oxidase complex in neutrophils and other phagocytes has been implicated in killing activity [24]. However, in non-phagocytic cells, the production of ROS triggered by a variety of ligands may be important as second messengers. Ligands such as several growth factors and cytokines induce production of ROS by ?broblasts, endothelial and other cell types [17]. This process is relevant depending upon the amount and duration of ROS modulation. SAA stimulated [3H]-thymidine uptake into DNA (Fig. 2a), directly indicating an increase in the relative rate of DNA synthesis, implying that SAA promotes Swiss 3T3 cell proliferation. These observations were con?rmed in the cell growth curve experiment (Fig. 2b) after 6–8 days of incubation. Fibroblast proliferation and ROS production induced by SAA were correlated events, as indicated by the inhibitory effect of the antioxidant agents, N-acetyl-lcysteine (NAC) and a-tocoferol, on cell proliferation (Fig. 2c). ROS activates cell proliferation by several pathways, as follows: (i) through the action of protein tyrosine phosphatases (PTPs). PTPs control the phosphorylation state of numerous signal-transducing proteins and are therefore involved in the regulation of cell proliferation, differentiation, survival, metabolism and motility. The catalytic region of PTPs includes cysteines, which are susceptible to oxidative inactivation. Thus, ROS decrease phosphatase activity that enhances protein tyrosine phosphorylation,


? 2010 The Authors Clinical and Experimental Immunology ? 2010 British Society for Immunology, Clinical and Experimental Immunology, 163: 362–367

SAA effects on ?broblasts
(a) [3H]-thymidine incorporation (%)






1 SAA (μg/ml)


(b) Control + SAA
### ###

25 Cells number (×104)/ml 20 15 10 5 0

*** *** *** **


** ***


4 Days



(c) [3H]-thymidine incorporation (%) 150


### ###





+ α-tocoferol

Fig. 2. (a) Percentage of [3H]-thymidine incorporation into 3T3 cells treated with 1 and 10 mg/ml serum amyloid A (SAA). **P < 0·01 for comparison between control and SAA treatment. (b) Growth curves of 3T3 ?broblasts in the absence and presence of SAA (15 mg/ml). *Signi?cant differences in relation to day 0; #between the control and SAA treatment curves. (c) Inhibitory effect of the anti-oxidants N-acetyl-l-cysteine (NAC) (10 mM) and a-tocoferol (400 mM) on [3H]-thymidine incorporation in 3T3 cells treated with SAA (1 ng/ml). The results are representative of three experiments performed in triplicate. ***P < 0·001 for comparison between control and SAA treatment; ###P < 0·001 for comparison between SAA and SAA plus anti-oxidant treatments.

thereby in?uencing signal transduction. (ii) Treatment of cells with hydrogen peroxide leads to phosphorylation and activation of p38 mitogen-activated protein (MAP) kinase. This probably occurs due to the activation of upstream signalling pathways of extracellular-regulated kinase (ERK1/2), or it may be an indirect effect of the inhibition of phosphatase activity by ROS. (iii) ROS can regulate intracellular and plasma membrane ion channels. Such regulation of ion channels may occur either directly or through ROS-sensitive signalling systems. Cytosol-free Ca+2 concentrations act as important intracellular messenger systems [17]. The effect of SAA on cell proliferation has been shown previously on human ?broblast-like synoviocytes. Moreover, SAA stimulated the proliferation, migration and tube formation of endothelial cells [25]. Further studies are needed to identify the speci?c receptor(s) involved in SAA-induced ROS production and ?broblast proliferation. Regulation of the intracellular redox state by growth factor-induced changes of NADPH oxidase activity is thought to have an important impact on redox-sensitive signalling cascades. Activation of growth factor-stimulated signalling cascades by low levels of ROS results in increased cell cycle progression. For example, the proliferative state of ?broblasts is associated closely with intracellular ROS levels. Low ROS levels lead to cell growth arrest, as induced by contact inhibition. Conversely, overproduction or insuf?cient scavenging of ROS can result in enhanced oxidative stress and membrane and DNA damage, which have been implicated in cancer initiation and promotion, apoptosis and necrosis [17]. High concentrations of SAA associated with increased microvascular permeability observed in some diseases [26] may de?ne speci?c loci, such as the interstitial space and body cavities, for SAA action. In these sites, SAA may activate neutrophils and macrophages and promote ?broblast proliferation. Furthermore, it has to be considered that local production of SAA, especially by endothelial cells, adipocytes, macrophages and own ?broblasts, may supply SAA even without a remarkable in?ammatory process. SAA serum levels are normally very low (1–3 mg/ml). In circulation, SAA associates predominantly with the third fraction of high-density lipoproteins (HDL3). SAA associated with HDL3 is not considered a proin?ammatory agent [1]. In in?ammatory foci, SAA may dissociate from HDL3 and be active. The concentration of free SAA in an in?ammatory site is not known. Nevertheless, we showed that SAA is active on ?broblasts in concentrations where it is possible to ?nd SAA. After the dissolution of HDL, SAA is absorbed by macrophages and in lysosomes it is cleaved by cathepsins. An impaired SAA degradation process leads to accumulation of the amino acid intermediate. The acid environment of lysosomes facilitates the formation and polymerization of these intermediates. After deposition of accumulated intermediates in the extracellular space, glycosaminoglycans, serum amyloid P (SAP) and lipid components bind to the ?brils

? 2010 The Authors 365 Clinical and Experimental Immunology ? 2010 British Society for Immunology, Clinical and Experimental Immunology, 163: 362–367

E. Hatanaka et al.

and confer resistance to proteolysis. In normal circumstances, SAA is degraded completely. In fact, any alteration in the genes responsible for maintaining the normal process or that persistently elevates the SAA serum level may in?uence the formation of AA amyloidosis. A large part of human AA proteins isolated from amyloid deposits derives from SAA1. AA amyloidosis is, in part, in?uenced by genetic factors. For example, Japanese patients with rheumatoid arthritis show a higher association of SAA1 polymorphisms and AA amyloidosis, speci?cally C13T and C2995T polymorphisms [27]. At the same concentration of SAA, ?broblasts synthesize more ROS than neutrophils. SAA alone does not trigger ROS production in neutrophils, but augments the maximum ROS production rate when a second stimulus is added. This effect de?nes SAA as a priming agent of neutrophils [6]. Neutrophils play an essential role in host defences against microbial pathogens and their anti-microbial arsenal is composed of ROS. In phagocytes, superoxide is generated mainly by the reaction of oxygen and NADPH through the NADPH oxidase complex. Superoxide anions and hydrogen peroxide (H2O2) generated by NADPH oxidase give rise to other ROS that are strong cytolytic agents, such as hypochlorous acid [formed by the action of myeloperoxidase (MPO) released from neutrophil granules] and hydroxyl radicals. The ?nding that SAA primes neutrophils suggests that SAA has a concerted mode of action, driving a more powerful response of innate host defence. In fact, neutrophils are classic and more potent cell producers of ROS than ?broblasts. Fibroblast functions involve synthesis of the extracellular matrix and collagen. These cells play a critical role in wound healing. Fibroblasts are not classic cells ‘programmed’ to produce ROS. In ?broblasts, ROS are signalling molecules for other cell functions, such as differentiation, proliferation, death, senescence and production of cytokines. Fibroblast proliferation and ?brogenesis are important factors that lead to complications of various diseases, such as atherosclerosis, rheumatic arthritis, diabetic nephropathy and retinopathy [28]. Data from this study highlight SAA as an important inducer of ROS production and proliferation of ?broblasts. This effect may be critical during physiological processes such as would healing, for example. However, it can be deleterious in promoting uncontrolled in?ammation in proliferative diseases that involve ?broblast dysfunction, such as ?brosis [25]. This is more relevant in pathological conditions with a persistent increase of SAA serum levels, such as diabetes [9], rheumatic diseases [7] and cancer [8].

The authors declare that there is no con?ict of interest.

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All authors thank Funda??o de Amparo à Pesquisa do Estado de S?o Paulo, Brasil (FAPESP) and Conselho Nacional do Desenvolvimento Cientí?co e Tecnológico, Brasil (CNPq) for grants and fellowships.

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? 2010 The Authors 367 Clinical and Experimental Immunology ? 2010 British Society for Immunology, Clinical and Experimental Immunology, 163: 362–367