Although the presence of an elevated level of serum amyloid A (SAA) has been regarded as a cardiovascular risk factor, the role of SAA on the progress of atherosclerosis has not been fully elucidated. In the present study, we investigated the effect of SAA on the production of CCL2, an important mediator of monocyte recruitment, and the mechanism underlying the action of SAA in human monocytes. The stimulation of human monocytes with SAA elicited CCL2 production in a concentration-dependent manner. The production of CCL2 by SAA was found to be mediated by the activation of NF-κB. Moreover, the signaling events induced by SAA included the activation of ERK and the induction of cyclooxygenase-2, which were required for the production of CCL2. Moreover, SAA-induced CCL2 induction was inhibited by a formyl peptide receptor-like 1 (FPRL1) antagonist. We also found that the stimulation of FPRL1-expressing RBL-2H3 cells induced CCL2 mRNA accumulation, but the vector-expressing RBL-2H3 cells combined with SAA did not. Taken together, our findings suggest that SAA stimulates CCL2 production and, thus, contributes to atherosclerosis. Moreover, FPRL1 was found to be engaged in SAA-induced CCL2 induction, and cyclooxygenase-2 induction was found to be essential for SAA-induced CCL2 expression. These results suggest that SAA and FPRL1 offer a developmental starting point for the treatment of atherosclerosis.
Atherosclerosis is the result of an inflammatory macrophage and lymphocyte response to the invading pathogenic lipoproteins in arterial walls (1). Accumulating evidence indicates that macrophages promote arterial lesions and both initiate and facilitate the progression of atherosclerosis. For example, hypercholesterolemic macrophage-deficient mice are extremely resistant to atherosclerosis development (2). CCL2 is a prototype of the CC chemokine subfamily and exhibits the highest chemotactic activity toward monocytes (3). A previous report demonstrated that the overexpression of CCL2 contributes to the development of atherosclerosis in mouse models. Moreover, a deficiency in either CCL2 or its high affinity receptor, CCR2, was found to decrease atheroma and monocyte numbers in vascular lesions (4, 5). Furthermore, the therapeutic gene transfer of dominant negative CCL2 reduced the development of atherosclerosis in apolipoprotein E-null mice (6).
Serum amyloid A (SAA)3 is a major acute phase protein that is released to the circulation in response to infection or injury (7). Several studies have shown that SAA is triggered after liver cells have been stimulated in a proinflammatory environment by the action of several cytokines such as IL-1β (8, 9). Moreover, concentrations of SAA have been reported to be elevated some 1,000-fold during acute phase reactions vs normal levels (8). SAA, which possesses cytokine-like properties, has been reported to play a number of immunomodulatory roles. Past studies have reported SAA to induce proinflammatory cytokine and chemokine production in several cell types such as rheumatoid synoviocytes, intestinal epithelial cells, monocytes, and neutrophils (10, 11, 12).
Furthermore, it has been suggested that SAA may be involved in the metabolism of high density lipoproteins (HDL) and to impede the protective function of HDL on the development of atherosclerosis (13). Recently, Badolato et al. demonstrated that SAA is a potent chemoattractant for human leukocytes such as monocytes and neutrophils (14). Additionally, Su et al. demonstrated that SAA cell surface receptors selectively stimulate formyl peptide receptor-like 1 (FPRL1), thus resulting in Ca2+ mobilization and cell migration (15). Su et al. also showed that radiolabeled SAA specifically bonded to 293 human phagocyte and FPRL1-transfected cells, thus demonstrating that FPRL1 is a SAA-specific receptor (15). However, the roles of SAA and FPRL1 in atherosclerosis have not been clearly elucidated.
In this study, we investigated whether SAA induces CCL2 production in human monocytes. We then further investigated the involvement of FPRL1 in SAA-induced CCL2 production, as well as the signaling pathways involved in this process.
Materials and Methods
Recombinant human SAA (endotoxin level < 0.1 ng/μg) was purchased from Peprotech. FBS and RPMI 1640 medium were purchased from Invitrogen. Histopaque-1077, l-glutamine, antibiotic-antimycotic solution (10,000 IU/ml penicillin, 10 mg/ml streptomycin, and 25 μg/ml amphotericin B), and cycloheximide (CHX) were purchased from Sigma-Aldrich. Actinomycin D (ActD), pertussis toxin (PTX), 2′-amino-3′-methoxyflavone (PD98059), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), and N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS398) were purchased from Calbiochem. (E)-3-[4(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082) was purchased from Biomol. Rabbit anti-human Abs to ERK, phospho-ERKs, and phospho-p38 kinase were purchased from Cell Signaling Technology, whereas the rabbit anti-human Abs to cyclooxygenase-2 (COX-2) were purchased from Cayman Chemical. HRP-conjugated Abs to rabbit IgG were purchased from Kirkegaard & Perry. l-α-Phosphatidylcholine from egg yolk, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) were purchased from Sigma-Aldrich. Enterokinase and the pET30a(+) expression vector, as well as Escherichia coli BL21 (DE3), were purchased from Roche and Novagen, respectively.
Isolation of human peripheral blood monocytes
Peripheral blood was collected from healthy adult donors using citrate as an anticoagulant. PBMC were separated on a Histopaque-1077 gradient. After two washings with Hank’s buffered saline solution without Ca2+ and Mg2+, the PBMC were suspended in RPMI 1640 medium containing 10% FBS and incubated for 60 min at 37°C to allow sufficient time for the monocytes to attach to the culture dish. The attached monocytes were then collected as described previously (16). The isolated cells were then used immediately.
FPRL1-expressing RBL-2H3 cells and vector-transfected RBL-2H3 cells were maintained, as described previously (17), at ∼1 × 106 cells/ml under standard incubator conditions (humidified atmosphere, 95% air, 5% CO2, 37°C).
Cytokine measurement was performed as previously described (18). The monocytes (3 × 105 cells/0.3 ml) were placed in RPMI 1640 medium containing 5% FBS in 24-well plates and kept in a 5% CO2 incubator at 37°C. After stimulation, the cell-free supernatants were collected, centrifuged, and measured for CCL2 by the enzyme-linked immunosorbent assay (BD Biosciences) as per the vendor’s instructions.
Monocytes (1 × 106 cells) were stimulated with 100 nM SAA for the indicated times (0, 1, 3, and 6 h). In addition, mRNA was isolated with the assistance of a QIAshredder and an RNeasy kit (Qiagen). Further, mRNA, Moloney murine leukemia virus reverse transcriptase, and pd(N)6 primers (Invitrogen) were used to obtain cDNA. One microgram of the cDNA and TaqMan real time primers and probes were used for amplification. A set of primers and probes were used for each gene tested (Applied Biosystems). The assay identifiers used for the genes are Hs00234140_ml for CCL2 and Hs99999905_m1 for GAPDH. All PCRs were conducted in a TaqMan Universal PCR master mix (Applied Biosystems) at 900 nM for each primer and 250 nM for each probe. The sequence-specific amplification was detected by increasing the fluorescent signal of FAM (reporter dye) during the amplification cycles of the ABI Prism 7000 real-time PCR system (Applied Biosystems). A PCR was performed for 15 s at 95°C and for 1 min at 60°C for 50 cycles, followed by the thermal denaturation protocol. The amplification of human GAPDH was used in the same reaction of all samples as an internal control. Next, a gene-specific mRNA was normalized to GAPDH RNA. The expression of each mRNA was determined using the 2−ΔCT threshold cycle method (19).
Stimulation of cells with SAA for Western blot analysis
Human monocytes (2 × 106) were stimulated with the indicated quantity (100 nM) of SAA at predetermined times. After stimulation, the cells were washed with serum-free RPMI 1640 medium and lysed in lysis buffer (20 mM HEPES (pH 7.2), 10% glycerol, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 1 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mM PMSF). Next, detergent insoluble materials were pelleted by centrifugation (12,000 × g for 15 min at 4°C), and the soluble supernatant fraction was removed and stored at either −80°C or used immediately. Lastly, the protein concentrations in the lysates were determined using the Bradford protein assay reagent.
Electrophoresis and Western blot analysis
Proteins were separated in 10% SDS-polyacrylamide gel and the proteins were blotted onto a nitrocellulose membrane that was then blocked by incubating with TBST (Tris-buffered saline with 0.05% Tween 20) containing 5% nonfat dry milk. Subsequently, the membranes were incubated with specific Abs and washed with TBST. The Ag-Ab complexes were visualized after incubating the membrane with 1/5000 diluted goat anti-rabbit IgG Ab combined with HRP and detected by ECL.
Vector- or FPRL1-expressing RBL-2H3 cells (1 × 106 cells) were stimulated with SAA (1 μM) and adjusted to a total volume of 0.2 ml for several time periods. The primers used for the RT-PCR analysis have been reported previously (20). The sequences of the primers used included the following: rat CCL2 (453-bp product), 5′-CTCAGCCAGATGCAGTTA-3′ (forward) and 5′-TGGAAGGGAATAGTGTAAT-3′ (reverse); rat actin (250-bp product) 5′-ATGGATGATGATATCGCCGCG-3′ (forward) and 5′-TCTCCATGTCGTCCCAGTTG-3′ (reverse). We ran 30 PCR cycles at 94°C (denaturation, 1 min), 62°C (annealing, 1 min), and 72°C (extension, 1 min). The PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.
Expression and purification of apoA-I
Human apolipoprotein A-I (apoA-I) was expressed and purified using the pET30 expression system (Novagen) and by Ni2+-nitrilotriacetic acid column chromatography according to a previous report (21).
Synthesis of reconstituted HDL and HDL-conjugated SAA
Discoidal reconstituted HDL was prepared with purified apoA-I (at least 95% purity) via sodium cholate dialysis with a POPC:cholesterol:apoA-I:sodium cholate molar ratio of 95:5:1:150, as previously described (22). HDL-SAA was prepared with apoA-I and SAA via sodium cholate dialysis with a POPC:cholesterol:apoA-I:SAA:sodium cholate molar ratio of 95:5:1:1:150. All HDL showed similar range of residual endotoxin levels between 3.1 and 3.3 endotoxin units/ml based on endotoxin quantification using a commercially available test kit (BioWhittaker).
Chemotaxis assays were performed using multiwell chambers (Neuroprobe) (16). Briefly, prepared human monocytes were suspended in RPMI 1640 medium at a concentration of 1 × 106 cells/ml, and 25 μl of the suspension were placed onto the upper well of a chamber that was separated by a 5-μm polyhydrocarbon filter from agonists contained in the lower well. After incubation for 2 h at 37°C, nonmigrated cells were removed by scraping them out and cells that had migrated across the filter were dehydrated, fixed, and stained with hematoxylin (Sigma-Aldrich). The stained cells in five randomly chosen high power fields (HPF) (×400) in that well were then counted (16).
The results are expressed as the means ± SE. The Student’s t test was used to compare individual treatments with their respective control values. Statistical significance was set at p < 0.05.
SAA stimulates CCL2 production in human monocytes
To investigate the effect of SAA on CCL2 production in human monocytes, the cells were stimulated with SAA (100 nM) at various times (0, 1, 4, 8, 16, 24, and 36 h). As shown in Fig. 1,A, SAA induced CCL2 production in a time-dependent manner and showed maximal activity at 24 h after stimulation. We also tested the relationship of CCL2 production as a result of the influence of SAA. SAA was found to induce CCL2 production in a concentration-dependent manner, with a maximal activity rate at 10 nM for SAA (Fig. 1,B). These results suggest that SAA stimulates CCL2 in both a time- and concentration-dependent manner. Because the SAA used in the present study was recombinant SAA produced in E. coli, we examined the possible contribution of LPS to SAA-induced CCL2 production by using polymyxin B (a potent inhibitor of LPS). However, SAA-induced CCL2 production was unaffected by polymyxin B (Fig. 1,C), thus indicating that SAA is not mediated by LPS during CCL2 production. We also examined the effect of SAA on the accumulation of CCL2 mRNA transcripts via real time-PCR. As shown in Fig. 1 D, the stimulation of human monocytes with SAA (100 nM) caused CCL2 mRNA transcript accumulation in a time-dependent manner. Moreover, the level of this transcript increased significantly after stimulating the cells for 1–6 h with SAA. These results indicate that CCL2 induction by SAA requires transcriptional activation and de novo protein synthesis.
SAA stimulates CCL2 production via NF-κB activity in human monocytes
To investigate the mechanism involved in SAA-induced CCL2 production, we pretreated human monocytes with the transcription inhibitor ActD or the protein synthesis inhibitor CHX. When human monocytes were pretreated with ActD or CHX before adding SAA, the SAA-induced CCL2 production was almost completely inhibited (Fig. 2 A).
Moreover, the expression of the CCL2 gene has also been reported to require the activation of NF-κB (23, 24). We previously reported via EMSA and Western blot analysis that the stimulation of human monocytes with SAA elicits the activation of NF-κB (25). To investigate the role of NF-κB on SAA-induced CCL2 production, we pretreated human monocytes with a NF-κB inhibitor, (i.e., BAY 11-7082) before adding SAA. As shown in Fig. 2 B, BAY 11-7082 (at 5 μM) blocked SAA-induced CCL2 production by 95%, thus indicating that NF-κB activation is essential for SAA-induced CCL2 production in human monocytes.
SAA-induced CCL2 production is PTX sensitive
A previous report demonstrated that SAA causes intracellular Ca2+ release and Ca2+ influx (15). Additionally, PTX-sensitive G proteins have been reported to play a role in SAA receptor-mediated Ca2+ signaling (26). In this study, we found that SAA induced CCL2 production (Fig. 1), and we consequently investigated the role of the sensitive G protein on SAA-induced CCL2 production in human monocytes. We found that SAA-induced CCL2 production was almost completely inhibited by preincubating cells with 100 ng/ml PTX for 24 h (Fig. 3). However, phorbol ester-induced CCL2 production was not significantly affected by PTX (data not shown). Thus, CCL2 production in response to SAA appears to be mediated by the PTX-sensitive G protein-linked receptor.
ERK regulates CCL2 production by SAA
MAPK has been reported to mediate extracellular signals to the nucleus in several cell types (27). When human monocytes were stimulated with 100 nM SAA for 5 min, the phosphorylation level of ERK dramatically increased (Fig. 4,A). Another important MAPK, p38 kinase, was also activated (Fig. 4,A). To examine the role of PTX-sensitive G proteins on SAA-induced MAPK activation, we pretreated human monocytes with 100 ng/ml PTX before SAA stimulation. Fig. 4 A shows that PTX completely blocked SAA-induced ERK and p38 kinase activation. These results suggest that PTX-sensitive G proteins participate in SAA receptor activation of the MAPK cascade.
To determine the role of individual MAPK on SAA-induced CCL2 production, we preincubated human monocytes with PD98059 (a selective MEK inhibitor) or with SB203580 (a selective p38 kinase inhibitor) before SAA treatment. As shown in Fig. 4 B, SAA-induced CCL2 production was dramatically inhibited by PD98059, but not by SB203580. Consequently, we demonstrated that MEK-dependent ERK, but not p38 kinase, is essential for SAA-induced CCL2 production.
SAA-induced COX-2 is involved in CCL2 production by SAA
We examined the effects of SAA on COX-2 expression by Western blotting with anti-COX-2 Ab. SAA was found to strongly stimulate COX-2 expression in a time-dependent manner. Moreover, COX-2 expression was detected 3 h after stimulation and increased up to 6–12 h after stimulation (Fig. 5,A). To examine the role of PTX-sensitive G proteins on SAA-induced COX-2 expression, we pretreated human monocytes with 100 ng/ml PTX before SAA stimulation. Fig. 5 B shows that PTX almost completely blocked SAA-induced COX-2 expression. These results suggest that SAA stimulates COX-2 expression via PTX-sensitive G proteins in human monocytes.
To determine the role of COX-2 on SAA-induced CCL2 production, we preincubated human monocytes with NS398, a selective COX-2 inhibitor, before SAA treatment. As shown in Fig. 5 C, SAA-induced CCL2 production was dramatically inhibited by NS398, thus demonstrating that COX-2 is essential for SAA-induced CCL2 production.
SAA stimulates CCL2 production via FPRL1
The finding that SAA (an FPRL1 agonist) elicited CCL2 induction in human monocytes led us to examine whether other formyl peptide receptor (FPR) family agonists also stimulate CCL2 expression. Thus, we investigated the effects of several FPR family agonists such as fMLF (28), MMK-1 (29), WKYMVm (Trp-Lys-Tyr-Met-Val-d-Met) (30), LL-37 (31), or F2L (32) on CCL2 expression. As shown in Fig. 6 A, none of these FPR family agonists, except for SAA, affected CCL2 induction in human monocytes, which suggests that CCL2 expression via FPRL1 is SAA selective. In a separate experiment, we observed all of the tested FPR family agonists, including SAA, stimulated chemotactic migration in human monocytes (data not shown).
To support our notion that SAA stimulates CCL2 production in human monocytes via FPRL1, we examined the effect of an FPRL1 antagonist, WRW4 (Trp-Arg-Trp-Trp-Trp-Trp) (17). The pretreatment of WRW4 dramatically inhibited SAA- but not LPS-induced CCL2 production in human monocytes (Fig. 6,B). To further support the notion that SAA stimulates CCL2 production via FPRL1 in human monocytes, we investigated the effects of SAA on CCL2 expression in FPRL1-expressing RBL-2H3 cells. As shown in Fig. 6 C, the stimulation of FPRL1-expressing RBL-2H3 cells but not vector-expressing RBL-2H3 cells with 1 μM SAA elicited CCL2 mRNA accumulation. The results indicate that SAA stimulates FPRL1, which results in CCL2 expression. The other tested FPRL1 agonists, except for SAA, failed to stimulate CCL2 mRNA accumulation in both vector- or FPRL1-expressing RBL-2H3 cells (data not shown). Taken together, we strongly suggest that SAA stimulate CCL2 production via FPRL1 in human monocytes.
HDL-conjugated SAA also stimulates CCL2 production in human monocytes
It has been previously reported that SAA is conjugated with HDL under pathophysiological conditions (33). To test whether the conjugated SAA also invokes a stimulatory effect on CCL2 production, we first purified apoA-I as described previously (22). The purity of apoA-I and SAA were at least 95%, as per densitometric analysis (Fig. 7,A). The reconstituted HDL preparations exhibited two major bands (97 and 98 Å) of POPC-HDL (Fig. 7,B, lane 1), as demonstrated previously (22, 34). This reconstituted HDL is a typical HDL containing only apoA-I, which showed in vitro antioxidant activity and in vivo anti-inflammatory activity in a hypercholesterolemic mice model (35, 36). However, HDL-SAA showed an additional band with a smaller particle size (71 Å), as well as two major bands (Fig. 7,B, lane 2). The appearance of smaller HDL (71 Å) by the addition of SAA might result from the displacement of apoA-I on HDL by SAA (37). The hallmark of acute phase HDL from infection and inflammation is an increase in SAA and a reduction in apoA-I content (38). Next, we investigated the effect of HDL-conjugated SAA and HDL alone on CCL2 production in human monocytes. As shown in Fig. 7,C, both SAA and HDL-conjugated SAA dramatically stimulated CCL2 production in human monocytes. However, HDL alone did not have a significant influence effect on the CCL2 production from human monocytes (Fig. 7,C). We also tested the chemotactic activity of HDL-conjugated SAA for monocytes. As shown in Fig. 7 D, both SAA and HDL-conjugated SAA stimulated chemotactic migration of human monocytes. HDL alone did not affect monocyte chemotaxis. These results are consistent with our finding that HDL-conjugated SAA stimulated CCL2 production in human monocytes.
In this study, we investigated the effect of SAA on the regulation of CCL2 from human monocytes. It was observed that SAA induced the concentration-dependent production of CCL2 in these monocytes. Moreover, this effect of SAA on CCL2 production was found to be associated with the accumulation of CCL2 mRNA, thus suggesting that SAA induces cytokine production at the transcriptional level. It was also found that SAA stimulates CCL2 production via NF-κB activation downstream of FPRL1 activation. Thus, this study provides the first evidence that SAA induces the production of CCL2 via FPRL1 activation.
SAA is regarded as a proinflammatory mediator and is highly elevated both in the circulation and locally in tissues during various pathologic conditions such as atherosclerosis (39). Previous reports suggest that an acute phase response is closely associated with lipoprotein abnormalities and that SAA, which is mainly associated with plasma HDL, is involved in the development of atherosclerosis (40, 41). In fact, SAA expression has been reported in human atherosclerotic lesions (41), and it has been suggested that SAA is involved in lipoprotein retention in atherosclerosis (42). It has also been demonstrated that oxidized low density lipoprotein induces acute phase SAA, thus suggesting that SAA participates in vascular injury and atherosclerosis (43). According to previous reports, low concentrations of SAA are associated with HDLs; however, free SAA is also found at higher concentrations of SAA in the circulation (44, 45). Marked increases in SAA have been used as an important indicator of the presence of inflammatory diseases such as atherosclerosis (46). However the direct role of SAA on the pathology of atherosclerosis is unclear. In this study, we demonstrated that SAA directly stimulates CCL2 production in monocytes. This result indicates that SAA up-regulation plays an important role by eliciting CCL2, an important chemokine. Current results have shown that HDL-SAA (apoA-I: SAA = 1:1 molar ratio) could induce the inflammatory process via the stimulation of CCL2. This result is consistent with a recent report that states that HDL lost its anti-inflammatory and antioxidant activity when SAA content increased in HDL during the acute inflammatory phase of HDL (47).
In this study, we also suggest for the first time that FPRL1, a chemoattractant receptor, plays a role in the production of CCL2 by SAA. SAA is an endogenous ligand of FPRL1 and was found to stimulate CCL2 production in human monocytes by activating ERK and NF-κB. We demonstrated that SAA-induced CCL2 production was completely inhibited by an FPRL1 antagonist (WRW4) in human monocytes (Fig. 6,B). Moreover, we subsequently found that CCL2 mRNA accumulation is induced by SAA in FPRL1-expressing RBL-2H3 cells, but not in vector-transfected RBL-2H3 cells (Fig. 6,C). These results strongly indicate that SAA stimulates the production of CCL2 via FPRL1. In the present study, we also found that the SAA-induced CCL2 expression is almost completely inhibited by PTX (Fig. 3). Because FPRL1 signaling is known to be mediated by PTX-sensitive G proteins, these results also support the notion that SAA stimulates FPRL1, which is coupled with PTX-sensitive G proteins, thus resulting in CCL2 expression.
In regard to the intracellular signaling mechanism involved in SAA-induced cytokine production, we found that ERK is essential for the expression of CCL2 caused by the activation of human monocytes by SAA (Fig. 4). A previous report has also demonstrated that ERK activity is essential for the expression of several proinflammatory cytokines, such as CCL2, by extracellular stimuli and that these stimuli include placenta growth factor (48). In addition, we found that SAA-induced CCL2 production is associated with COX-2 induction. As shown in Fig. 5, SAA was found to stimulate COX-2 induction via the PTX-sensitive G protein. Previously, Linton and colleague demonstrated that COX-2 promotes early atherosclerotic lesion formation in apolipoprotein E-deficient mice (49). They also showed that CCL2 production by proinflammatory stimuli (IL-1β and LPS) is reduced in COX-2-deficient mice (50), thus indicating the essential role of COX-2 for the recruitment of monocytes during the early atherosclerotic process. In the present study, we found that SAA-induced CCL2 production is dramatically inhibited by NS-398, a COX-2 selective inhibitor (Fig. 5 C). These results indicate that COX-2 induction is a prerequisite for the production of CCL2 by SAA.
In terms of the ligand selectivity of FPRL1-mediated CCL2 production, we found that SAA stimulated CCL2 production (Fig. 6,A), which was not the case for the rest of the other FPRL1 agonists examined (MMK-1, WKYMVm, and LL-37). In terms of the mechanisms involved in the differential effect of SAA or WKYMVm on the production of CCL2, we have already demonstrated that SAA does not inhibit 125I-labeled WKYMVm to bind FPRL1 (51), which suggest that SAA and WKYMVm bind to FPRL1 at different sites. Thus, we suggest that FPRL1 is differentially stimulated by these two ligands. Furthermore, this differential production of CCL2 may be mediated by the nonoverlapping binding of these two ligands to FPRL1. Previously Hancock et al. reported that the human antimicrobial peptide LL-37, which is a host-derived FPRL1 agonist, stimulates CCL2 induction in RAW 264.7 cells and whole human blood (52). In the present study, we did not observe any dramatic increases in CCL2 after treating human monocytes with LL-37 (Fig. 6 A). However, it is not currently clear what causes the differential effect of LL-37. To resolve this issue, investigations on the effects of several FPRL1 ligands on CCL2 production are required in various cell types.
In conclusion, we showed for the first time that SAA stimulates CCL2 production via FPRL1 in human monocytes. This stimulatory effect of SAA is accomplished through ERK-, NF-κB-dependent and COX-2-dependent pathways. Our results suggest that the production of CCL2 by FPRL1 activation of SAA might be involved as a part of the atherosclerotic effects and provide SAA and FPRL1 as potential therapeutic targets of the atherosclerosis.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by the Korea Science and Engineering Foundation Grant R01-2007-000-11241-0 funded by the Korean government (Ministry of Science and Technology) and by Korea Health 21 Research and Development Project Grant A060065 from the Ministry of Health and Welfare, Republic of Korea.
Abbreviations used in this paper: SAA, serum amyloid A; ActD, actinomycin D; apoA-I, apolipoprotein A-I; BAY 11-7082, (E)-3-[4(4-methylphenyl)-sulfonyl]-2-propenenitrile; CHX, cycloheximide; COX-2, cyclooxygenase-2; FPR, formyl peptide receptor; FPRL1, FPR-like 1; HDL, high density lipoprotein; NS398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; PD98059, 2′-amino-3′-methoxyflavone; PTX, pertussis toxin; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole.