Published online before print November 5, 2007
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* Department of Rheumatology and Inflammation Research, The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden;
CEA, DSV, iRTSV, Laboratoire Biochimie et Biophysique des Systèmes Intégrés, Grenoble, France;
CNRS, UMR 5092, Grenoble, France; and
Université Joseph Fourier, Grenoble, France
1 Correspondence: Sahlgrenska Academy, Göteborg University, Department of Rheumatology and Inflammation Research, Guldhedsgatan 10A, 41346 Göteborg, Sweden. E-mail: lena.i.bjorkman{at}vgregion.se
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and LPS from Gram-negative bacteria. The induced activity was inhibited by pertussis toxin, indicating the involvement of a GPCR. However, based on FPRL1-specific desensitization and use of FPRL1 antagonist WRW4, we found the SAA-mediated effects in neutrophils to be independent of FPRL1. Based on these findings, we conclude that SAA signaling in neutrophils is mediated through a GPCR, distinct from FPRL1. Future identification and characterization of the SAA receptor could lead to development of novel, therapeutic targets for treatment of RA.
Key Words: FPRL1 • G protein-coupled receptor • WRW4 • rheumatoid arthritis
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Approximately one decade ago, the acute-phase protein, serum amyloid A (SAA), was shown to be one of many endogenous molecules capable of inducing intracellular calcium transients and directional cell migration in monocytes and neutrophils. In addition, SAA could induce chemotaxis in vivo, shown by injection of SAA s.c. in mice, which resulted in recruitment of monocytes and neutrophils to the injection site [9 ]. SAA is produced primarily by hepatocytes, and the levels in plasma can rise 1000-fold in response to inflammation, infection, or tissue injury [10 11 12 ]. In blood, SAA normally circulates in low levels bound to high-density lipoproteins (HDL), which to a large extent, reduce its biological effects [10 , 13 ]. At highly elevated concentrations of SAA, however, the Apo lipoprotein A-I in HDL is displaced, allowing SAA to circulate in a lipid-free form [13 , 14 ]. SAA has been used as an important indicator for diagnosis and prognosis in inflammatory diseases such as rheumatoid arthritis (RA) [15 , 16 ], and the protein may reach concentrations of >0.6 mg/ml, corresponding to >50 µM in blood [17 18 19 ]. In certain groups of RA patients, the inflammation persists and becomes chronic; a serious consequence may be the development of amyloidosis, characterized by fibrillar deposits of SAA-derived amyloid A, leading to progressive destruction of organs and impairment of their functions [20 , 21 ]. Furthermore, it was demonstrated recently that SAA binds to outer membrane protein A of gram-negative bacteria [22 ] and by this function, as an innate-immune opsonin, representing a novel recognition protein [23 ].
The SAA-induced activation of neutrophils has been shown to be inhibited by PTX; i.e., the SAA-binding receptor belongs to the GPCR family [24 ]. The first clue to the identity of the SAA receptor came from experiments in which SAA selectively induced calcium mobilization and a migratory response in human embryonic kidney (HEK) cells overexpressing FPRL1, thus establishing SAA as the first naturally occurring chemotactic ligand for the earlier orphan FPRL1 [25 ]. Since then, several studies have confirmed that FPRL1 is a receptor for SAA [26 , 27 ], but also, other receptors have been suggested, such as CD36 and lysosomal integral membrane protein-II analogous-1 (CLA-1) [28 ] and soluble RAGE (s-RAGE) [29 ]. Some other receptors have been reported for SAA with reference to its role in lipid metabolism, the scavenger receptor class B type I (SR-BI) and ATP-binding-cassette transporter A1 (ABCA1), promoting cholesterol efflux [30 , 31 ]. One problem with the studies published so far about the SAA receptor identity is that they use cell lines transfected with the receptor candidate. This leaves a question open about the importance of this receptor for interaction with SAA in primary human leukocytes.
In the present study, we show that SAA activates human neutrophils, measured as mobilization of intracellular calcium and as activation of the NADPH-oxidase. However, by using a desensitization protocol and a specific FPRL1 antagonist, we can conclude that the SAA-induced signaling in neutrophils is initiated by a receptor distinct from FPRL1.
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, complement fragment 5a (C5a), and the formylated peptide N-fMLP (fMLF) were from Sigma Chemical Co. (St. Louis, MO, USA). Dextran and Ficoll-Paque were from Pharmacia (Uppsala, Sweden). Fura 2-AM was from Molecular Probes Inc. (Eugene, OR, USA) and SAA from PeproTech Inc. (Rocky Hill, NJ, USA); endotoxin level was less than 0.1 ng/µg (1 EU/µg). The hexapeptide WKYMVM was synthesized and HPLC purified by Alta Bioscience (University of Birmingham, UK); endotoxin levels were less than 0.1 ng/µg (1 EU/µg). The SAA was dissolved in water, and the other peptide agonists were dissolved in DMSO to 10–2 M and stored in –70°C until use. Further dilutions of all peptides were made in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca2+ (1 mM), and Mg2 + (1.5 mM, pH 7.3, KRG). The peptide QRLFQVKGRR (gelsoline residues 160–169, PBP10), prepared by solid-phase peptide synthesis and coupled to rhodamine, as described earlier [32
], was a gift from Dr. Paul Janmey (University of Pennsylvania, Philadelphia, PA, USA). The receptor antagonist WRWWWW (WRW4) was from GenScript Corp. (Piscataway, NJ, USA). Cyclosporin H was kindly provided by Novartis Pharma (Basel, Switzerland). The PE-conjugated mAb specific for CD11b/CD18 (anti-CR3 antibodies) were from Dako M741, a mouse mAb (GM1D6; ab26316) to FPRL1 was from Abcam (Cambridge, UK), and a FAM-labeled WKYMVM peptide was obtained from Phoenix Pharmaceuticals (Belmont, CA, USA).
Isolation of human neutrophils from buffy coats
Neutrophils were separated from buffy coats obtained from healthy individuals donating blood at the Sahlgrenska University Hospital in Göteborg (Sweden) by using dextran sedimentation and Ficoll-Pacque gradient centrifugation [33
]. The cells were washed and resuspended (5x106/ml) in KRG and stored on melting ice until use.
Differentiation of nontransfected HL-60 cells
Differentiation of nontransfected HL-60 cells was achieved as described previously [34
]. In short, the cells were grown in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, penicillin (100 IE/ml), and streptomycin (100 µg/ml). The cells were induced to differentiate by addition of DMSO to a final concentration of 1.3%, harvested after 6 days, washed, resuspended in KRG, and stored on ice until use.
Changes in cytosolic calcium in FPRL1 overexpressing nondifferentiated HL-60 cells and nontransfected differentiated HL-60 cells and in neutrophils
The stable expression of FPRL1 in undifferentiated HL-60 cells has been described previously [35
, 36
]. Transfected cells were cultured in the RPMI-1640 medium described above but also supplemented with G418 (1 mg/ml), and density was maintained below 2 x 106 cells/ml. The cells were passaged to a concentration of 5 x 105 cells/ml 
24 h prior to use in assays, and the transient change in intracellular Ca2+ was measured with Fura-2 as described earlier [37
]. In short, cells (HL-60 or neutrophils; 2x107/ml) were incubated with Fura 2-AM (2 µM) in calcium-free KRG, supplemented with BSA (0.1%) at room temperature for 30 min, and were from here on protected from light. Cells were then diluted to twice the original volume with RPMI-1640 culture medium without phenol red (PAA Laboratories GmbH, Austria) and centrifuged, washed once in KRG (with 1.0 mM Ca2+ from here on), and resuspended in KRG to a density of 2 x 107/ml. Cells with or without WRW4 were equilibrated for 5 min at 37°C, after which, the peptide/protein agonist was added. The fura-2 fluorescence was measured by a luminescence spectrometer (LS50B, Perkin Elmer Corp., Wellesley, MA, USA) using an excitation wavelength of 340 nm and an emission wavelength of 505 nm. The intracellular Ca2+ concentration was calculated as described previously [37
].
Neutrophil priming and NADPH-oxidase activity
Neutrophil NADPH-oxidase activity was determined using an isoluminol ECL technique [38
], which measures the release of superoxide anion, the primary oxygen metabolite generated by the assembled oxidase [39
]. The ECL activity was measured in a six-channel Biolumat LB 9505 (Berthold Co., Wildbad, Germany) by using 4 ml disposable polypropylene tubes with a 0.45-ml reaction mixture containing (5x105/ml) neutrophils, HRP (4U), and isoluminol (20 µM). Neutrophils were incubated at 37°C for 5–30 min in the presence or absence of a priming agent and/or inhibitors. After priming, the NADPH-oxidase activity was recorded immediately and continuously after addition of stimulus.
Determination of receptor exposure in TNF--primed neutrophils
The exposure of CD11b/CD18 (CR3) on the neutrophil cell surface was assessed by immunostaining and FACS analysis. Neutrophils were incubated with or without TNF- (10 ng/ml, 20 min, 37°C) and were paraformaldehyde-fixed, washed with FACSwash (PBS, 0.02% NaN3), incubated with anti-CR3 antibodies (10 ul to a cell pellet of 106), and analyzed by flow cytometry (FACScan, Becton Dickinson, Mountain View, CA, USA).
Detection of surface-exposed FPRL1
Duplicate cell samples were washed and resuspended in KRG (5x106/ml) and placed on ice. Regular (unlabeled) WKYMVM (10–6 M) was added to one sample of each type, and after 1 h incubation on ice, FAM-labeled WKYMVM (10–8 M) was added to all samples. Following 1 additional hour of incubation on ice, 104 cells were analyzed directly by flow cytometry, and the mean fluorescence was recorded. Specific peptide binding was quantified by subtracting the mean fluorescence of samples in the presence of excess unlabeled peptide. At no instances was the final DMSO concentration greater than 0.01%.
Desensitization of neutrophil receptors
For the induction of desensitization of neutrophils without accompanying NADPH-oxidase activation, cells were incubated with an agonist for 10 min at 15°C [40
]. Desensitized cells were transferred immediately to 37°C and equilibrated for 10 min, followed by stimulation during which ECL activity was recorded continuously.
Statistical analysis
Two-tailed, paired Student’s t-tests were performed for statistical evaluation of the data, and P values <0.05 were regarded as statistically significant.
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Figure 1. Effect of WRW4 on cytosolic calcium mobilization induced by WKYMVM or SAA in FPRL1-overexpressing, nondifferentiated HL-60 cells, which were analyzed for intracellular calcium mobilization by monitoring Fura-2 fluorescence upon stimulation with SAA (10 µM) in the absence (solid lines) or presence (dashed lines) of WRW4 (5x10–6 M). The cells were challenged with (A) WKYMVM (10–7 M) or (B) SAA (10 µM). The arrows indicate the time-point for addition of agonist; graphs are derived from one representative experiment out of three.
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Figure 2. NADPH-oxidase activity and surface expression of FPRL1. Resting neutrophils were stimulated by WKYMVM (10–7 M; solid line), SAA (10 µM; dashed line) (A). Neutrophils were preincubated in the absence (non-primed) or in the presence of TNF- (10 ng/ml; 20 min at 37°C) or LPS (10 µg/ml; 30 min at 37°C), after which they were stimulated with (B) WKYMVM (10–7 M) or (C) SAA (10 µM). The extracellular production of superoxide anions after stimulation was measured by isoluminol-amplified ECL (CL) and responses given as Mcpm (106 cpm). The figure is representative of experiments performed at least three times. A FACS histogram of the surface expression of CR3 after priming with TNF- at 37oC for 20 min is also shown (C, inset). (D) Cell samples (HL-60 cells or neutrophils pretreated as indicated) were placed on ice and incubated with fluorescently labeled WKYMVM (10–8 M) in the presence or absence of 100 times excess of unlabeled peptide. Quantification of fluorescent binding per cell was accomplished by flow cytometry, and specific binding was calculated by subtracting the values obtained for samples in the presence of unlabeled peptide; the figure depicts mean ± SD (n=3–4). MFI, Mean fluorescence intensity; HL60 dif., differentiated HL-60.
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, have mobilized their subcellular granules and by that, exposed new receptors on their cell surface. As an example, the surface expression of CR3 is shown in the inset of Figure 2C
. Provided that a triggering agonist operates through a receptor that is normally stored in the granules, primed cells will release increasing amounts of ROS when triggered with such an agonist. Neutrophils, pretreated with LPS for 30 min at 37°C or with TNF- for 20 min at 37°C, exhibited increased extracellular release of ROS in response to WKYMVM and SAA (Fig. 2B
and 2C
, respectively), suggesting that both agonists operate through a mobilizable receptor. Pretreating cells with SAA did not increase oxidant production by WKYMVM, indicating that SAA did not serve as a priming agent in this system (data not shown).
With regard to kinetics of the response, SAA and WKYMVM induced a rapid onset of ROS production, and the response peaked within a minute from addition of the agonists. There was, however, a clear kinetic difference in the termination of the responses (Fig. 2B
and 2C)
. When cells were challenged with SAA, the termination of the NADPH-oxidase response was much more rapid than the WKYMVM-induced response. Calculated as the t1/2 fall from the peak value of TNF--primed cells, the response following SAA stimulation terminated after 79 ± 11 s (mean±SD, n=7), which was significantly faster than that of WKYMVM (145±36 s; mean±SD, n=5, P<0.0005). We have shown this type of difference earlier between FPR and FPRL1 as compared with CXCR1/2 (the IL-8R) [42
], and this compelling difference in kinetics between the two stimuli (SAA and WKYMVM) thus raises the question of whether two different receptors are involved.
Varying surface expression of FPRL1 on neutrophils and HL-60 cells
To investigate the surface expression of FPRL1 on the different cell types, a fluorescently labeled WKYMVM peptide was used. By using flow cytometry, we found that the surface expression of FPRL1 increased substantially after TNF- priming (Fig. 2D)
. Furthermore, we investigated the FPRL1 expression on HL-60 cells and found that nondifferentiated HL-60 cells transfected with FPRL1 overexpressed the receptor, even compared with primed neutrophils. Nontransfected HL-60 cells differentiated into granulocyte-like cells expressed low levels of FPRL1 (Fig. 2D)
.
The SAA-induced ROS production is PTX-sensitive
FPRL1 belongs to the group of PTX-sensitive GPCRs [3
]. To determine the sensitivity of the SAA-induced ROS production to PTX, neutrophils were incubated with the toxin until the responses induced by fMLF or WKYMVM were blocked, and the response induced by PMA (that bypasses the PTX-sensitive G-protein) was unaffected (data not shown). Cells incubated with PTX were nonresponsive to SAA (Fig. 3
), indicating that the receptor responsible for SAA-induced ROS production in neutrophils is indeed a GPCR.
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Figure 3. Neutrophil NADPH-oxidase activity induced by SAA is PTX-sensitive. Neutrophils were preincubated for 120 min in the absence (–PTX) or presence of PTX (+PTX; 500 ng/ml), after which, the cells were activated with SAA (10 µM). The recordings of superoxide anion production started after 120 min, when the agonists were added. The release of extracellular superoxide anions was determined by isoluminol-amplified ECL and expressed in Mcpm (106 cpm). The figure shows a representative experiment out of three.
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(Fig. 2C)
. Neutrophils pretreated with SAA did not desensitize neutrophil responses induced by WKYMVM (data not shown). Taken together, we conclude that the receptor responsible for SAA-induced ROS production was distinct from FPRL1 by showing that the FPRL1 receptor was not able to desensitize the SAA receptor heterologously.
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Figure 4. Homologous and heterologous receptor desensitization in neutrophils (5x106/ml), which were incubated with SAA (10 µM), WKYMVM (10–7 M) or KRG for 10 min at 15°C to induce desensitization without NADPH-oxidase activation. (A) Cells desensitized by SAA (dashed line) and nondesensitized cells (Ctrl; solid line) were transferred to 37°C and equilibrated for 10 min before stimulation with SAA (10 µM). (B) WKYMVM-desensitized cells (dashed line) and nondesensitized cells (Ctrl; solid line) were transferred to 37°C and equilibrated for 10 min before stimulation with SAA (10 µM). The extracellular production of superoxide anions after stimulation was measured by isoluminol-amplified ECL, and responses were given as Mcpm (106 cpm). The figure shows a representative experiment out of three.
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Figure 5. Effects of FPRL1 inhibitors/antagonists in neutrophils. Neutrophils were activated by SAA (10 µM; open bars) or WKYMVM (10–7 M; solid bars), with or without prior incubation with PBP10 (1 µM) or WRW4 (5x10–6 M), and the extracellular release of superoxide anions was recorded continuously (A). Peak values were compared with control peak values (obtained without inhibitor), and results are summarized as percent of control; mean ± SD (n=3–6). The statistical significance is given for each condition compared with its control condition, i.e., the same stimulus in the absence of inhibitor (***, P<0.001; *, P<0.05; ns, not significant). Neutrophil intracellular calcium mobilization was analyzed by monitoring Fura-2 fluorescence upon stimulation with SAA (10 µM; B) or WKYMVM (10–7M; C) in the absence (solid lines) or presence (dashed lines) of WRW4 (5x10–6 M). The arrows indicate the time-point for addition of agonist. Graphs show representative experiments out of at least three.
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An antibody against the N-terminal domain of FPRL1 has been shown to inhibit IL-8 secretion from human neutrophils [26 ]. Hence, a mAb to FPRL1 was tested to elucidate its potential to inhibit ROS production induced by SAA. The WKYMVM -induced neutrophil NADPH-oxidase activity was totally inhibited by the FPRL1 antibody, and at the same concentration, it also partially inhibits the SAA-mediated response. However, the antibody also markedly inhibits the C5a (neutrophil chemoattractant, non-FPRL1 agonist) [46 ]-induced ROS production at this concentration; hence, the FPRL1 antibody used did not help out in the determination of receptor specificity for SAA (data not shown).
SAA fails to induce NADPH-oxidase activity and calcium responses in differentiated, nontransfected HL-60 cells
To determine if the receptor responsible for SAA-induced NADPH-oxidase activation was present on DMSO-differentiated (neutrophil-like), nontransfected HL-60 cells, equipped with FPR and FPRL1, as well as a functioning NADPH-oxidase [47
], we challenged these cells with SAA or WKYMVM. A robust, oxidative burst was induced by WKYMVM, while stimulation with SAA gave no response at all (Fig. 6
). WKYMVM also induced a rise in intracellular calcium, which was inhibited by WRW4, but when challenging cells with SAA, no such calcium increase was detected (Fig. 6
, inset), suggesting that the activating SAA receptor is not present in differentiated, nontransfected HL-60 cells.
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Figure 6. Lack of NADPH-oxidase activation and calcium mobilization in response to SAA in nontransfected, differentiated HL-60 cells. DMSO-differentiated, nontransfected HL-60 cells were activated by WKYMVM (10–8 M; solid line), WKYMVM (5x10–9 M; solid, bold line), or SAA (10 µM; dashed line), and the extracellular release of superoxide anions was recorded by ECL, expressed in Mcpm (106 cpm). The graph shows one experiment out of two with identical results. Intracellular calcium mobilization induced in DMSO-differentiated HL-60 cells was analyzed by monitoring Fura-2 fluorescence upon stimulation with WKYMVM (10–7 M) in the absence (solid line) or presence (dashed line) of WRW4 (5x10–6 M; inset, upper diagram) or with SAA (10 µM) in the absence of WRW4 (inset, lower diagram); the arrows indicate the time-point for addition of agonist.
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B, resulting in matrix-metalloproteinase-9 (MMP-9) up-regulation in THP-1 cells and human monocytes in a PTX-sensitive manner, and stimulating FPRL1-transfected Chinese hamster ovary cells with SAA resulted in MMP-9 up-regulation [27
]. The possibility of receptor candidates other than FPRL1 has been proposed in SAA-mediated cell-signaling events. One recent study demonstrated that WKYMVm (lowercase m, a D-methionine) and SAA differentially stimulated the production of two distinct lipid mediators in neutrophils, implying that another receptor could be involved [48
]. We have also recently shown that SAA inhibited apoptosis of human neutrophils independently of FPRL1 [49
]. Another study showed that CLA-1, expressed in HeLa cells, could function as an endocytic SAA receptor by affecting SAA-triggered IL-8 secretion [28
]. Other receptors implicated are s-RAGE, SR-BI, and ABCA1 transporter [29
, 31
, 50
].
The reports that identify FPRL1 as a functional SAA receptor are based mainly on experiments with cell lines transfected with FPRL1, i.e., not with primary human leukocytes. Our experiments with transfected HL-60 cells expressing FPRL1 confirm earlier findings showing that SAA is an agonist for this receptor, and we add to this that the FPRL1 antagonist WRW4 [41
] inhibits the SAA response induced in these cells. In neutrophils, however, SAA seems to use another receptor to activate the cells, a suggestion based on several findings. The FPRL1 antagonist WRW4 had no effect on SAA-induced activation, whereas the response induced by WKYMVM (a potent FPRL1 agonist) [36
] was totally inhibited. The possibility that the difference in WRW4 sensitivity between WKYMVM and SAA in neutrophils was a result of different binding sites on FPRL1 was ruled out by the finding that the antagonist inhibited SAA signaling in FPRL1-transfected HL-60 cells. In addition, we could see a consistent difference in kinetics between the WKYMVM-induced and the SAA-induced responses, again implying the involvement of a receptor distinct from FPRL1. In attempts to characterize the neutrophil receptor for SAA further, we performed receptor desensitization experiments. It is known that neutrophils given sequential homologous stimulations become totally or partially desensitized to the second addition of stimulus if the first addition was sufficient to trigger oxidase activation [51
, 52
]. In this study, we could show that the release of ROS induced by SAA was inhibited completely when the cells were stimulated previously by SAA, thereby limiting activation and allowing the inflammatory event to be brought to an end. Neutrophils desensitized with the FPRL1 agonist WKYMVM were still able to respond to SAA. When assessing differentiated, nontransfected HL-60 cells for intracellular calcium responses, WKYMVM induced a rapid increase in intracellular calcium, whereas SAA did not. Clearly, these cells do express FPRL1, but the receptor number, as shown by our experiments with the fluorescently labeled WKYMVM, is probably too low to transduce a measurable signal using SAA as the triggering agonist. When performing our experiments, we found that 10 µM SAA induced a clear, oxidative burst consistently, especially when cells were primed by LPS or TNF-. The fact that 10 µM SAA is way below the SAA concentrations that can be reached in vivo during pathological inflammatory conditions suggests that SAA could be a powerful effector protein during inflammatory disease states. Therefore, we did not exceed the concentration of SAA beyond 10 µM in attempt to reach a plateau level of ROS production.
It is obvious from our results that SAA has the potential to bind and activate cells through FPRL1, but this is not the receptor used/involved when neutrophils are activated by this agonist. A similar type of receptor preference/involvement has been described for the peptide WKYMVm, an agonist that preferentially uses FPRL1. It has the potential to activate neutrophils, also through FPR, but this receptor is not used unless signaling through FPRL1 is blocked [37 ]. The receptor with the highest affinity is possibly the one preferentially used by agonists that can bind to two different receptors, and according to this, SAA should have higher affinity for the nonidentified receptor than for FPRL1. This, however, remains to be determined when the SAA receptor has been identified.
SAA is considered to be a powerful, proinflammatory mediator, as its concentration can increase 100- to 1000-fold in circulation during different inflammatory conditions, for example, RA [17 ]. Levels in the order of >0.6 mg/ml SAA are not extraordinary to detect during the active phase of RA. In our study, we have challenged cells with 10 µM SAA corresponding to a concentration of 0.117 mg/ml, a concentration somewhat lower than those found under a pathological, inflammatory condition.
Apart from serving as an opsonin for Gram-negative bacteria [22
, 23
], several immunomodulatory roles of SAA have been reported. SAA has been suggested to possess cytokine-like properties stimulating the production of proinflammatory cytokines from THP cells [53
]. Some studies report that SAA plays an important role in the synovial matrix degradation of articular cartilage and bone. This could be accomplished by up-regulation of adhesion molecules and MMPs from fibroblast-like synoviocytes and chondrocytes, leading to the progressive destruction of joints that is characteristic for RA, thus suggesting an attractive connection between SAA, as a mediator of synovial inflammation, and the early development of erosive RA [11
, 54
, 55
]. SAA triggers NF-B activation in intestinal epithelial cells, thereby inducing IL-8 gene expression, pointing to another potential role for SAA in initiating and maintaining inflammation [56
]. In addition, we showed recently that SAA, purified from plasma from RA patients, or in recombinant form suppressed apoptosis of human neutrophils, thereby delaying the termination of the inflammatory response [49
].
When assessing SAA and its ability to activate the NADPH-oxidase, we found SAA to be a relatively weak inducer of the oxidative burst in neutrophils, compared with WKYMVM. However, priming of the cells with TNF- or LPS did result in a marked potentiation of the ROS production. The ability to prime the response with these secretagogues is indicative of a reserve pool of the receptor responsible for SAA-induced oxidative burst, present in mobilizable granules. The SAA receptor most probably has the same subcellular distribution as many other neutrophil chemoattractant receptors, including the FPR family members. As revealed by subcellular fractionation studies, these receptors are localized mainly in the gelatinase and specific granules, storage organelles that are mobilized to the cell surface by priming agents such as LPS and TNF- [36
, 57
]. This is in contrast to the chemoattractant IL-8, whose oxidative response is not primed by TNF- treatment [42
]. With this in mind, SAA could likely act as a powerful mediator of an oxidative burst in an inflammatory setting, where the cells have been primed by the extravasation process and/or stimulation of inflammatory mediators, especially as pathological concentrations of SAA exceed the concentrations we have been using in the current study.
The cell-permeable peptide PBP10, with a sequence corresponding to the PIP2-binding region of gelsolin [32 ], can distinguish between FPR and FPRL1, as it selectively blocks FPRL1-mediated activation [45 ]. We detected a significantly reduced NADPH-oxidase activity by WKYMVM in the presence of PBP10, which was in full agreement with earlier studies from our group [45 ]. The SAA-induced ROS production was also inhibited by PBP10, although not to the same extent as the WKYMVM response. Apparently, the PBP10-sensitive pathway is not only used by FPRL1 but is also essential for the SAA receptor. Although PBP10 inhibits the SAA-induced activity, the receptor desensitization results presented and the lack of effects of the FPRL1-specific antagonist strongly suggest that the signal activating the NADPH-oxidase in SAA-stimulated neutrophils is transduced through a receptor distinct from FPRL1. The precise mechanism by which PBP10 interferes with signaling by some GPCRs but not by others remains to be determined, but it is plausible that the signaling intracellular domains of the PBP10-sensitive receptors, i.e., FPRL1 and the SAA receptor, are in some way structurally similar.
In summary, the present study shows that SAA interacts with a receptor distinct from FPRL1 for the activation of the NADPH-oxidase in human neutrophils. SAA is a true ligand of FPRL1, as shown clearly when this receptor is overexpressed on the cell surface of transfected cells. In neutrophils, the receptor activated by SAA is, however, not identical to FPRL1. SAA plays a key role in the modulation of immune responses, particularly in RA, by contributing to the destructive immune process that leads to joint destruction, and may eventually result in the development of amyloidosis. It has also been reported to be the most reliable parameter to reflect changes in disease status in RA patients, thus indicating a crucial role for SAA in the pathogenesis of RA. Based on these facts, it seems vital to further elucidate the receptor identity and signaling pathways used by SAA, information/knowledge that may disclose novel drug targets and/or therapeutic strategies for the treatment of RA.
Received June 12, 2007; revised September 19, 2007; accepted September 19, 2007.
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B-dependent signal transduction pathway Arthritis Rheum. 54,105-114[CrossRef][Medline]B and proinflammatory gene expression in human and murine intestinal epithelial cells Eur. J. Immunol. 35,718-726[CrossRef][Medline]This article has been cited by other articles:
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