SR-A ligand and M-CSF dynamically regulate SR-A expression and function in primary macrophages via p38 MAPK activation

Background Inflammation is characterized by dynamic changes in the expression of cytokines, such as M-CSF, and modifications of lipids and proteins that result in the formation of ligands for Class A Scavenger Receptors (SR-A). These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined. To address these questions, SR-A expression and function were examined in resident mouse peritoneal macrophages incubated with M-CSF or the selective SR-A ligand acetylated-LDL (AcLDL). Results M-CSF increased SR-A expression and function, and required the specific activation of p38 MAPK, but not ERK1/2 or JNK. Increased SR-A expression and function returned to basal levels 72 hours after removing M-CSF. We next determined whether prolonged incubation of macrophages with SR-A ligand alters SR-A expression. In contrast to most receptors, which are down-regulated by chronic exposure to ligand, SR-A expression was reversibly increased by incubating macrophages with AcLDL. AcLDL activated p38 in wild-type macrophages but not in SR-A-/- macrophages, and p38 activation was specifically required for AcLDL-induced SR-A expression. Conclusions These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes. In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK. In this way, SR-A may modulate inflammatory responses by enhancing macrophage uptake of modified protein/lipid, bacteria, and cell debris; and by regulating the production of inflammatory cytokines, growth factors, and proteolytic enzymes.

Background SR-A is a multifunctional macrophage receptor that is upregulated during monocyte differentiation into macrophages, and is further increased in pathological conditions such as diabetes [1][2][3]. SR-A is also highly expressed by macrophages in atherosclerotic lesions and Alzheimer's plaques [4][5][6]. In contrast, decreased SR-A expression is associated with increased susceptibility to bacterial infection, progression of prostate cancer, and enhanced cytokine production [7][8][9]. Such results suggest important and complex roles for SR-A in modulating immune function and inflammation.
Macrophage differentiation and recruitment during inflammation is mediated by changes in the local environment and the secretion of cytokines/chemokines such as M-CSF. M-CSF, which is produced by many cell types, is a cytokine that plays an essential role in monocyte-macrophage functions including membrane ruffling, cell migration, and the production of inflammatory mediators [reviewed in [10,11]]. Because of its role in the development of monocyte/macrophage cells, M-CSF is thought to play important roles in immune function and inflammatory diseases [reviewed in [12]]. For example, M-CSF is thought to promote atherosclerosis by increasing macrophage viability/differentiation, low-density lipoprotein (LDL) receptor-mediated lipoprotein uptake, and the expression of macrophage SR-A [12][13][14]. SR-A promotes foam cell formation by binding and internalizing modified lipoproteins [e.g., acetylated LDL (AcLDL), oxidized LDL (oxLDL)], but not native lipoproteins [15]. SR-A has also been associated with additional macrophage functions including cell adhesion to modified extracellular matrix, phagocytosis/clearance of apoptotic cells, and the modulation of macrophage activation and cytokine production [16][17][18][19][20]. Thus, increased SR-A expression may play an important part in the effects of M-CSF on immune function and inflammation. Taken further, the ability to inhibit M-CSF-induced SR-A expression may have important therapeutic implications. However, the cellular pathways that couple M-CSF binding to increased SR-A expression are not known.
In addition to increased secretion of M-CSF, local inflammation results in the modification of proteins, alterations in the extracellular matrix (ECM), and tissue damage. Such modifications result in the formation and accumulation of SR-A ligands. Many receptors including receptor-tyrosine kinases (e.g., insulin receptors), G protein-coupled receptors (e.g., β-adrenergic receptors), and nutrient receptors (e.g., LDL receptors) are down-regulated by prolonged exposure to ligand. This negative feedback is mediated by activation of intracellular signaling pathways that regulate receptor expression. Although it might be of particular importance in diverse inflammatory conditions, the effect of modulating the concentration of SR-A ligand in tissue on SR-A expression in resident macrophages is not known.
SR-A gene expression is under the control of a proximal promoter in combination with an upstream enhancer element [2,21,22]. Binding of the transcription factor AP-1 to this upstream enhancer element has been shown to be sufficient to direct specific macrophage SR-A expression [1,22]. Activation of AP-1 in inflammatory cells is primarily regulated by the mitogen activated protein kinases (MAPK), in particular via c-Jun phosphorylation by JNK and ATF2 phosphorylation by p38 MAPK [23]. Roles for both of these MAPKs in regulating SR-A expression in elicited macrophages has been suggested [24,25].
It has been suggested that different agents used to elicit resident peritoneal macrophages can alter macrophage populations, their regulation by intracellular signals, and macrophage responses e.g., superoxide production, chemokine generation, and Ab-dependent cellmediated cytolysis [26,27]. In this study, we used isolated resident peritoneal macrophages to examine the intracellular signaling pathways involved in regulating SR-A expression and function. We also examined whether chronic exposure to SR-A ligand alters SR-A expression in macrophages. Our results demonstrate that cytokine and SR-A ligand reversibly enhance SR-A expression and function via activation of p38 MAPK and the subsequent induction of SR-A transcription.

Cell culture and treatment
Resident (non-elicited) mouse peritoneal macrophages (MPMs) were harvested by peritoneal lavage with icecold sterile saline from male NIH Swiss mice (Harlan; Indianapolis, IN), and from C57Bl/6 and SR-A-/-mice on a C57Bl/6 background ( Figures 1C and 4B; Jackson Laboratory, Bar Harbor, ME). Animal care and use for all procedures were done according to protocols reviewed and approved by the Institutional Animal Care and Use Committee at University of Arkansas for Medical Sciences and the University of Kentucky. Isolated macrophages were cultured as previously described [28]. Briefly, peritoneal exudates were incubated overnight at 37°C and non-adherent cells removed by gently washing with PBS. Adherent macrophages were maintained in DMEM containing antibiotics and FBS (10%) for 48 hrs prior to use in experiments. MPMs were then treated with agonists in the presence or absence of specific inhibitors as described in figure legends. Following treatment, MPMs were washed with ice-cold PBS and cell lysates prepared by incubating cells in MBST/OG buffer (25 mM MES; 150 mM NaCl; 60 mM octylglucopyranoside; 1% Triton X-100; pH 6.4) containing protease and phosphatase inhibitors (Sigma, St. Louis, MO) for 45 min on ice. Cell lysates were centrifuged at 13,000 rpm for 15 min and the pellets were discarded. Protein concentration of supernatant was determined using the BioRad DC assay using BSA as a standard (Hercules, CA).

Western blot analysis
Equal amounts of cell lysate protein were resolved by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). Proteins were detected by immunoblotting with specific primary antibodies followed by incubation with speciesspecific HRP-conjugated secondary antibodies. Bands were visualized by chemiluminescence, images captured with a Kodak Image Station 4000 MM Pro, and band intensities quantified using Kodak 1D image analysis software.

Flow Cytometry
To assess SR-A-mediated lipoprotein uptake and surface accessible SR-A, cultured MPMs were preincubated for 2 hrs in serum-free DMEM and then incubated for 2 hrs at 37°C with Alexa 488 -AcLDL(2.5 μg/ml).

M-CSF induces SR-A expression and AcLDL association
Macrophage differentiation and recruitment into inflammatory sites are associated with increased SR-A expression. SR-A expression is regulated by both transcriptional and post-transcriptional processing [1,3,14,25,[28][29][30]. M-CSF is involved in both monocyte/ macrophage differentiation and recruitment during inflammation, and has previously been shown to enhance SR-A expression in elicited macrophages via increased transcription [14]. However, the intracellular signals that couple M-CSF to enhanced SR-A expression have not been defined.
To examine the signaling pathways that regulate SR-A expression, the effect of M-CSF on SR-A expression was examined in non-elicited, resident MPMs. Non-elicited, resident MPMs were used because of the potential for eliciting agents to alter macrophage phenotype and regulation by intracellular signals. Culturing isolated MPMs with M-CSF resulted in the concentration-( Figure 1A To confirm that increased SR-A expression is correlated with enhanced SR-A function, we examined the ability of M-CSF to stimulate fluorescently-labeled AcLDL (Alexa 488 -AcLDL) association with MPMs. Results shown in Figure 1C show that a blocking SR-A monoclonal antibody (2F8) and an excess of SR-A competitor (fucoidan) reduced AcLDL association with macrophages isolated from C57Bl/6 or NIH-Swiss mice to a level similar to that obtained using macrophages isolated from SR-A-/-in a C57Bl/6 background. These results demonstrate the specificity of this asssay for assessing SR-A function. M-CSF treatment induced a time-dependent increase in AcLDL association with MPMs that was maximal (2.4-fold) at 24 hrs ( Figure 1D), with no further increase at longer incubation times (data not shown). M-CSF stimulation of SR-A function was linearly correlated (Pearson r = 0.97; p = 0.007) with the effect of M-CSF on SR-A expression ( Figure 1E) indicating that in resident MPMs SR-A function is limited, at least in part, by the level of receptor expression. These results suggest that by increasing SR-A expression M-CSF, produced for example by endothelial cells or lymphocytes in an atherosclerotic plaque or other inflammatory sites, can increase the uptake of modified lipoprotein and other scavenger receptor ligands.

M-CSF-stimulates SR-A expression by upregulating SR-A mRNA and protein synthesis which requires p38 MAPK activation
Many effects of M-CSF including macrophage migration, differentiation, survival, and cytokine production are mediated, in part, via activation of MAPKs, a family of kinases that include ERK1/2, JNK, and p38 [34][35][36]. Activation of MAPKs, in particular JNK and p38 MAPK, regulates the activity of several transcription factors including AP-1 [23]. The binding of AP-1 to an upstream enhancer element is sufficient to direct specific macrophage SR-A expression in inflammatory cells [1,21,22]. Therefore, we examined M-CSF-dependent MAPK activation and whether MAPK activation was required for M-CSF-induced SR-A expression. For this, resident MPMs were treated with M-CSF and the activation of ERK, p38, and JNK assessed by immunoblotting with phospho-specific MAPK antibodies. As shown in Figure 2A

M-CSF (data not shown). To determine if activation of MAPK was specifically required for M-CSF-induced SR-
A expression and function, the ability of M-CSF to stimulate SR-A expression and AcLDL association was assessed in MPMs pretreated with specific inhibitors of p38 MAPK (SB203580), JNK (SP600125), and MEK1 (PD98059), which inhibits ERK1/2 activation. Inhibiting JNK or ERK1/2 activation had no effect on either M-CSF-induced SR-A expression ( Figure 2B) or M-CSFinduced uptake of modified lipoprotein ( Figure 2C). In contrast, pretreating macrophages with SB203580 inhibited both M-CSF-induced SR-A expression and modified lipoprotein uptake ( Figure 2C). Together, these data define a specific requirement for activation of p38 MAPK, but not ERK1/2 and JNK, in M-CSF-induced SR-A expression and function.

M-CSF stimulates SR-A expression and AcLDL association in reversible manner
Increased SR-A expression and function following M-CSF treatment suggests that regulating SR-A expression in resident macrophages is an adaptive response to changes in the local inflammatory environment. Inflammation is a dynamic process in which the production of cytokines such as M-CSF changes as inflammation resolves over time. To test whether the enhanced SR-A expression and function induced by M-CSF is reversible, we examined SR-A protein and AcLDL uptake following removal of M-CSF using the incubation scheme depicted in Figure 3A. The increase in SR-A expression, as quantified by western blotting ( Figure 3B the pretreated levels 72 hr after M-CSF removal (t 1/2 ≈ 24 hr). To determine if SR-A expression was still responsive to M-CSF, previously treated MPMs were restimulated with M-CSF for another 30 hrs. Similar to naïve MPMs, SR-A expression and function were increased by the restimulation with M-CSF. As summarized in Figure 3D, M-CSF-induced proportional changes in SR-A expression and function in both naïve and previously treated MPMs. Together, these data demonstrate that in resident macrophages SR-A expression and function is dynamically regulated by M-CSF.

AcLDL stimulates SR-A expression in reversible manner via activation of p38 MAPK
In addition to cytokines, inflammatory settings are characterized by accumulation of SR-A ligands including oxidized lipoproteins (e.g., modified LDL), necrotic cell debris, and modified ECM. Because many receptors are down-regulated by chronic exposure to ligand, we tested whether SR-A expression was decreased by ligand. In contrast to our hypothesis, incubating macrophages with an SR-A selective ligand (AcLDL; 50 μg/ml) for 24 hr increased SR-A protein expression ( Figure 4A). As demonstrated for M-CSF, enhanced SR-A expression returned to the pretreated levels 72 hr after AcLDL was removed, and pre-treating MPMs with a specific p38 MAPK inhibitor (SB203580) blocked AcLDL-induced SR-A expression. In contrast, inhibitors of ERK1/2 and JNK did not affect the ability of AcLDL to enhance SR-A expression. To confirm that incubating resident MPMs with AcLDL induced SR-A-dependent MAPK activation, the ability of AcLDL to activate MAPK in wild-type and SR-A deficient resident MPMs was examined. As shown in Figure 4B, treating macrophages with AcLDL (50 μg/ml, 10 min) induced phosphorylation of p38 MAPK and ERK1/2 in wild-type but not in SR-A deficient macrophages. In contrast, p38 MAPK and ERK1/2 phosphorylation were similarly increased in both wild type and SR-A deficient MPMs treated with M-CSF. JNK phosphorylation was not detected in any treatment group (data not shown). Together, these data indicate that ligand binding to SR-A is positively coupled to SR-A expression via specifically activating p38 MAPK.
Regulating SR-A expression in vivo may be relevant to many inflammatory disorders. For example, M-CSF plays important roles in inflammation and immunity. M-CSF increases anti-tumor and anti-infective functions of macrophages, whereas M-CSF deficiency decreases atherosclerosis and impairs osteoclast development [11,37]. The extent to which these effects depend on altered SR-A expression remains to be defined. However, the possibility that regulating SR-A expression modulates inflammatory responses is suggested by the many studies showing that decreased SR-A expression is associated with reduced atherosclerosis, increased susceptibility to infection, disease progression in prostate cancer, and dysregulation of bone development [8,9,[38][39][40]. Like SR-A, activation of p38 MAPK is important for many inflammatory processes including the production of TNFα, IL-1β, and other cytokines [41,42]. In addition, inhibiting p38 MAPK has shown potential benefit in the treatment of inflammatory disease [43,44]. Our findings suggest that the effects of M-CSF and p38 MAPK on immune and inflammatory processes may be mediated, in part, by modulating SR-A expression.

Conclusions
Our results indicate that resident tissue macrophages adapt to changes in their local environment by modulating SR-A expression and function. Such modulation may involve the local secretion of M-CSF, which increases SR-A expression via activating p38 MAPK. Unlike many receptor systems which are down-regulated by ligand, ligand binding to SR-A up-regulates SR-A expression by activating p38 MAPK. Increased SR-A expression might modulate inflammation by enhancing macrophage uptake and clearance of modified protein/ lipid, bacteria, and cell debris. In addition, SR-Amediated p38 MAPK activation may regulate the production of inflammatory cytokines, growth factors, and proteolytic enzymes, and therefore modulate the progression of many inflammatory disorders [18][19][20]. This may be of particular importance in settings where SR-A ligands accumulate such as atherosclerosis, diabetes, and Alzheimer's disease.