Leukocyte Phenotype Is Altered in Peripheral Arterial Disease

Abstract

Peripheral arterial disease (PAD) is characterized by low-grade systemic inflammation. Monocytes and monocyte-derived macrophages (MDMs) play a central role in vascular inflammation and its resolution. We hypothesize that impaired resolution in PAD contributes to adverse clinical outcomes. In a cross-sectional study, we profiled serum cytokines, phagocytic activity of leukocytes, monocyte cell surface markers, and gene expression of MDM from healthy individuals (n ≥ 10) and patients with stable claudication (n ≥ 10). Leukocyte phagocytosis of fluorescently labeled Escherichia coli and monocyte surface markers were determined by flow cytometry. MDMs were cultured from peripheral blood monocytes isolated by density gradient centrifugation. MDM cytokine production and gene expression, before and after stimulation with lipopolysaccharide, were determined by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. Patients with PAD had elevated serum high-sensitivity C-reactive protein (3.7 vs 0.6 mg/L; P = .004) and interleukin (IL) 6 (5.1 vs 1.1 pg/mL; P = .01) and trended toward higher levels of monocyte chemoattractant protein 1 and lower adiponectin compared with healthy individuals. Circulating monocytes and polymorphonuclear cells (PMNs) from PAD patients had reduced phagocytic activity for E. coli (monocytes, >30% [P < .001]; PMNs, >25% [P < .01]). Flow cytometry demonstrated a higher proportion of the proinflammatory intermediate monocyte subset (CD14++16+, 1.8-fold; P = .04) in PAD subjects. MDMs from PAD patients retain their intrinsic inflammatory program, producing more IL-6 (>4-fold; P = .03) and IL-1β (>10-fold; P = .04) and demonstrating increased expression of M1 genes (tumor necrosis factor α, monocyte chemoattractant protein 1, CXCL10) and decreased expression of M2 genes (CCL17, MRC1) vs healthy individuals (Table). Clinically stable PAD patients have elevated serum inflammatory markers compared with healthy individuals. Circulating monocytes and PMNs in patients with PAD have reduced phagocytic activity and a greater proportion of the proinflammatory intermediate monocyte subset. MDMs from PAD patients preserve their elevated inflammatory state in culture. These data demonstrate a heightened inflammatory and impaired resolution phenotype in PAD that has potential implications for disease progression and response to interventions.TableMonocyte-derived macrophage (MDM) gene expression of the peripheral arterial disease (PAD) cohort vs healthy individualsGeneMl or M2PAD patients relative to healthy individualsExpression fold changeP valueTNF-α (veh)Ml2.71.02aTNF-α (LPS)0.81.33MCP-1 (veh)Ml1.92.05aMCP-1 (LPS)1.91.01aiNOS (veh)Ml1.34.26iNOS (LPS)1.04.94CXCL10 (veh)Ml7.87.04aCXCL10 (LPS)1.8129IL-10 (veh)M21.50.19IL-10 (LPS)2.72.009aCCL17 (veh)M20.48.09CCL17 (LPS)022.14MRC1 (veh)M20.52.09MRC1 (LPS)0.30.01aCCL17, Chemokine (C-C motif) ligand 17; CXCL10, C-X-C motif chemokine 10; IL-10, interleukin 10; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1; MRC1, mannose receptor C type 1; TNF-α, tumor necrosis factor α; veh, vehicle.Expression fold change was calculated as 2ˆ(–ΔΔCT) and represents the fold change in the average expression of the target gene within the PAD cohort (n = 10) vs the average expression of the target gene within the cohort of healthy individuals (n = 10), normalized to the housekeeping gene (HPRT). MDMs were stimulated with lipopolysaccharide to mimic the occurrence of an acute inflammatory event or vehicle for 24 hours before assessment of gene expression.aP < .05 by unpaired Student t-test. Open table in a new tab