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Abstract
Objective—Syndecan-1 (Sdc-1) is a member of a family of cell surface proteoglycans, which has been reported to participate in the regulation of events relevant to tissue repair and chronic injury responses, including cell–substrate interactions, matrix remodeling, and cell migration. In this study, we report the functional significance of Sdc-1 in polarized macrophage populations and its role in adhesion and motility events relevant to resolution of the inflammatory program.
Approach and Results—Macrophage Sdc-1 expression is associated with differentiated M2 macrophages with high intrinsic motility, and Sdc-1 deficiency is characterized by impaired migration and enhanced adhesion. Leukocyte infiltration and emigration were examined in a thioglycollate-induced model of peritonitis in Sdc-1+/+ and Sdc-1−/− mice. Although the infiltration of inflammatory cells was similar in both cohorts, a significant delay in the lymphatic clearance of Sdc-1−/− macrophages was observed. Moreover, we observed enhanced inflammation and greater burden of atherosclerotic plaques in ApoE−/−Sdc-1−/− mice maintained on a Western diet.
Conclusions—These results demonstrate that defective motility in Sdc-1−/− macrophages promotes a persistent inflammatory state with relevance to the pathogenesis of atherosclerosis.
Introduction
A persistent population of activated lesional macrophages has been linked to the progression of atherosclerosis. Unrestrained macrophage activation plays a vital role in pathogenic remodeling and plaque destabilization that can culminate in rupture and acute coronary syndromes. Intensive research efforts are focused on understanding the regulatory mechanisms that signal resolution programs characteristic of a healthy inflammatory response and a return to tissue macrophage homeostasis. Nonphlogistic clearance of apoptotic cells has been suggested as a key event that drives macrophage resolution via anti-inflammatory signaling and enhanced egress of macrophages from inflammatory foci.1,2 Indeed, secondary necrosis observed in advanced atherosclerotic plaques may be a signature of failed resolution programs in sessile lesional macrophages that do not efficiently clear apoptotic debris and likewise do not propagate signals to silence inflammation and exit the vascular wall.3–6 Such observations have renewed interest to understand the macrophage motility dynamics, as they relate to a persistent inflammatory state within the vessel wall with the hope of developing new strategies to limit macrophage burden in atherosclerosis.
Plaque regression has been documented in numerous animal studies and remains the ultimate therapeutic goal in the treatment of human atherosclerosis.7–10 Recent investigations using a mouse surgical transplant model of plaque regression have suggested that dramatic improvements in plasma lipoprotein profiles can induce monocyte-derived cells to emigrate out of the inflamed vascular wall and that this process correlates with reduced plaque burden.11,12 Cell tracking studies identified egress depots in the iliac and hepatic lymph nodes, suggesting dissemination through draining lymph nodes, as well as reentry into the blood stream.13 Consistent with this hypothesis of failed macrophage clearance in chronically inflamed vascular lesions, Moore et al15 have reported the expression of inhibitory guidance cues, netrin-1, and semaphorin 3E, in atheroma macrophages.14 Moore et al15 offered a paradigm of competing trafficking signals to explain the macrophage retention, where neuronal guidance molecules secreted by activated macrophages may serve as a stop signal to inhibit the activation of the actin cytoskeleton and override the chemokine-mediated macrophage emigration from the atherosclerotic wall. Conversely, data have been reported in an apoE complementation model of regression, where reduced macrophage burden is strongly correlated with decreased monocyte recruitment and apoptotic turnover rather than migratory egress.16 Collectively, these reports emphasize the complexity in dissecting the macrophage resolution response and provide motivation for continued investigations of inflammatory effectors that govern macrophage retention in atherosclerosis.
In this study, we report the functional significance of the cell-surface heparan sulfate proteoglycan, syndecan-1 (Sdc-1), on macrophage adhesion and motility events relevant to inflammatory resolution. We have previously reported macrophage Sdc-1 as an endogenous modulator of the inflammatory response in abdominal aortic aneurysm, and we speculated that Sdc-1-expressing macrophages represented an alternatively activated regulatory population in the chronically inflamed wall.17 Anti-inflammatory functions of Sdc-1 are often attributed to the sequestration of inflammatory mediators by pendant glycosaminoglycans;18 however, Sdc-1 has also been reported to regulate cell adhesion and migration.19–21 Macrophage Sdc-1 protein expression is governed through the cAMP/protein kinase A signaling cascade, which is a signaling program reported to inhibit inflammation in macrophages.22–25 However, there have been no previous reports examining Sdc-1 expression as a function of macrophage polarization. Moreover, predicting the anticipated role of Sdc-1 in macrophage motility is not intuitive because Sdc-1 has been reported to both inhibit and augment motility in cell- and tissue-dependent contexts.21,26–28 We demonstrate that Sdc-1 expression is specific to differentiated M2 macrophages with high intrinsic motility and that Sdc-1 deficiency is characterized by impaired migration and enhanced extracellular matrix adhesion. In a self-resolving model of inflammation, we examined both infiltration and emigration of macrophages in wild-type (Sdc-1+/+) and Sdc-1 deficient (Sdc-1−/−) mice. Significantly, we observed no difference in the kinetics of leukocyte influx or the magnitude of the inflammatory response; however, a significant difference in the resolution response was observed with delayed lymphatic clearance of Sdc-1−/− macrophages. We further examined the relevance of macrophage Sdc-1 in apolipoprotein E (ApoE−/−) deficient mice maintained on a Western diet. Sdc-1−/−ApoE−/− mice were characterized by the presence of an enhanced population of inflammatory macrophages and a greater burden of atherosclerotic plaques. Collectively, these results support the notion that defective motility of mature macrophage populations may promote a persistent inflammatory state and suggest that Sdc-1 expression is part of a broad program to regulate the trafficking of alternatively activated macrophages.
Methods
Materials and Methods are available in the online-only Data Supplement.
Results
Sdc-1 Expression on Polarized Macrophage Populations
Macrophage activation has been operationally divided into 2 subsets: classically activated proinflammatory M1 and alternatively activated M2. M2 polarization encompasses a broad spectrum of macrophage activation, and additional subdivisions have been suggested to clarify functional phenotypes, such as M2a to distinguish traditional Th2-type (IL-4 and IL-13) polarization and M2c to distinguish anti-inflammatory (IL-10, TGF-β, and glucocorticoid) polarization.29 Cell-surface marker expression, biochemical signature, and phagocytotic capacity of Sdc-1 positive macrophages were characterized for M1, M2a, and M2c standard populations generated from elicited peritoneal macrophages. Flow cytometry was used to measure the cell-surface expression of MHCII, CD86, and programmed death ligand 2 (PD-L2), 3 markers that have been previously reported to associate with M1 or M2 polarization.30,31 M1-polarized cells displayed characteristically high expression of the antigen presenting molecule, MHCII, and the coactivating molecule, CD86 (Figure 1A). M2a-polarized cells were most easily characterized as MHCII+ with high expression of the PD-L2, a Th2-stimulated molecule that inhibits T-cell proliferation.32 M2c does not positively correlate with MHCII, CD86, or PD-L2 consistent with reports that IL-10 deactivates macrophages.33,34 Sdc-1-expressing macrophages, which can be enriched after treatment with a membrane-permeant cAMP analogue or adenosine-induced activation of endogenous adenyl cyclase (Figure 1B; media control presented in Figure I in the online-only Data Supplement), displayed the strongest similarity to M2c with MHCIIlow, CD86low, and PD-L2− signatures.
Sdc-1 expression on polarized macrophage populations. A, Sdc-1-expressing macrophages were examined against standard populations for surface expression of M1 (MHCII and CD86) and M2a (PD-L2) markers, and data are displayed in histogram format against the IgG control (gray) and graphed as mean fluorescent intensity (MFI). B, Sdc-1 expression can be induced on F4/80 positive macrophages after exposure to 6-bnz-cAMP or adenosine. C, Elevated M1-induced inducible nitric oxide synthase activity was characterized via Griess reaction. D, Arginase activity was measured in cell lysates and reported as fold increase over unstimulated control macrophages. E, IL-10 was measured via conditioned media ELISA. F, Percent phagocytosis was characterized in polarized populations by fluorescent bead uptake. G, Sdc-1 expression is not induced under standard M1, M2a, or M2c conditions. H and I, Sdc-1 expression is induced after efferocytosis of apoptotic macrophages. H, Data plotted as histogram (Sdc-1, white) against IgG control (gray) and as MFI. I, Quantitative polymerase chain reaction analysis of Sdc-1 mRNA in Sdc-1 wild-type and knockout macrophages +/− efferocytosis. Data are representative of triplicate sample mean±SEM, *P<0.05, and **P<0.01.
Macrophage populations were further assayed for soluble mediators, with elevated nitrite levels observed in the M1 population (Figure 1C), whereas arginase activity was enhanced in M2a macrophages (Figure 1D). A significant increase in IL-10 production was observed for Sdc-1-expressing macrophages when compared with M1- or M2a-polarized populations. However, M2c-polarized macrophages secreted the highest level of IL-10 (Figure 1E). Finally, bead phagocytosis, which has been reported to be upregulated in alternatively activated M2 macrophages,35 was found to be significantly elevated in all populations when compared with M1 (Figure 1F). Characterization of Sdc-1 expression in polarized standard populations suggests that Sdc-1 protein expression is not induced within the context of a primary Th1 (IFN-γ) or Th2 (IL-4) inflammatory response (Figure 1G) but instead may be displayed in those macrophages that have received a deactivation response, typified by decreased antigen presentation (MHCIIlow) and increased immunosuppression (IL-10+). Induction of Sdc-1 was not observed with IL-10 treatment (Figure 1G); however, additional protocols commonly reported to suppress or deactivate macrophages were examined, including exposure to TGFβ or glucocorticoids, as well as efferocytosis-mediated deactivation.36,37 Significantly, upregulation of Sdc-1 protein and mRNA was observed after efferocytosis of apoptotic macrophages (Figure 1H and 1I; Figure II in the online-only Data Supplement), suggesting a physiological program for expression that is consistent with deactivation and inflammatory resolution.
Leukocyte Chemoattractive Potential of Sdc-1 Macrophages
In vitro phenotypic analysis suggests that macrophage Sdc-1 expression is not consistent with a classically activated M1 subtype, and subsequent quantitative real-time reverse transcription polymerase chain reaction studies confirmed this distinction. A higher level of proinflammatory genes and chemokines was observed in M1 versus Sdc-1-enriched macrophages (Tables I and II). Given the effect that leukocyte recruitment has on inflammatory outcome, we examined the chemoattractive potential of conditioned media generated from M1 or Sdc-1-enriched populations toward purified circulating monocytes or activated CD4 T-cells. Significantly, conditioned media from Sdc-1 macrophages elicited significantly reduced chemotaxis of monocytes and CD4 T-cells (Figure 2A and 2B; controls presented in Figure III in the online-only Data Supplement). These results suggest that although M1 macrophages actively contribute to the progression of inflammation, Sdc-1-expressing macrophages should correlate with reduced leukocyte influx in vivo.
mRNA Expression Profile of Proinflammatory Genes in M1 vs Sdc-1+
mRNA Chemokine Expression Profile in M1 vs Sdc-1+
Sdc-1 macrophages display reduced leukocyte chemoattractive potential. Leukocyte chemoattractive potential was measured by transwell chemotaxis of murine-circulating monocytes (A) or murine-activated CD4+ T-cells (B) to conditioned media generated from M1 or Sdc-1 macrophages. Cells that migrated to the lower chamber were counted and reported as % migrated, n=5, mean±SEM, *P<0.05, and **P<0.01.
Distinct Motility Behavior in Polarized Macrophage Populations
Decreased migratory capability may contribute to macrophage retention in chronic inflammatory environments. Given that Sdc-1 has been traditionally studied in the context of cell adhesion and migration, we examined the functional consequence of macrophage Sdc-1 deficiency on motility and adhesion in polarized populations. Motility was first investigated by characterizing the migration of Sdc-1+/+ and Sdc-1−/− macrophages toward fetal bovine serum using a Boyden chamber assay. Cells were specifically induced toward an M1 or Sdc-1+ phenotype. We observed dramatically dampened motility in classically activated Sdc-1+/+ and Sdc-1−/− M1 macrophages, with no significant difference noted between genotypes (Figure 3A). However, when the migration of Sdc-1+/+ and Sdc-1−/− macrophages was examined under conditions to induce a Sdc-1+ phenotype through the addition of adenosine to basal media, we observed a significant reduction in the motility of Sdc-1−/− macrophages (Figure 3A; Figure IV in the online-only Data Supplement). Given our previous results suggesting the absence of Sdc-1 expression on M1-polarized macrophages, the lack of a functional role for Sdc-1 in M1 macrophage motility is not surprising. Instead, our results suggest that Sdc-1 functionally contributes to the motility of specifically induced M2 macrophage populations. Sdc-1+/+ and Sdc-1−/− motility experiments were repeated using conditioned media generated from apoptotic macrophages as a surrogate to chemotactic signals generated in the atherosclerotic lesion environment.38–41 A significant reduction in the motility of Sdc-1−/− macrophages was noted toward apoptotic conditioned media, suggesting that Sdc-1 deficiency may limit the efficiency of efferocytosis processes in chronic inflammatory environments by restricting the movement toward apoptotic cells (Figure 3B and controls in Figure V in the online-only Data Supplement). We examined motility-dependent efferocytosis using a Boyden chamber assay. Violet-labeled Sdc-1+/+ or Sdc-1−/− macrophages were seeded in the upper chamber, and CFSE green–labeled apoptotic macrophages were provided as the chemotactic stimulant in the bottom chamber. We quantified the total number of migrated cells and the total number of migrated cells with engulfed green apoptotic cells. As we observed in our migration experiments with apoptotic conditioned media, a significant reduction in the migration events was observed for Sdc-1−/− macrophages; consequently, we also observed a significant reduction in efferocytosis events for Sdc-1−/− macrophages (Figure 3C). When efferocytosis efficiency is examined under static conditions, we observed no difference between Sdc-1+/+ and Sdc-1−/− macrophages, which is consistent with our hypothesis that defective motility in Sdc-1−/− macrophages may promote a persistent inflammatory state.
Role of Sdc-1 in the motility of polarized macrophages. A, Migration index to serum was calculated for M1-induced Sdc-1+/+ and Sdc-1−/− and for Sdc-1-induced (+adenosine) Sdc-1+/+ and Sdc-1−/− macrophages. Sdc-1+/+ macrophages display significantly enhanced motility compared with Sdc-1−/− and M1-polarized cells. B, Sdc-1−/− shows a significantly reduced motility response to apoptotic cell conditioned media. C, In an assay of migration-dependent efferocytosis, Sdc-1−/− shows significantly reduced efferocytosis events, and control bars represent migration events with basal media on top and bottom. D, Increased adhesion was observed in Sdc-1−/− and M1 macrophages compared with Sdc-1+/+. Actin cytoskeletal staining (red) reveals increased formation of cell protrusions in Sdc-1+/+ (E) vs Sdc-1−/− (F) macrophages, nucleus stained with DAPI (blue); scale bar, 10 μm. Data are representative of triplicate samples, mean±SEM, *P<0.05, and **P<0.01.
The adhesion profiles of Sdc-1+/+ and Sdc-1−/− macrophages were investigated in comparison with wild-type M1-induced macrophages. M1-induced macrophages displayed the greatest adhesion, with increased adhesion noted for Sdc-1−/− when compared with Sdc-1+/+ macrophages (Figure 3D). Subsequent staining of the actin cytoskeleton revealed impaired ability of Sdc-1−/− macrophages to form cell protrusions (Figure 3E and F), consistent with previous reports that Sdc-1 can regulate cell protrusions that may be functionally important in migration by mediating outward extension of cell edges.42,43 We confirmed that Sdc-1−/− macrophages polarize to the expected M1 or M2 phenotype when incubated with Th1 or Th2 cytokines, thus the only phenotypic defect observed in Sdc-1−/− macrophages was reduced motility and enhanced adhesion (Figure VI in the online-only Data Supplement). Collectively, these results suggest that Sdc-1 expression modulates motility in mature-polarized macrophage populations and may limit macrophage retention in chronic inflammatory environments.
Sdc-1 Contributes to the Resolution of Inflammatory Responses
We used a thioglycollate-induced murine model of peritonitis as an example of self-resolving inflammation to investigate the role of Sdc-1 in leukocyte infiltration and resolution. Cell counts, neutrophil and macrophage populations, and Sdc-1 expression were recorded during a 12-day time frame. At the induction of peritonitis, Sdc-1 expression was not detected on peritoneal macrophages. However, 24 to 48 hours after onset of peritonitis, Sdc-1 macrophages were abundant and then decreased over the 12-day time course (Figure 4A). Analysis of total cell counts revealed no difference in the kinetics or magnitude of the initial inflammatory response between Sdc-1+/+ and Sdc-1−/− mice (Figure 4B, time, 0–2 days). Indeed, the evolution and resolution of the neutrophil response were identical in Sdc-1+/+ and Sdc-1−/− mice (Figure 4C). Significantly, the resolution of the late inflammatory response was delayed in Sdc-1−/− mice, with a greater number of macrophages detected at 8 to 12 days (Figure 4D). There was no difference in the magnitude or kinetics of macrophage influx between Sdc-1+/+ and Sdc-1−/− mice; however, the time interval required for a 50% to 25% reduction in the number of peritoneal macrophages was significantly prolonged in Sdc-1−/− mice (1.7 versus 4.7 days; P<0.05; Figure 4E; Figure VII in the online-only Data Supplement).44 This result is consistent with a delayed clearance of macrophages from the peritoneal cavity in the absence of Sdc-1 expression. Adoptive transfer was used to examine macrophage emigration into the lymphatic system during the resolution phase of peritonitis. Fluorescently labeled Sdc-1+/+ and Sdc-1−/− macrophages were introduced into the peritoneal cavity at day 3, and mesenteric lymph or spleen was harvested 18 hours post adoptive transfer. Significantly, an increase in lymphatic trafficking in Sdc-1+/+ macrophages was noted (Figure 3F). Thus, in vitro and in vivo migration studies suggest that Sdc-1 expression may be an important modulator of macrophage motility and, specifically, may be an important functional component of macrophage emigration and resolution.
Sdc-1 contributes to macrophage resolution in thioglycollate-induced peritonitis. A, Macrophage Sdc-1 expression was monitored using flow cytometry over the time course of peritonitis. Total cell count (B), polymorphonuclear (PMN) count (C), and macrophage count (D) reveal an altered macrophage resolution response in Sdc-1−/− (knockout [KO]) peritonitis vs Sdc-1+/+ (wild-type [WT]). E, Resolution curve in WT vs KO peritonitis, and time interval for a 50% to 25% reduction (designated with black arrows for WT and gray arrows for KO) was significantly prolonged in KO (1.7 vs 4.7 days, analysis curve detailed in Figure I in the online-only Data Supplement). F, Adoptively transferred Sdc-1+/+ (WT) cells traffic more efficiently from the peritoneal cavity to the lymph and spleen compared with Sdc-1−/− (KO). n=4 to 5 mice per time point; data are mean±SEM and *P<0.05.
Sdc-1 Limits Atherosclerotic Lesion Formation
To examine the role of Sdc-1 in atherosclerosis, ApoE−/−Sdc-1−/− mice were generated17 and the presentation of atherosclerosis was compared with that observed in ApoE−/− mice when both cohorts were maintained on a Western diet. At 8 weeks, brachiocephalic lesion area was quantified in consecutive tissue sections, and the inflammatory profile was examined using standard immunohistochemistry.45–48 We did not observe Sdc-1 staining in native brachiocephalic sections (data not shown), but by 8 weeks, Sdc-1 expression was clearly evident in macrophage positive areas around lesional plaques and within the adventitial tissue (Figure 5A and B; staining control in Figure VIII in the online-only Data Supplement). Although serum cholesterol levels were identical in both cohorts, a significant increase in atherosclerotic lesion area was measured in ApoE−/−Sdc-1−/− mice when compared with ApoE−/− mice (88.03±8.66 versus 57.40±8.08; P<0.05; Figure 5C; Figure IX in the online-only Data Supplement). Consistent with enhanced lesion formation, we observed enhanced expression of the macrophage M1 marker, inducible nitric oxide synthase, and greater necrotic area within Apoe−/−Sdc-1−/− lesions (Figure 5D and 5E).
Sdc-1 is protective in atherosclerosis. A, Immunohistochemical staining of Sdc-1 in 8-week Western diet–induced lesions. B, Macrophage positive staining observed in the regions of Sdc-1 staining. C, A significant increase in lesion area was measured at 8 weeks in ApoE−/−Sdc-1−/− mice vs ApoE−/−, n=19 per genotype. D, Enhanced inducible nitric oxide synthase (iNOS) staining was observed in ApoE−/−Sdc-1−/− lesions, representative of 3 mice per genotype. E, A significant increase in necrotic area (arrow) was observed in ApoE−/−Sdc-1−/− lesions, representative of n=10 per genotype. F, Sdc-1 positive staining was observed in macrophage positive zones of human plaque samples. Scale bars, 200 μm. Data are mean±SEM and *P<0.05.
To explore the relevance of our findings in human atherosclerosis, we examined human carotid plaque sections for macrophage Sdc-1 staining. Atherosclerotic plaques were harvested during the course of carotid endarterectomy from symptomatic patients having reported a recent stroke or transient ischemic attack. Immunohistochemical staining revealed colocalization of macrophages and Sdc-1 positive staining within identical regions of the lesion (Figure 5F). To further investigate Sdc-1 expression in human macrophages, we examined polarization and motility in THP1 macrophages in vitro. Notably, Sdc-1 expression was consistent with M2 polarization and an enhanced motility profile (Figure X in the online-only Data Supplement).
Discussion
Recent findings suggest that the resolution of inflammation involves both efferocytosis and emigration of monocyte-derived cells out of sites of inflammation through nearby lymphatic vessels.1,49 However, both processes may be impaired in atherosclerosis, which accounts for macrophage accumulation within plaques.4,50 In this regard, the mechanism for impeded efferocytosis and emigration from plaques remains a topic of intense interest for potential therapeutic intervention. We have determined that macrophage Sdc-1 expression is associated with anti-inflammatory M2 polarization and enhanced motility in both murine and human macrophages. Sdc-1−/− macrophages displayed a significant reduction in motility and increased adhesion, and in vivo, Sdc-1 macrophage deficiency resulted in delayed egress of macrophages in a model of self-resolving peritonitis and enhanced atherosclerotic lesion formation in ApoE−/−Sdc-1−/− mice. These data highlight a functional role for Sdc-1 in the motility of alternatively activated macrophage populations and reinforce the significant relationship of the dynamics of macrophage motility to responses that underlie the resolution of inflammation.
In adult tissue, Sdc-1 expression is restricted to noncirculating plasma cells and epithelial cells. Expression can be induced in additional cell types, such as endothelial cells, smooth muscle cells, fibroblasts, and macrophages.17,18 There are limited studies documenting in vivo expression and functional relevance of Sdc-1 expression in macrophages. Indeed, Sdc-1 expression is not observed in circulating monocytes or resident macrophages; however, Sdc-1 mRNA is upregulated on macrophage elicitation.51 Protein expression is governed via cAMP/protein kinase A as a post-transcriptionally regulated event, which has also been reported in keratinocytes, mesenchymal cells, and epithelial cells.52–54 The molecular mechanisms governing this nontraditional regulation of expression have yet to be elucidated, but presumably, on-demand expression would allow macrophages to rapidly tailor cell surface properties to meet specific functional requirements. Agents that elevate intracellular cAMP, such as E-type prostaglandins, membrane permeable cAMP analogues, or adenosine, promote Sdc-1 protein expression in macrophages. Nonphlogistic clearance of apoptotic cells, or efferocytosis, is a key process regulating macrophage M2 inflammatory polarization and resolution. Indeed, there are numerous reports detailing macrophage immune silencing as the consequence of efferocytosis. Macrophage engulfment of apoptotic cells can trigger the secretion of soluble anti-inflammatory mediators, such as IL-10, TGF-β, adenosine, and prostaglandin E2.55,56 Moreover, efferocytosis has been suggested to modulate macrophage motility and promote emigration,1 and our studies confirm that efferocytosis induces Sdc-1 protein expression. Given that the only phenotypic defect in Sdc-1−/− macrophages was reduced motility and enhanced adhesion, it is plausible that efferocytosis-induced Sdc-1 expression contributes to the emigration program of macrophages and resolution of inflammation.
Traditionally, Sdc-1 has been studied in the context of cell adhesion and migration in mesenchymal and epithelial cell types. Because a membrane-anchored proteoglycan was expressed on the basolateral surface of epithelial cells, Sdc-1 supports cell contact with extracellular matrix proteins and modulates cytoskeletal organization, leading to changes in cell shape and adhesion. Sdc-1 has been reported to augment epithelial migration in skin and cornea but inhibits migration in the lung by slowing focal adhesion disassembly.21,27,57 Both overexpression and gene knockout of Sdc-1 reduce migration in a wound-healing assay, suggesting that an optimal balance of surface expression is required to support the locomotion of mesenchymal and epithelial cells.28,58 There are limited reports documenting a functional role of Sdc-1 in adhesion or motility of leukocytes, which can use amoeboid migration without adherence to matrix components.59,60 Macrophages display intermediate migration speed and are capable of both amoeboid and mesenchymal migration. They do not exhibit large focal complexes consistent with their requirement for rapid migration responses. Sdc-1 has been linked to the formation of fascin-dependent cell protrusions, which can guide migration events.43 Fascin spikes, which have been reported in dendritic cells, are dynamic extensions of the plasma membrane that can extend and retract quickly and assemble in the absence of focal contacts.42 The morphological absence of cell protrusions is evident in Sdc-1−/− macrophages treated with agents that induce high cell surface Sdc-1 expression, such as membrane permeable cAMP analogues or adenosine. This morphological distinction coupled with a motility defect that is observed in the absence of Sdc-1 expression suggests that expression is critical in modulating cytoskeletal dynamics for this specific subset of M2 macrophages. Given the capacity of macrophages to engage in both integrin-mediated adhesion-dependant motility and adhesion-independent interstitial migration, future efforts will focus on examining the contribution of Sdc-1 within the context of both motility paradigms.
Recent investigations have highlighted the potential significance of heterogeneous populations of specialized macrophages in the initiation and resolution of inflammatory responses. One of the defining features of chronic inflammation is the persistence of activated macrophages. In atherosclerotic plaques, activated lesional macrophages possess a potent repertoire of proinflammatory mediators to sustain a sessile M1 profile and hinder the evolution of resolution programs. We have demonstrated that Sdc-1 expression on macrophages is associated with an anti-inflammatory M2 polarization and enhanced motility in both murine and human macrophages. We further demonstrated a role in macrophage resolution and in limiting atherosclerotic macrophage burden. Given the pathological role of macrophage trapping in chronic inflammation, it is critical to define the endogenous modulators of macrophage resolution. The macrophage Sdc-1 expression program represents a bridge between efferocytosis, polarization, and motility and may be fundamental to the clearance of inflammatory macrophages.
Sources of Funding
This work was funded by National Institutes of Health grant HL060903 (E.L. Chaikof) and a fellowships from the American College of Surgeons (S.V. Smith), and the American Heart Association (J. Angsana).
Disclosures
None.
Footnotes
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304720/-/DC1.
- Nonstandard Abbreviations and Acronyms
- ApoE
- apolipoprotein E
- PD-L2
- programmed death ligand 2
- Sdc-1
- syndecan-1
- Received November 18, 2013.
- Accepted December 14, 2014.
- © 2014 American Heart Association, Inc.
References
Significance
Atherosclerosis is a chronic inflammatory disease that is distinguished by the persistence of inflammatory macrophages in vascular lesions. There is intense interest in understanding why the macrophage population fails to resolve in atherosclerotic plaques, and recent findings suggest that macrophage migration out of the site of inflammation may be an important process to trigger the return to tissue homeostasis. Our data show that the cell-surface heparan sulfate proteoglycan, syndecan-1, modulates motility and adhesion in mature-polarized macrophage populations. In vivo, we observed that this motility defect translated to reduced macrophage trafficking during inflammatory resolution and enhanced atherosclerotic plaque formation. Collectively, these results support the notion that defective motility of mature macrophage populations may promote a persistent inflammatory state and suggest that syndecan-1 is part of a broad program to regulate resolution phase trafficking.
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