Fractalkine preferentially mediates arrest and migration of CD16+ monocytes

Abstract

CD16+ monocytes represent 5-10% of peripheral blood monocytes in normal individuals and are dramatically expanded in several pathological conditions including sepsis, human immunodeficiency virus 1 infection, and cancer. CD16+ monocytes produce high levels of proinflammatory cytokines and may represent dendritic cell precursors in vivo. The mechanisms that mediate the recruitment of CD16+ monocytes into tissues remain unknown. Here we investigate molecular mechanisms of CD16+ monocyte trafficking and show that migration of CD16+ and CD16- monocytes is mediated by distinct combinations of adhesion molecules and chemokine receptors. In contrast to CD16- monocytes, CD16+ monocytes expressed high CX3CR1 and CXCR4 but low CCR2 and CD62L levels and underwent efficient transendothelial migration in response to fractalkine (FKN; FKN/CX3CL1) and stromal-derived factor 1 alpha (CXCL12) but not monocyte chemoattractant protein 1 (CCL2). CD16+ monocytes arrested on cell surface-expressed FKN under flow with higher frequency compared with CD16- monocytes. These results demonstrate that FKN preferentially mediates arrest and migration of CD16+ monocytes and suggest that recruitment of this proinflammatory monocyte subset to vessel walls via the CX3CR1-FKN pathway may contribute to vascular and tissue injury during pathological conditions.

Figure 1.

Phenotypic analysis of human monocyte subsets. (A) PBMCs were stained with FITC anti-CD14 and PC5 anti-CD16 mAbs. Monocytes were gated according to size, granularity, and CD14 expression. Three subsets of monocytes were identified: CD14^high CD16⁻, CD14^high CD16⁺, and CD14^low CD16⁺. Results are representative of experiments performed with cells from 20 different donors. (B) PBMCs were stained with FITC anti-CD14, PE anti-CD56, and PC5 anti-CD16 mAbs, and CD14⁺ monocytes were analyzed for CD16 and CD56 expression. Results are representative of four experiments performed with cells from different donors. (C and D) PBMCs were stained with FITC anti-CD14, PC5 anti-CD16, and the indicated mAbs, and the phenotype of each monocyte subset was analyzed by flow cytometry. Values represent the percentage of positive cells (mean ± SD, n = 9). *, P < 0.05, Student's t test (CD16⁺ vs. CD16⁻ monocytes).

Figure 2.

Expression of CX3CR1 mRNA in CD16⁻ and CD16⁺ monocytes. Total RNA from CD16⁻ and CD16⁺ monocytes was reverse transcribed and serial dilutions of the RT product were subjected to PCR amplification using CX3CR1 and β globin primers. The optical density of each PCR band was determined using Eagle Sight software (Stratagene). Results are representative of three experiments performed using monocytes from different donors.

Figure 3.

Chemotactic migration of CD16⁻ and CD16⁺ monocytes. (A) Monocytes were stained with PC5 anti-CD16 mAb and placed in the upper chamber of transwells. Chemokines were placed in the bottom chamber. After 2.5 h, CD16⁻ and CD16⁺ monocytes that migrated into the bottom chamber were counted by FACS^®. The IM (mean ± SEM, n = 3–8) at optimal chemokine concentrations, 50 ng/ml for MCP-1, MIP-1α, and SDF-1α, and 10 ng/ml for FKN, is shown. (B) Confluent pHUVEC and iHUVEC monolayers were analyzed for junction expression of VE-cadherin, β-catenin, and JAM-1 molecules. Results are representative of two independent experiments. (C) Monocytes were assessed for their ability to cross a confluent iHUVEC monolayer in response to optimal concentrations of chemokines. After 4 h, CD16⁻ and CD16⁺ monocytes that migrated into the bottom chamber were counted by FACS^®. The IM (mean ± SEM, n = 6) is shown. *, P < 0.05, Student's t test (CD16⁺ vs. CD16⁻ monocytes).

Figure 4.

Firm arrest of CD16⁺ monocytes onto endothelial cells under flow. (A) iHUVEC were incubated with 40 ng/ml TNF-α and 50 ng/ml IFN-γ (ST-HUVEC) for 5 h (top) or transfected to stably express FKN (bottom, KN-HUVEC). Cells were harvested, stained with anti-CD62E, anti–VCAM-1, and anti-FKN mAbs, and analyzed by FACS^®. Results are representative of three independent experiments. (B) Monocytes were labeled with FITC anti-CD16 mAb and perfused over unstimulated (NS-HUVEC), ST-HUVEC, and FKN-HUVEC monolayers at 0.5 dynes/cm² flow rate. The number of total monocytes arrested onto endothelial cells (mean ± SD, n = 4) is shown. *, P < 0.05, Student's t test (FKN-HUVEC or ST-HUVEC vs. NS-HUVEC). (C) The percentage of CD16⁺ monocytes in the input fraction was compared with that in the fraction arrested onto FKN-HUVEC and ST-HUVEC (mean ± SD, n = 4). *, P < 0.05, Student's t test (arrested vs. input fraction). (D) Monocytes were incubated with soluble FKN (1 μg/10⁶ cells for 10 min at 4°C) and perfused over FKN-HUVEC and ST-HUVEC. Results are representative of two experiments performed with monocytes from different donors.