Abstract
The acute-phase proteins, C-reactive protein and serum amyloid A (SAA), are biomarkers of infection and inflammation. However, their precise role in immunity and inflammation remains undefined. We report in this study a novel property of SAA in the differential induction of Th1-type immunomodulatory cytokines IL-12 and IL-23. In peripheral blood monocytes and the THP-1 monocytic cell line, SAA induces the expression of IL-12p40, a subunit shared by IL-12 and IL-23. SAA-stimulated expression of IL-12p40 was rapid (≤4 h), sustainable (≥20 h), potent (up to 3380 pg/ml/106 cells in 24 h), and insensitive to polymyxin B treatment. The SAA-stimulated IL-12p40 secretion required de novo protein synthesis and was accompanied by activation of the transcription factors NF-κB and C/EBP. Expression of IL-12p40 required activation of the p38 MAPK and PI3K. Interestingly, the SAA-induced IL-12p40 production was accompanied by a sustained expression of IL-23p19, but not IL-12p35, resulting in preferential secretion of IL-23, but not IL-12. These results identify SAA as an endogenous ligand that potentially activates the IL-23/IL-17 pathway and present a novel mechanism for regulation of inflammation and immunity by an acute-phase protein.
In mammals, the acute-phase response is a systemic reaction to infection and tissue injury that protects host by isolating pathogens, minimizing tissue damage, and promoting healing (1, 2). A hallmark of the acute-phase response is the rapid increase in production of acute-phase proteins (APPs),3 including serum amyloid A (SAA) and C-reactive protein (CRP). Secretion of these APPs by hepatocytes results in a marked increase (up to 1000-fold for SAA; higher for CRP) in their plasma concentrations above the basal level, reaching as high as 80 μM or 1 mg/ml for SAA (3, 4). The dramatic change in APP production is a clinical marker for inflammatory diseases, including atherosclerosis (5, 6), rheumatoid arthritis (7), and Crohn’s disease (8). Recent studies have shown that changes in CRP concentration may predict the risk of unstable angina, recurrent coronary heart disease, and other cardiovascular disorders (9, 10). However, despite these exciting developments and recent advancement in understanding the role of CRP in immunomodulation (11, 12), little is known about the precise roles of SAA in inflammation and immunity.
Acute-phase SAA, also known as inducible SAA, is a 104-aa protein encoded by the SAA1 and SAA2 alleles in humans (3). Bacterial products such as LPS and inflammatory cytokines, including IL-1β and IL-6, induce acute-phase SAA expression in hepatocytes (13). Once released to the circulation, the newly synthesized SAA is incorporated into high density lipoprotein (HDL) (14), thus altering HDL metabolism and cholesterol transport (15). At elevated concentrations, SAA dissociates from HDL and displaces apoA-I, generating lipoprotein fractions that contain primarily lipid-poor apoA-I and SAA (15, 16). The lipid-poor SAA also is produced by inflammatory macrophages and synoviocytes at sites of chronic inflammation (17, 18, 19) and is responsible for its biological functions through binding to the SAA receptors (20, 21, 22). SAA also binds to T lymphocytes and platelets, and interacts with Tanis, heparin, heparan sulfate, and certain glycoproteins, although whether these binding events lead to transmembrane signaling remains to be tested (23, 24, 25, 26). The initial elevation of SAA is followed by a gradual decline in its plasma concentration after 72 h, eventually returning to baseline after 5–7 days (1). The change in plasma SAA concentration parallels the initiation of acute inflammatory response and its resolution. The kinetics of SAA production also matches the time course of transition from innate to specific immune responses, raising the possibility that SAA serves a function in the development of adaptive immunity (27).
Several recent studies have shown that SAA possesses cytokine-like activity and can stimulate the production of IL-8 (28, 29), matrix metalloproteinases (30), cytokines such as TNF-α and IL-1β, and cytokine receptor antagonists, including IL-1R antagonist and soluble TNFR type II (31). We previously have shown that neutrophils respond to SAA stimulation with synthesis and release of IL-8, a process that requires de novo protein synthesis and transcriptional regulation (28). These findings have led us to investigate whether SAA can induce immunoregulatory cytokine expression in the responding cells. Our results, reported in this study, indicate that SAA differentially induces the heterodimeric cytokines IL-12 and IL-23, which play an important role in the development of cell-mediated immune response (32). Although IL-12 and IL-23 share a common p40 subunit and both are Th1-type cytokines, they promote two distinct immunological pathways that have separate, but complementary functions. IL-12 is required for antimicrobial responses to intracellular pathogens, and IL-23 is potentially important for recruitment and activation of inflammatory cells that are required for the induction of chronic and autoimmune inflammation and granuloma formation (32). These findings suggest a potential mechanism for SAA to regulate autoimmune inflammatory responses through induction of IL-23.
Materials and Methods
Reagents
Recombinant human apo-SAA was obtained from PeproTech. The endotoxin content was <0.1 ng/μg protein. Double-stranded consensus oligonucleotides for NF-κB and C/EBP were purchased from Promega and Santa Cruz Biotechnology, respectively. Complementary oligonucleotides for the NF-κB half-site (33) within the IL-12p40 promoter region (5′-TAAAATTCCCCCAGAATGTTTTG-3′) were synthesized, purified, and annealed before use. Inhibitors for protein kinases were obtained from Calbiochem and Sigma-Aldrich. Abs against Akt and p38 MAPK were purchased from Cell Signaling Technology and Santa Cruz Biotechnology, respectively. The p85 dominant-negative (Δp85) and myristoylated p110α constructs were described previously (34). The myristoylated Akt and kinase-dead Akt constructs were prepared, as described previously (35). Construction of the p38 MAPK plasmids used in this study was described in a previous publication (36).
Cell preparation and culture
Human peripheral blood monocytes were prepared from fresh, heparinized venous blood by Ficoll-Hypaque density-gradient centrifugation. Blood drawing followed a protocol approved by the Institutional Review Board at University of Illinois. Purified monocytes (∼93% CD14+ by flow cytometry) were kept in nonadherent condition in RPMI 1640 containing 0.5% FBS and maintained at 37°C. THP-1 (American Type Culture Collection; TIB 202) cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 0.05 mM 2-ME, 10 mM HEPES, 2 mM l-glutamine, 100 IU/ml penicillin, and 50 μg/ml streptomycin.
Measurement of secreted cytokines
Monocytes or THP-1 cells (5 × 106 cells/ml/sample) were placed in the culture medium and kept in a 5% CO2 atmosphere at 37°C with or without stimulants. After stimulation with 1 μM (11.4 μg/ml) SAA for the indicated time period, cell-free supernatants were collected by centrifugation at 1000 × g for 30 s and assayed for different cytokines with ELISA kits for human IL-12p40 (BioSource International), IL-12p70 (BioSource International), and IL-23 (Bender MedSystems). The cell pellets were quickly frozen in liquid N2 and stored at −80°C for total RNA extraction.
RNA extraction and RT-PCR
THP-1 cells (5 × 106) were stimulated with 1 μM SAA in a total volume of 1 ml cell suspension for different periods of time. Total RNA was isolated with a RNeasy isolation kit obtained from Qiagen. RT-PCR was performed to check the steady state mRNA level for IL-12p40. After 40 cycles, an expected 370-bp fragment was identified in agarose gel. The housekeeping gene fragment of G3PDH (452 bp) was used for verification of equal loading and RT-PCR efficiency.
Real-time quantitative PCR was performed using ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The reaction mixture contains 2× iTaq SYBR Green Supermix with ROX (Bio-Rad), 20 pmol forward and reverse primers, and 1 μg of cDNA from THP-1 cells treated with or without SAA. Sample loading was normalized by ROX. The thermocycling program was 40 cycles of 95°C for 30 s, 60°C for 45 s, and 72°C for 45 s, with an initial cycle of 95°C for 2 min. Accumulation of PCR products was detected by monitoring the increase in fluorescence of SYBR Green after each cycle. A dissociation (melting) curve was constructed in the range of 60°C to 95°C, to eliminate interference from dimerized primers. All data were analyzed with the ABI PRISM 7000 SDS software (version 1.1). The slope of the standard curve (copy number, dilution factor vs threshold cycle (Ct)) was correlated directly to the PCR efficiency, efficiency (η) = (10(−1/slope)) – 1. The copy number of gene in each sample was within the linear range of the standard curve. Relative levels of mRNA for IL-23p19, IL-12p35, and IL-12p40 were determined by using the Ct value and the formula: fold increase = ((1 + ηtarget)ΔCt target (unstimulated − stimulated))/((1 + ηG3PDH)ΔCt G3PDH (unstimulated − stimulated)).
The sequences of the oligonucleotides used in this study are as follows: 5′-TGTTCCCCATATCCAGTGTGG-3′ and 5′-CTGGAGGCTGCGAAGGATTT-3′ for p19; 5′-GATAAAACCAGCACAGTGGAGGC-3′ and 5′-GGGAGGATTTTTGTGGCACAGT-3′ for p35; 5′-AGAGGCTCTTCTGACCCCCAAG-3′ and 5′-CTCTTGCTCTTGCCCTGGACCTG-3′ for p40; and 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for G3PDH.
EMSA
Nuclear extracts were isolated using the method of Dignam et al. (37) with minor modifications (38). For each sample, 4 × 106 cells were used. Radiolabeling of the NF-κB, C/EBP, and NF-κB half-site probes (33), binding reactions, electrophoresis, and autoradiography was conducted, as described previously (38).
Transfection and luciferase reporter assay
THP-1 cells (107) were transfected with NF-κB luciferase reporter (5 μg), pCMVβ (0.5 μg), and other expression constructs, as indicated in the text and figure legends. Transient transfection was performed using DEAE-dextran (Sigma-Aldrich). Briefly, cells were washed twice with suspension cell transfection buffer solution (STBS) (25 mM Tris-HCl (pH 7.5), 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4.7H2O, 0.7 mM CaCl2.2H2O, and 0.5 mM MgCl2.6H2O), and collected through centrifugation. DNA constructs (with total DNA of 10 μg) were diluted in 350 μl of STBS, and then mixed with 350 μl of 0.8 mg/ml DEAE-dextran in STBS. The mixture was immediately added to the cell pellet, mixed by resuspension, and incubated at 37°C for 20 min. Cells were then washed with STBS twice, resuspended in 10 ml of culture medium, and grown for 48 h. Cells were stimulated with 1 μM SAA for 5 h and lysed with reporter lysis buffer (Promega). The expressed luciferase activity was measured with a Femtomaster FB12 luminometer (Berthold Detection Systems). The luciferase activity was normalized against the coexpressed β-galactosidase activity, determined with luminescence reagent from BD Biosciences, and expressed as relative luciferase activity. Unless otherwise indicated in the figure legends, all data were collected from three independent experiments, each in duplicate. Normalized data were plotted using Prism software (version 4.0; GraphPad).
Kinase assay
Whole cell extracts were prepared from nonstimulated and SAA-stimulated THP-1 cell samples. The activation of the p38 MAPK and Akt was determined by Western blotting using anti-phospho Abs to detect the phosphorylated kinases of interest.
Results
SAA stimulates IL-12p40 secretion
To investigate a possible role of SAA in the induced expression of immunomodulatory cytokines, we determined IL-12p40 production by peripheral blood monocytes. IL-12p40 is a protein subunit shared by IL-12 and IL-23, which are heterodimeric cytokines having different, but complementary immunomodulatory functions (32). Human peripheral blood monocytes were incubated with or without 1 μM human rSAA for up to 24 h, and samples were taken at various time points. The IL-12p40 secreted into the culture medium was detected with ELISA. As shown in Fig. 1⇓A, the IL-12p40 basal level remained stable over the entire incubation period. SAA induced a substantial increase in IL-12p40 secretion that was detectable after 4 h of stimulation. At the end of the incubation period, the IL-12p40 concentration in the culture medium reached 2550–3880 pg/ml/106 cells.
SAA stimulates secretion of IL-12p40. A, Induction of IL-12p40 secretion in SAA-stimulated human peripheral blood monocytes. Approximately 2 × 106 cells were incubated with or without human rSAA at 37°C for the indicated periods of time. The secreted IL-12p40 was determined with ELISA. Data shown are means ± SD from one of the three experiments. Individual differences at the 24-h time point varied from 2580 ± 42 to 3380 ± 69 pg/ml/106 cells among three blood donors. B, Secretion of IL-12p40 by SAA-stimulated THP-1 cells. Approximately 5 × 106 cells were stimulated with or without 1 μM SAA at 37°C for different periods of time, and the secreted IL-12p40 was measured with ELISA. Data shown are means ± SEM of three experiments. A total of 1 pg of IL-12p40 equals to 2.5 × 10−5 pmol of the same protein.
To overcome variability among blood donors, especially in studies of SAA-induced signaling, we examined THP-1 for induced IL-12p40 production (Fig. 1⇑B). THP-1, a human monocytic cell line, responded to SAA stimulation with a similar pattern of IL-12p40 production, except for a lower basal level. This cell line was used in subsequent experiments to investigate the mechanism of induction.
Because rSAA was applied to blood monocytes and THP-1 cells that could respond to LPS with IL-12p40 production (39), the effect of contaminating LPS in the SAA preparation on IL-12p40 expression was determined. SAA was incubated with polymyxin B, an amphiphilic cyclic polycationic peptide that forms complex with LPS and prevents LPS-induced proinflammatory cytokine production (40, 41), before it was applied to the THP-1 cells. As shown in Fig. 2⇓, polymyxin B (0.1–10 μg/ml) did not significantly alter SAA-induced IL-12p40 expression. Under the same experimental conditions, polymyxin B potently inhibited the LPS-induced IL-12p40 expression. This result supports the notion that the observed induction of IL-12p40 secretion is a primary function of SAA and not caused by contaminating LPS in the sample.
Effects of polymyxin B on SAA- and LPS-induced IL-12p40 expression. SAA (1 μM) or LPS from Escherichia coli strain 0111:B4 (100 ng/ml) were incubated with different amounts of polymyxin B (PMB) at 37°C for 1 h before application. THP-1 cells (1 × 106/sample) were stimulated with the PMB-incubated SAA or LPS at 37°C for 8 h. The IL-12p40 secreted to the culture medium was determined using ELISA. The amounts of IL-12p40 induced by 1 μM SAA and 100 ng/ml LPS were similar (data not shown). Changes in IL-12p40 concentration were shown as percentage of the control, determined in the absence of PMB. Data shown are means ± SEM from three experiments, each with duplicate measurements.
SAA-stimulated IL-12p40 secretion requires de novo protein synthesis and transcriptional activation
The SAA-stimulated IL-12p40 secretion was blocked by cycloheximide, an inhibitor of protein synthesis, and by actinomycin D, an inhibitor of transcription (Fig. 3⇓A). At the mRNA level, SAA caused accumulation of the IL-12p40 transcript, which was inhibited by actinomycin D, but potentiated by cycloheximide (Fig. 3⇓B). The SAA-induced accumulation of IL-12p40 transcript was observed as early as 2 h after stimulation (Fig. 3⇓C). This relatively fast response suggests that the induction is a primary effect of SAA and not secondary to another cytokine induced by SAA. The IL-12p40 transcript continued to accumulate for ∼16 h, after which the level of the transcript began to decline. Consistent with previous reports that expression of IL-12p40 is regulated at the transcriptional level (39), SAA was found to activate the transcription factors NF-κB in stimulated THP-1 cells. Oligonucleotide sequences corresponding to the canonical NF-κB binding site (Fig. 4⇓A) or the NF-κB half-site (Fig. 4⇓B) found in the IL-12p40 promoter (33) were able to bind NF-κB in SAA-stimulated cells. Under the same experimental condition, LPS (1.25 μg/ml) also induced binding to the NF-κB half-site (Fig. 4⇓B). An additional experiment was conducted to identify whether C/EBP, a transcription factor known to work in synergy with NF-κB for the induction of IL-12p40 gene expression (42), could be induced by SAA. As shown in Fig. 4⇓C, both SAA and LPS could stimulate C/EBP binding to the specific DNA sequence.
SAA-induced IL-12p40 secretion requires de novo protein synthesis. A, Effects of transcriptional and translational inhibitors on IL-12p40 secretion. THP-1 cells were pretreated with or without cycloheximide (100 μM) or actinomycin D (10 μg/ml) for 30 min before SAA (1 μM) stimulation. After 8 h, the IL-12p40 in the cell-free medium was detected with ELISA. B, RT-PCR results showing changes in IL-12p40 transcript level using cell pellets from the above experiments. A representative figure from three similar experiments is shown. C, Time-dependent changes in IL-12p40 transcript level in SAA-stimulated THP-1 cells. Cell pellets collected from unstimulated and SAA-stimulated samples (Fig. 1⇑B) were used for preparation of RNA and cDNA. Real-time PCR was performed, and the relative concentrations of the IL-12p40 transcript were presented as fold changes. Data shown are means ± SD from one of the three experiments with similar results.
SAA activates transcription factors NF-κB and C/EBP. EMSA was performed to determine the binding of NF-κB to the canonic sequence (A), and the NF-κB half-site within the IL-12p40 promoter (B) in unstimulated and SAA-stimulated (1 μM) THP-1 cells. LPS was used at a concentration of 1.25 μg/ml. C/EBP binding in SAA- and LPS-stimulated THP-1 cells is shown in C, with the same ligand concentrations. The concentration of the unlabeled (cold) probes used for competition was 100-fold in excess in B and C. Representative autoradiographs from at least three experiments are shown. NS, nonspecific binding.
Activation of p38 MAPKs and phosphoinositide 3-kinases is required for SAA-induced IL-12p40 expression
The signaling mechanism for SAA-stimulated IL-12p40 expression was investigated in THP-1 cells. MAPK and PI3K are important signaling components for cytokine expression. To determine whether the SAA-induced IL-12p40 expression involves these kinases, we first examined whether the relevant pharmacological inhibitors for the kinases could reduce IL-12p40 production. Among the inhibitors examined, the p38 MAPK inhibitor, SB202190, and the PI3K inhibitor, LY294002, both markedly reduced IL-12p40 mRNA level (Fig. 5⇓, A and B) and IL-12p40 secretion (Fig. 5⇓C). In contrast, the MAPK kinase inhibitor U0126 did not significantly affect IL-12p40 induction (data not shown). The combined use of both inhibitors produced additional inhibition of IL-12p40 secretion (Fig. 5⇓C). Stimulation of THP-1 cells with SAA resulted in a time-dependent phosphorylation of p38 MAPK as well as Akt, a serine/threonine kinase downstream of PI3K (Fig. 6⇓, A and B). As expected, LY294002 blocked Akt phosphorylation (data not shown), and did not affect the induced p38 MAPK phosphorylation (Fig. 6⇓A). Surprisingly, treatment with SB202190 blocked SAA-induced Akt phosphorylation (Fig. 6⇓B). This result seems to suggest that p38 MAPK is upstream of the PI3K-Akt pathway in the SAA-stimulated cells. However, additional experiments using dominant-negative constructs of p38 MAPK produced only a moderate inhibition in SAA-induced NF-κB reporter expression (Fig. 6⇓C). Moreover, the inhibitory effect of SB202190 could not be reversed by the constitutively active constructs of p110α and Akt (Fig. 6⇓D). Taken together, these results suggest that PI3K and p38 MAPK may be activated in parallel by SAA, and that the observed inhibition of Akt phosphorylation by SB202190 can be a nonspecific effect of the inhibitor. Alternatively, the observed discrepancy may result from the inability of the dominant-negative constructs of p38α MAPK and p85 to inhibit other members of the p38 MAPK family and the PI3K family of enzymes that play a role in SAA-induced signaling. In comparison, pharmacological inhibitors typically target multiple members of an enzyme family and can produce a more potent inhibition.
Roles of the p38 MAPK and PI3K in SAA-induced p40 expression. A and B, Effect of pharmacological inhibitors in the SAA-induced increase of the IL-12p40 transcript. THP-1 cells were treated for 30 min with either SB202190 or LY294002 (20 μM each) before SAA (1 μM) stimulation. The results from conventional RT-PCR (upper panel) and quantitative RT-PCR (lower panel) are shown. C, Secretion of IL-12p40 from THP-1 cells treated before SAA stimulation with either SB202190 or LY294002 or both, before SAA stimulation. The IL-12p40 level in culture medium was determined 8 h after SAA stimulation. Data shown are means ± SEM from three experiments.
Phosphorylation of p38 and Akt. A and B, Western blots showing phosphorylation of p38 and Akt after SAA stimulation and the effect of the pharmacological inhibitors LY294002 (A) and SB202190 (B). The inhibitors were used at 20 μM each, and SAA was used at 1 μM. Blots shown are representative of at least three experiments. C, The effect of dominant-negative constructs on SAA-induced NF-κB luciferase reporter activity was examined in transfected THP-1 cells. The cells were transfected, as described in Materials and Methods, with 6 μg of DNA for each of the dominant-negative constructs, as indicated, or with a vector control. Transfected cells were stimulated with SAA (1 μM) for 5 h. Induced luciferase activity was determined, as described in Materials and Methods, and expressed as percentage of relative luciferase activity (RLA). The activity measured in SAA-stimulated, vector-controlled cells was set as 100%. D, Similar to C, but the effects of constitutively active p110α and Akt were determined in the absence (CTL) and presence of SB202190 (20 μM, 30-min pretreatment). SB202190 was present during the 5-h stimulation. The induced RLA in control sample (with SAA, but not SB202190) were set as 100%. Data for all experiments are presented as the means ± SD from three experiments, each with duplicate measurements.
SAA preferentially stimulates IL-23 secretion by THP-1 cells and human blood monocytes
These results support the notion that stimulation of IL-20p40 expression is a direct effect of SAA, and not a secondary effect mediated through another cytokine that is induced by SAA, or through the contaminating LPS. The potent induction of IL-12p40 raised the possibility that SAA might stimulate secretion of cytokines bearing this subunit. To determine which cytokine was induced by SAA, we first measured the relative concentrations of the transcripts for IL-23p19 and IL-12p35 with quantitative RT-PCR. In THP-1 cells, SAA caused a marked (∼12-fold) increase in the IL-23p19 transcript, and a less potent increase in the IL-12p35 transcript, after 2 h of stimulation (Fig. 7⇓A). The accumulation of the IL-23p19 transcript continued over a 16-h period and eventually reached a level 35-fold above baseline. In comparison, the increase in the IL-12p35 transcript was transient and less potent. Thus, after 4 h of SAA stimulation, the IL-23p19 transcript became the predominant species of the two subunits. This difference was also reflected at the protein level as determined by ELISA. The concentration of the IL-23 heterodimer in unstimulated THP-1 cells was 76 pg/ml/106 cells, ∼8-fold higher than that of the IL-12 heterodimer. Secretion of IL-23 was detectable after 4 h of SAA stimulation and continued to increase for up to 20 h, reaching a concentration of 650 pg/ml/106 cells (Fig. 7⇓B). SAA also induced IL-23 secretion from peripheral blood monocytes, which produced IL-23 at a concentration of 562 pg/ml/106 cells after 24 h of stimulation (Fig. 7⇓C). In contrast, no induction of IL-12 secretion was observed in SAA-stimulated blood monocytes and THP-1 cells (Fig. 7⇓C), although these cells are known to produce IL-12 in response to other stimuli such as the Staphylococcus aureus Cowan I strain (43).
Preferential induction of IL-23 expression by SAA. A, Time-dependent changes in the transcripts of IL-12p35 and IL-23p19 in SAA (1 μM)-stimulated THP-1 cells, as determined by real-time PCR. Results shown are means ± SEM from three experiments. Secretion of IL-12 and IL-23 by THP-1 (B) and by human blood monocytes (C). The cell-free medium was collected after SAA (1 μM) stimulation for the indicated periods of time. The levels of the two cytokines were determined with ELISA, as detailed in Materials and Methods. Data shown in B are means ± SEM from three experiments. Shown in C is a representative set of data. Individual differences, from 522.2 ± 5.2 to 780 ± 6.9 pg/ml/106 cells, were observed among three different donors at the 24-h time point for IL-23 secretion.
Discussion
Results presented in this study demonstrate that SAA not only stimulates the expression of proinflammatory cytokines as reported in recent literature (28, 29, 31, 44), but also induces cytokines primarily responsible for the modulation of lymphocyte functions. The SAA-stimulated IL-12p40 secretion is rapid and potent, and the kinetics of induction is similar to that of LPS and S. aureus Cowan I strain (39). Polymyxin B, which negates the cytokine-inducing effect of LPS, has no significant inhibition on SAA-induced IL-12p40 production. Collectively, these results support the notion that stimulation of IL-20p40 expression is a direct effect of SAA, and not a secondary effect mediated through another cytokine that is induced by SAA, or through the contaminating LPS. The SAA-induced IL-12p40 expression requires de novo protein synthesis and is regulated at the transcriptional level. Like most proinflammatory cytokines, SAA potently activates the transcription factor NF-κB within the first 2 h after its application. SAA also stimulates the DNA-binding activity of C/EBP, a transcription factor that binds to a specific site in the promoter region of IL-12p40 (42). Possible regulatory actions may occur at the posttranscriptional level, including alteration of the stability of the induced gene transcripts. Taken together, these results suggest that SAA is an endogenous ligand for the induced expression of IL-12p40 in monocytic cells.
The signaling mechanisms responsible for SAA-induced cytokine expression and the receptors involved have not been fully delineated. A number of proteins have been shown to interact with SAA (20, 21, 24, 25, 45). Among these proteins, formyl peptide receptor-like 1 (FPRL1), receptor for advanced glycation end products, and to some extent CD36 and LIMP II analogous-1 are cell surface receptors with known transmembrane signaling capability. Although receptor for advanced glycation end products preferentially binds fibrillar SAA, and the transcriptional regulatory property of CD36 and LIMP II analogous-1 has not been fully characterized, published studies have shown that FPRL1 is able to mediate SAA’s effect in the induction of IL-8 (28) and matrix metalloproteinase-9 (46). FPRL1 is a G protein-coupled receptor that can mediate activation of PI3K and MAPKs. Thus, an understanding of the involvement of these kinases may help to delineate the transmembrane signaling property of SAA. It is known that activation of the p38 MAPK is critical to LPS-induced IL-12 production (47, 48). Likewise, p38 MAPK plays a critical role in SAA-induced IL-12p40 expression (Fig. 4⇑). There are, however, differences in the pathways triggered by LPS vs SAA that lead to IL-12 expression. The LPS-induced IL-12 production is negatively regulated by PI3K (reviewed in Ref. 49). In contrast, the SAA-induced IL-12p40 expression is accompanied by an activation of Akt, a kinase downstream of PI3K. Inhibition of PI3K significantly reduced IL-12p40 production in SAA-stimulated cells (Fig. 5⇑). The same treatment, however, potentiated IL-12 production in LPS- and IFN-γ-stimulated dendritic cells (50). This latter finding was supported by several recent studies showing a role of PI3K in the inhibition of LPS-induced IL-12 production (51, 52, 53). In contrast, our results indicate that activation of PI3K and p38 MAPK in SAA-stimulated cells is not mutually dependent or exclusive (Fig. 6⇑), suggesting that these events can occur in parallel. Furthermore, we now have shown that a commonly used p38 MAPK inhibitor can produce a possibly nonspecific effect on the activation of PI3K either directly or indirectly (Fig. 6⇑), suggesting the limitation of using this inhibitor in certain studies. The observed difference among studies using a dominant-negative construct and those using a pharmacological inhibitor may also be due to the fact that a pharmacological inhibitor can target multiple members of an enzyme family, and a dominant-negative construct usually blocks a particular member of the enzyme family. It is possible that other members of the p38 MAPK and PI3K families also play a role in SAA-induced signaling, and these signaling events may not be affected by the dominant constructs used in the study. Although these findings clearly demonstrate a difference between SAA-stimulated and LPS-induced IL-12 expression, it also suggests the complexity of the signaling mechanisms that control the expression of IL-12 in response to various agonists.
A potentially important observation made in this study was the preferential induction of IL-23 as opposed to IL-12 in SAA-stimulated THP-1 cells and peripheral blood monocytes. How SAA, an endogenous factor, differentially regulates IL-12 and IL-23 is unclear at present. Because IL-12 and IL-23 share the same p40 subunit, differential expression of IL-12p35 and IL-12p40, as well as IL-12p35 and IL-23p19, is responsible for the preferential formation of IL-12p70, IL-23, and IL-12 homodimer. A number of published studies have compared the expression of IL-12p40 with that of IL-12p35, and have shown that NF-κB is required for the expression of both IL-12p40 and IL-12p35. However, optimal expression of IL-12p40 also requires C/EBP activation (42), whereas induction of IL-12p35 expression requires IFN-γ in addition to LPS (54). The role of IFN-γ in IL-12p35 expression is also suggested by the observation that IFN-γ can restore the defective expression of IL-12p35 gene in neonatal dendritic cells (55), through interaction of IFN regulatory factor-1 with an inverted IFN regulatory factor element within the IL-12p35 promoter (54). The SAA induction of IL-12p40, but not IL-12p35, is reminiscent to that of PGE2, a Th2-promoting factor that selectively induces IL-12p40 expression and IL-12p40 homodimer secretion (56). Unlike PGE2, SAA stimulation results in IL-23p19 expression that leads to the production of IL-23, a heterodimer consisting of p19 and p40. Because the p19 promoter has not been characterized, it is presently difficult to compare IL-12p35 expression with that of IL-23p19. It will be of significant interest to learn how IL-23p19 is regulated at the transcriptional level.
The differential regulation of IL-23 vs IL-12 by SAA may have important biological consequences. Although IL-23 shares certain functional properties with IL-12 and is a critical cytokine to bridge innate immunity and adaptive immunity (32), differences between the two cytokines exist. IL-12 has been widely accepted as an important regulator of Th1 immune response. It enhances innate immunity by stimulating NK cells and a selected group of T cells to produce IFN-γ (57), which is an important factor for microbial killing, particularly for elimination of intracellular pathogens. Unlike IL-12, IL-23 is known to induce activated and CD4+ T cells to secret IL-17. This IL-23-dependent CD4+ T cell population, termed the Th-IL-17 cells, displays a gene expression profile distinct from that of the prototypic Th1 and Th2 cells (58, 59). In contrast to IFN-γ, IL-17 is the prototype of a new group of cytokines involved in the proliferation, maturation, and migration of neutrophils. It has been detected in the sera and tissues of patients with rheumatoid and Lyme arthritis, multiple sclerosis, systemic lupus erythematosus, and asthma, suggesting its involvement in the development of various autoimmune diseases (60, 61). Thus, the IL-23-driven Th-IL-17 cells are essential for mediating tissue destruction and inducing autoimmunity, as seen in rheumatoid arthritis, in which a rise of SAA concentration has been reported (7, 30). The IL-23-induced IL-17 production also is involved in leukocyte proliferation, as IL-17 stimulates the production of G-CSF and contributes to granulopoiesis (62). A recent study has shown that increased granulocyte counts in peripheral blood, commonly seen after infection, can be attributed to the increased production of IL-23 and IL-17 triggered by phagocytosis of apoptotic neutrophils (63). To date, there is no experimental evidence suggesting that SAA released to blood circulation actually stimulate granulopoiesis, and this possibility remains to be investigated in future studies. However, it is likely that SAA derived from macrophages and synoviocytes contributes to local inflammation such as in arthritic joints, where induction of the IL-23-IL-17 pathways can greatly exacerbate symptoms through leukocyte activation.
Both IL-12 and IL-23 are produced by inflammatory macrophages and activated dendritic cells within hours after their encounter with pathogens (32). However, induction of IL-23 expression continues after clearance of microbial products. This difference suggests the presence of endogenous ligands for IL-23 expression; however, few endogenous factors for IL-23 production have been identified to date. Results reported in this study demonstrate that SAA, which is induced by both microbial products and inflammatory cytokines such as IL-6 and IL-1β, is a candidate of endogenous ligand for IL-23 production. Thus, SAA produced by hepatocytes during acute-phase response, and by macrophages and synoviocytes in chronic inflammatory conditions, may be an important endogenous mediator for the development of autoimmune inflammatory diseases such as rheumatoid arthritis.
A recent study using a SAA1-driven luciferase transgene demonstrates that SAA expression in hepatocytes could be induced within 4 h after LPS stimulation (64), indicating that SAA is an early signal generated by the invading microbes. Our results show that SAA is able to stimulate IL-23 production at a relatively low concentration of 1 μM, which is a fraction of its peak plasma concentration. Thus, the acute-phase SAA that dissociates from HDL (15), and particularly, SAA produced by macrophages and other cells at sites of chronic inflammation can possibly reach a concentration in the lower micromolar range for its cytokine-inducing activity. We hypothesize that the first wave of innate immune response, induced by microbial products such as LPS, may be propelled by a second wave of response stimulated by endogenous factors such as SAA. The two-step activation in immune cells may lead to a sustained production of selected immunomodulatory cytokines. Furthermore, cell-mediated immune response promoted by the Th-IL-17 cells may be triggered by SAA through induction of IL-23, which can lead to the development of chronic inflammation and autoimmune diseases.
In summary, this study identifies SAA as an endogenous ligand for stimulation of IL-12p40 synthesis and secretion. Our results also demonstrate that SAA differentially regulate the expression of IL-12 and IL-23, through mechanisms that involve preferential induction of IL-23p19. This and other recent developments suggest that APPs such as SAA can play an active role in the regulation of inflammation and immune responses.
Acknowledgments
We thank H. Sang for technical assistance, J. Han for providing the p38 MAPK plasmids, N. Cheng for helpful discussions, and L. Hricisak for critically reading the manuscript.
Disclosures
The authors have no financial conflict of interest.
Footnotes
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.
↵1 This work was supported in part by National Institutes of Health Grant AI040176 and by a Biomedical Science Grant from Arthritis Foundation. R.H. is a recipient of a Scientist Development Grant from the American Heart Association.
↵2 Address correspondence and reprint requests to Dr. Richard D. Ye, Department of Pharmacology, University of Illinois, 835 South Wolcott Avenue, Chicago, IL 60612. E-mail address: yer{at}uic.edu
↵3 Abbreviations used in this paper: APP, acute-phase protein; CRP, C-reactive protein; Ct, threshold cycle; FPRL1, formyl peptide receptor-like 1; HDL, high density lipoprotein; SAA, serum amyloid A; STBS, suspension cell transfection buffer solution.
- Received October 27, 2005.
- Accepted June 30, 2006.
- Copyright © 2006 by The American Association of Immunologists
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