gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

CXCL4 downregulates the atheroprotective hemoglobin receptor CD163 in human macrophages

Meeting Contribution

  • corresponding author C. A. Gleissner - University of Heidelberg, Germany; La Jolla Institute for Allergy & Immunology, La Jolla/California, U.S.A.
  • I. Shaked - University of Heidelberg, Germany
  • C. Erbel - University of Heidelberg, Germany
  • D. Böckler - University of Heidelberg, Germany
  • H. A. Katus - University of Heidelberg, Germany
  • E. Ley - University of Heidelberg, Germany

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf16

DOI: 10.3205/10dgaf16, URN: urn:nbn:de:0183-10dgaf161

Published: March 23, 2011

© 2011 Gleissner et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

CXCL4 is a platelet-derived chemokine that promotes macrophage differentiation from monocytes. Deletion of the PF4 gene that encodes CXCL4 reduces atherosclerotic lesions in Apoe-/- mice. We sought to study effects of CXCL4 on macrophage differentiation with possible relevance for atherogenesis. Flow cytometry for expression of surface markers in MCSF- and CXCL4-induced macrophages demonstrated virtually complete absence of the hemoglobin scavenger receptor CD163 in CXCL4-induced macrophages. mRNA for CD163 was downregulated as early as two hours after CXCL4. CD163 protein reached a minimum after three days, which was not reversed by treatment of cells with MCSF. The CXCL4 effect was entirely neutralized by heparin, which bound CXCL4 and prevented CXCL4 surface binding to monocytes. Similar to recombinant CXCL4, releasate from human platelets also downregulated CD163. CXCL4-differentiated macrophages were unable to upregulate the atheroprotective gene heme oxygenase-1 in response to hemoglobin-haptoglobin complexes. Immunofluorescence of human coronary atherosclerotic plaques demonstrated presence of both CD68+CD163+ and CD68+CD163- macrophages suggesting that the CXCL4-induced macrophage phenotype exists in atherosclerotic lesions in vivo. We conclude that CXCL4 may promote atherogenesis by suppressing CD163 in macrophages, which are then unable to upregulate the atheroprotective enzyme heme oxygenase-1 in response to hemoglobin.


Introduction

Atherogenesis is characterized by monocytes entering the subendothelial space where they differentiate into macrophages. These macrophages represent a major cellular component of the atherosclerotic lesion and promote plaque development by secreting numerous inflammatory mediators like proteases, cytokines and chemokines. The resulting inflammatory milieu leads to recruitment of smooth muscle cells and additional immune cells. All these functions make the plaque macrophage an interesting target for prevention and therapy of atherosclerotic disease [1].

There is clear evidence for the presence of phenotypically and functionally different macrophages within human atherosclerotic lesions. Waldo et al. described the presence of different macrophage sets within atherosclerotic plaques defined by specific surface markers as well as differential ability to take up oxidized low density lipoprotein (oxLDL) [2]. Similarly, Bouhlel et al. demonstrated the presence of M1 and M2 markers in lesions, suggesting that plaque macrophages do not represent a uniform entity [3].

Macrophage differentiation from monocytes is induced by macrophage colony-stimulating factor (MCSF). The platelet-derived chemokine CXCL4 has also been demonstrated to promote macrophage differentiation. Interestingly, presence of CXCL4 in atherosclerotic lesions correlates with clinical parameters in humans [4]. Furthermore, deletion of the PF4 gene encoding CXCL4 results in reduced lesion size in atherosclerotic Apoe-/- mice [5].

Based on the important role of macrophages in atherosclerosis as well as the accumulating evidence that CXCL4 may be an important player in atherogenesis, we hypothesized that macrophages differentiated under the influence of this chemokine may display specific characteristics relevant for atherogenesis. In a gene expression and surface marker screening we found that CXCL4-induced macrophages completely lack expression of CD163, a scavenger receptor for hemoglobin and hemoglobin-haptoglobin complexes [6]. CD163 engagement has been described to induce up-regulation of heme oxygenase-1, an enzyme that protects from atherosclerosis. Here, we test the hypothesis that CXCL4 suppresses CD163 expression, thus generating a pro-inflammatory macrophage type in atherosclerotic lesions.


Methods

Monocyte-derived macrophages: Human peripheral blood was obtained with approval from the institutional review board from healthy volunteer donors. Briefly, monocytes were isolated from human peripheral blood by gradient centrifugation and subsequent negative bead isolation. Monocytes were cultured for six days in the presence of 100 ng/mL MCSF or 1 µmol/L CXCL4

Real-time RT-PCR: RNA was isolated using columns including a DNAse-step followed by reverse transcription. Real-time PCR was performed in duplicates on a Light Cycler 480 using GAPDH as housekeeping gene. Product specificity was assessed by melting curve analysis or agarose gel. Primer sequences were obtained from primer bank (http://pga.mgh.harvard.edu/primerbank/).

Flow cytometry: For flow cytometry, cells were stained with antibodies against CD11b (clone ICRF44), CD14 (clone M5E2), CD45 (clone 2D1), CD163 (clone GHI/61c), CXCR3 (clone 2Ar1), heme oxygenase-1 (clone HO-1-1) or appropriate isotype controls. Surface binding of CXCL4 to monocytes was assessed by exposing freshly isolated monocytes to CXCL4 (1 µmol/L) for 10 min at 4°C in the presence or absence of 2 U/mL heparin. Subsequently, cells were washed and stained with a FITC-labeled antibody against CXCL4 (clone 170138). For intracellular staining, cells were fixed with 2% paraformaledhyde and subsequently stained in buffer containing saponin and the appropriate antibodies.

Platelets: Platelets were isolated as described previously from platelet-rich human plasma by sepharose column and activated for 10 minutes with TRAP-7 (10 µmol/L) and ADP (1 mmol/L). Activation was confirmed by flow cytometric assessment of CD62P (P-selectin) positivity by FACS (clone AK-4). Platelet-free supernatants were added to freshly isolated monocytes so that each macrophage was treated with the releasate of 1000 platelets. Controls were treated with the same amount of elution buffer.

Hemoglobin: Macrophages were cultured for six days with either MCSF or CXCL4 as described. At the time of monocyte isolation, red blood cell lysates and plasma of individual donors were kept and stored at –20°C. On day 6 after monocyte isolation, macrophages were incubated with a final concentration of 500 µg/mL autologous hemoglobin and 20% autologous serum to ensure sufficient amounts of haptoglobin. After four hours, cells were harvested and HMOX1 gene and protein expression were measured by real-time PCR as described above.

Immunofluorescence: Human coronary arteries from patients with cardiovascular disease were obtained with approval by the institutional review board post mortem from the University of Virginia Department of Pathology/Tissue bank (Charlottesville, VA). Immunofluorescence was performed to determine the colocalization of CD163 and CD68. Briefly, coronary arteries were embedded in paraffin and 5 µmol/L sections were prepared. After heat-induced antigen retrieval using antigen unmasking solution, sections were incubated with antibodies against CD68 (clone KP-1) and CD163 (clone C-16). KP-1 was FITC labeled, C-16 was visualized with a Texas red-labeled anti-goat secondary. DAPI was used as nuclear stain.


Results

To study the differential effects of MCSF and CXCL4 on monocyte-macrophage differentiation, human peripheral blood monocytes were cultured with 100 ng/mL MCSF or 1 µmol/L CXCL4 for six days. These dosages had previously been demonstrated to induce macrophage differentiation. When studying gene and protein expression of various macrophage surface receptors, we found CD163 to be differentially expressed in macrophages differentiated with MCSF or CXCL4. As described previously, MCSF significantly increased CD163 at the mRNA and surface expression level during monocyte-macrophage differentiation. By contrast, CXCL4 significantly downregulated CD163 at the mRNA and protein level. Accordingly, CD163 was virtually absent in CXCL4-induced macrophages.

To assess whether CXCL4-induced downregulation of CD163 is reversible, monocytes were treated with either MCSF or CXCL4 for three days and then switched to CXCL4 or MCSF, respectively. These experiments showed that MCSF-induced CD163 up-regulation as seen on day 3. This was reversed when cells were switched to CXCL4. By contrast, MCSF was unable to significantly induce CD163 expression once monocytes had been exposed to CXCL4 for three days.

Heparin is known to specifically bind and inactivate CXCL4 [7]. We therefore reasoned that adding heparin to the culture medium may prevent CXCL4 effects on CD163 expression. We first studied whether CXCL4 surface binding to monocytes was affected by heparin. In fact, heparin completely abrogated surface binding to monocytes at 4°C as assessed by flow cytometry.

We then treated monocytes with MCSF for three days to induce robust CD163 expression. After this, cells were kept in medium alone or switched to CXCL4 ± heparin (2 U/mL). Presence of heparin in the culture medium completely abrogated CXCL4-dependent downregulation of CD163. These findings suggest that CXCL4 binding to heparan-sulfate expressing surface receptors is responsible for CD163 downregulation. In endothelial cells, CXCL4 has been reported to bind to the chemokine receptor CXCR3B, however, CXCR3 was undetectable by real-time RT-PCR or flow cytometry in monocytes and macrophages.

Platelet releasate contains high amounts of CXCL4, but also a variety of other chemokines. To test whether the CXCL4 concentrations resulting from platelet degranulation in the context of the platelet releasate were able to down-regulate CD163, monocytes were cultured with MCSF for three days to induce robust CD163 expression. On day three, cells were treated with cell-free releasate of ADP- and TRAP-7-activated platelets. Platelet activation was verified by flow cytometric assessment of CD62P (P-selectin) surface expression. Platelet releasate, but not control buffer significantly reduced CD163 gene expression as early as 2 hours after addition supporting the physiological relevance of our findings with recombinant CXCL4.

Engagement of CD163 by hemoglobin-haptoglobin (Hb-Hp) complexes has been described to induce heme oxygenase-1, an enzyme linked to atheroprotection [8], [9], [10]. Accordingly, CD163 expressing macrophages have been demonstrated to exert anti-inflammatory effects in response to CD163 engagement. We therefore assessed whether CXCL4-induced loss of CD163 on macrophages suppressed heme-oxygenase-1. MCSF- and CXCL4-induced macrophages were treated with Hb-Hp complexes for four hours. After this period, HMOX1 gene expression was assessed by real-time RT-PCR. As expected, MCSF-induced CD163+ macrophages robustly upregulated HMOX1 after exposure to Hb-Hp, whereas CXCL4-induced CD163- macrophages were not able to upregulate HMOX1. This supports the notion that CXCL4-induced downregulation of CD163 is functionally relevant and prevents an important anti-inflammatory mechanism relevant in macrophages.

To assess whether our in vitro finding of CXCL4-induced modulation of macrophage phenotype was relevant in vivo, we studied macrophage CD163 expression in post mortem samples of coronary arteries from patients with cardiovascular disease. Macrophages were detected by immunofluorescence staining for CD68 (green) and CD163 (red). Indeed, we found that some, but not all macrophages within atherosclerotic lesions expressed CD163, demonstrating macrophage heterogeneity with respect to CD163 expression in vivo (see Figure 1 [Fig. 1]).


Discussion

Here, we show that macrophages differentiated under the influence of the platelet chemokine CXCL4 lose surface expression of CD163. The physiologic relevance of CXCL4-induced loss of CD163 was demonstrated by (1) the ability of releasate from activated platelets to downregulate CD163, (2) the inability of CD163- macrophages to respond to exposure to hemoglobin-haptoglobin complexes by upregulating heme oxygenase-1 (HMOX-1), and (3) demonstration of both CD163+ and CD163- macrophages within atherosclerotic lesions in patients.

In 2000, Scheurer et al. demonstrated that platelet-derived CXCL4 prevents monocyte apoptosis and promotes differentiation toward macrophages [11]. Presence of CXCL4 within atherosclerotic lesions has been associated with clinical parameters including lesion grade and presence of symptoms, suggesting an important role of this chemokine in atherogenesis [4]. Sachais et al. recently demonstrated that lack of CXCL4 in mice results in reduced lesion formation in the Apoe-/- model [5], thus showing that CXCL4 has a net pro-atherogenic effect.

Several biological effects of CXCL4 may be important for plaque development. CXCL4 promotes recruitment of monocytes towards the arterial wall through formation of heterodimers with CCL5 (RANTES). Blocking the formation of CXCL4-CCL5 heterodimers significantly reduces lesion size in the Apoe-/- mouse model of atherosclerosis [12]. In addition, CXCL4 binds to oxidized LDL and mediates its binding to endothelial cells of the vascular wall. Finally, macrophages differentiated under the influence of CXCL4 express high levels of surface HLA-DR and completely lack CD86 [12]. Here, we show that CXCL4-induced macrophages completely lose CD163 expression and that this macrophage phenotype is found in atherosclerotic lesions. The downregulation of CD163 expression in monocyte-derived macrophages may explain a mechanism by which CXCL4 exerts pro-inflammatory effects in atherosclerosis. CD163 is an important scavenger receptor for hemoglobin-haptoglobin complexes and, with lower affinity, for uncomplexed hemoglobin [6]. Decreased expression of CD163 on peripheral blood mononuclear cells has been demonstrated in patients with a specific haptoglobin genotype (Hp 2-2), which is know be associated with a significantly increased risk of cardiovascular disease in diabetic patients [12], [13]. Engagement of CD163 results in upregulation of HMOX-1, an enzyme, which has anti-oxidative and anti-inflammatory effects. HMOX-1 is atheroprotective, which was demonstrated by knocking out the Hmox1 gene in mice, resulting in increased lesion size in both Apoe-/- and Ldlr-/- mice [8], [9]. Bone marrow transplant experiments have clearly shown that heme oxygenase-1 in hematopoietic cells, which includes monocytes and macrophages, is responsible for this effect [14].

In summary, we present evidence for a novel CD163- pro-inflammatory macrophage phenotype that is induced by the platelet-chemokine CXCL4 and is present in atherosclerotic lesions in vivo. This work adds to the growing body of evidence that atherosclerotic plaque macrophages do not represent a homogeneous entity and are composed of phenotypically and functionally distinct subsets and suggests CXCL4 as favorable target for atheroprotective therapeutic or prophylactic interventions.


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