CXCL4 downregulates the atheroprotective hemoglobin receptor CD163 in human macrophages

Abstract

Rationale: 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.

Objective: We sought to study effects of CXCL4 on macrophage differentiation with possible relevance for atherogenesis.

Methods and results: Flow cytometry for expression of surface markers in macrophage colony-stimulating factor (M-CSF)- 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 2 hours after CXCL4. CD163 protein reached a minimum after 3 days, which was not reversed by treatment of cells with M-CSF. The CXCL4 effect was entirely neutralized by heparin, which bound CXCL4 and prevented CXCL4 surface binding to monocytes. Pretreatment of cells with chlorate, which inhibits glycosaminoglycan synthesis, strongly inhibited CXCL4-dependent downregulation of CD163. Similar to recombinant CXCL4, releasate from human platelets also reduced CD163 expression. CXCL4-differentiated macrophages were unable to upregulate the atheroprotective enzyme heme oxygenase-1 at the RNA and protein level in response to hemoglobin-haptoglobin complexes. Immunofluorescence of human atherosclerotic plaques demonstrated presence of both CD68+CD163+ and CD68+CD163- macrophages. PF4 and CD163 gene expression within human atherosclerotic lesions were inversely correlated, supporting the in vivo relevance of CXCL4-induced downregulation of CD163.

Conclusions: 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.

Figure 1

CXCL4 downregulates CD163 during macrophage differentiation. A, Histograms of CD163 surface expression as measured by flow cytometry in freshly isolated monocytes and macrophages differentiated for 6 days with M-CSF (100 ng/mL) or CXCL4 (1 µmol/L), respectively. All cells are derived from the same donor. Dotted line indicates isotype control. B, CD163 gene expression relative to GAPDH as determined by real-time PCR in freshly isolated monocytes or macrophages differentiated with M-CSF or CXCL4 as described above. Means+SEM (n = 3 to 8). *P<0.05, **P<0.01. C, Summary of CD163 background-corrected mean fluorescence intensity in monocytes and M-CSF– or CXCL4-induced macrophages. Means+SEM (n = 3 to 5). **P<0.01, ***P<0.001. D, Soluble CD163 was measured by ELISA in macrophage cell culture supernatants after 6 days exposure to M-CSF or CXCL4. The dotted line indicates the detection limit of the assay.

Figure 2

Dose response and time course of CD163 expression during macrophage differentiation with CXCL4. A, Dose response of CD163 background-subtracted mean fluorescence intensity in monocytes treated with CXCL4 for 3 days. Means±SEM (n = 3). *P<0.05, **P<0.01 vs concentration 0. B, Time course of CD163 background-subtracted mean fluorescence intensity in monocytes and M-CSF– or CXCL4-induced macrophages. Means±SEM (n = 3 to 6). *P<0.05, **P<0.01, ***P<0.001 vs time 0.

Figure 3

CXCL4 actively downregulates CD163. A, Histograms of CD163 surface expression in freshly isolated monocytes and macrophages treated for 6 days with M-CSF (100 ng/mL) or CXCL4 (1 µmol/L), switched from M-CSF to CXCL4 or from CXCL4 to M-CSF on day 3 as indicated. B, Line graph indicating CD163 surface expression as determined by flow cytometry in monocytes/macrophages treated with M-CSF for 6 days (solid squares) or switched from M-CSF to CXCL4 on day 3 (open squares). Means±SEM (n = 3). **P<0.01. C, Line graph indicating CD163 surface expression as determined by flow cytometry in monocytes/macrophages treated with CXCL4 for 6 days (solid circles) or switched from CXCL4 to M-CSF on day 3 (open circles). Means±SEM (n = 3).

Figure 4

CXCL4-induced downregulation of CD163 requires surface glycosaminoglycans (GAG). A, CXCL4 (1 µmol/L) was added to freshly isolated human peripheral blood monocytes for 10 minutes at 4°C in presence and absence of 2 U/mL heparin, which binds to CXCL4, as well as to surface GAGs. Surface binding of CXCL4 was assessed by flow cytometry using a FITC-labeled anti-CXCL4 antibody. B, Monocytes were cultured with M-CSF (100 ng/mL) for 3 days and then switched to medium alone, CXCL4 (1 µmol/L), or CXCL4 plus heparin (2 U/mL). After 3 additional days, CD163 surface expression was assessed by flow cytometry. Representative histograms of 4 experiments summarized as bar graph in C. Means±SEM (n = 4). *P<0.01. D, Monocytes were cultured with M-CSF (100 ng/mL) for 3 days, treated for 4 hours with 10 mmol/L potassium chlorate or control to inhibit GAG synthesis, and then switched to CXCL4 (1 µmol/L, gray bars) or kept in M-CSF (open bars). After 3 additional days, CD163 surface expression was assessed by flow cytometry. Means±SEM (n = 3). *P<0.01.

Figure 5

Releasate from activated platelets induces CD163 downregulation in macrophages. Platelets were gated by forward and side scatter and CD41 (top row). Monocytes were cultured with M-CSF (100 ng/mL) for 3 days to induce robust CD163 expression and then treated with releasate from platelets activated with a combination of TRAP-7 and ADP resulting in robust P-selectin expression (bottom row). Controls were treated with elution buffer only (middle row). After 2 hours, CD163 gene expression was assessed by real-time PCR and normalized for GAPDH. Means+SEM (n = 3 to 6). **P<0.01.

Figure 6

CD163⁻ macrophages do not upregulate heme oxygenase-1 in response to Hb-Hp complexes. Macrophages were differentiated from monocytes for 6 days with M-CSF (100 ng/mL) or CXCL4 (1 µmol/L) and exposed to 500 µg/mL autologous hemoglobin in culture medium supplemented with 20% autologous serum providing sufficient amounts of haptoglobin (Hb-Hp). A, After 4 hours, HMOX1 gene expression was measured by real-time RT-PCR. Means±SEM (n = 5 to 6). **P<0.01. B, Intracellular heme oxygenase-1 protein expression was assessed by flow cytometry after 18 hours of exposure to Hb-Hp. Dotted line indicates isotype control; fine line, no Hb-Hp; bold line, Hb-Hp. The results of 4 independent experiments are summarized as a bar graph in C. *P<0.01 (n = 4).

Figure 7

Negative correlation between PF4 and CD163 in human atherosclerotic plaques. A, Coronary artery sections from patients with cardiovascular disease were obtained post mortem. Paraffin sections were stained for the macrophage marker CD68 (FITC) and CD163 (phycoerythrin). DAPI was added as nuclear stain. Vascular lumen is indicated by *. B, Magnification of a CD68⁺CD163⁺ (yellow arrow in A, top row) and CD68⁺CD163⁻ macrophage (green arrow in A, bottom row). Scale bars: 20 µm. C, RNA was isolated from carotid atherosclerotic plaques immediately after carotid endatherectomy. Expression of CD163 and PF4 message was measured by real-time PCR and normalized to GAPDH.