CX3CL1 up-regulation is associated with recruitment of CX3CR1+ mononuclear phagocytes and T lymphocytes in the lungs during cigarette smoke-induced emphysema

Abstract

CX3CR1 is expressed on monocytes, dendritic cells, macrophages, subsets of T lymphocytes, and natural killer cells and functions in diverse capacities such as leukocyte adhesion, migration, and cell survival on ligand binding. Expression of the CX3CL1 gene, whose expression product is the sole ligand for CX3CR1, is up-regulated in human lungs with chronic cigarette smoke-induced obstructive lung disease. At present, it is unknown whether CX3CL1 up-regulation is associated with the recruitment and accumulation of immune cells that express CX3CR1. We show that mice chronically exposed to cigarette smoke up-regulate CX3CL1 gene expression, which is associated with an influx of CX3CR1+ cells in the lungs. The increase in CX3CR1+ cells is primarily comprised of macrophages and T lymphocytes and is associated with the development of emphysema. In alveolar macrophages, cigarette smoke exposure increased the expression of both CX3CR1 and CX3CL1 genes. The inducibility of CX3CR1 expression was not solely dependent on a chronic stimulus because lipopolysaccharide up-regulated CX3CR1 in RAW264.7 cells in vitro and in mononuclear phagocytes in vivo. Our findings suggest a mechanism by which macrophages amplify and promote CX3CR1+ cell accumulation within the lungs during both acute and chronic inflammatory stress. We suggest that one function of the CX3CR1-CX3CL1 pathway is to recruit and sustain divergent immune cell populations implicated in the pathogenesis of cigarette smoke-induced emphysema.

Figure 1

Histological and systemic changes attributable to chronic cigarette smoke exposure. A and B: Representative images of mouse lungs after 24 weeks of air (A) or cigarette smoke exposure (B). The samples were stained using the modified Gills technique. C: Environmental tobacco smoke exposure is associated with an increase in airspace enlargement by 24 weeks. There is an increase in mean linear intercept (Lm) measured after 24 weeks in the cigarette smoke (CS)-exposed mice (31.75 μm) when compared to the air-exposed controls (29.54 μm). *P = 0.014, n = 5 to 7 mice per group. D: The average weights of the cigarette smoke-exposed mice (white circles) were significantly lower than the average weights of the air-exposed mice (black circles), P < 0.001 (analysis of variance repeated measurements). The smoke- and air-exposed mice averaged 30.0 g at t = 0. The air-exposed mice steadily gained weight and averaged 46.2 g after 24 weeks. The smoke-exposed mice did not lose or gain a significant amount of weight and averaged 27.6 g after 24 weeks. Error bars at each time point indicate ±SEM n = 12 mice in each group for exposures up to 12 weeks. n = 7 mice in each group for exposures from 13 to 24 weeks. Original magnifications, ×200.

Figure 2

CX3CR1⁺ cells in the lungs of chronic cigarette smoke-exposed mice and air-exposed controls. A and B: Representative images used for the quantitation of CX3CR1⁺ cells in mouse lungs after 12 weeks of either air exposure (A) or cigarette smoke exposure (B). The arrows indicate CX3CR1 immunostaining of discrete, mononuclear cells within the parenchyma. C: Rabbit IgG control immunostaining of murine lung section after 12-week cigarette smoke exposure. D: CX3CR1⁺ cells as a percentage of total cells per high-powered field in lung sections from air-exposed controls and cigarette-exposed mice after 12 weeks (12.7 ± 0.8 versus 35.7 ± 9) and 24 weeks (9.6 ± 0.7 versus 21.5 ± 1) of exposure. *P < 0.001. Gray bars, air-exposed controls; black bars, cigarette smoke-exposed group. n = 50 random hpf of lung parenchymal sections examined from five animals per condition at 12 weeks, n = 20 random hpf examined from five animals per condition at 24 weeks. Original magnifications, ×400.

Figure 3

MAC3⁺, CX3CR1⁺, and CD11c⁺ cells in murine lungs after cigarette smoke exposure. A: MAC3 immunostaining (red channel). B: CX3CR1 immunostaining (green channel). C: Co-localization of MAC3 and CX3CR1 (merged channels); white arrows. D: Absence of immunostaining in studies using rat and rabbit IgG control antibodies. E: CD11c immunostaining (red channel). F: CX3CR1 immunostaining (green channel). G: White arrows indicate cells that co-localize with CD11c and CX3CR1 (merged channels). H: Absence of immunostaining in studies using Armenian hamster IgG and rabbit IgG control antibodies. I: CD11c expression (red channel). J: MAC3 expression (green channel). K: Co-localization of CD11c and MAC3 (white arrows, merged channels). Yellow arrows indicate cells that express MAC3 (green) but do not express CD11c. L: Absence of immunostaining in studies using Armenian hamster IgG and rat IgG control antibodies. Original magnifications, ×400.

Figure 4

CD68⁺ human alveolar macrophages express CX3CR1. A: CD68 immunostaining identifies macrophages in human lung tissue (white arrows, red channel) (uninflated lung sections from human explants). B: CX3CR1 immunostaining (white arrows, green channel). C: Co-localization of CX3CR1 and CD68 on human alveolar macrophages (white arrows, merged channels). D: Absence of immunostaining in studies using rabbit IgG and mouse IgG control antibodies. Original magnifications, ×400.

Figure 5

CD3⁺ cells but not Gr-1⁺ or pro-SPC⁺ cells express CX3CR1 in murine lungs. A: CD3 immunostaining (red channel). B: CX3CR1 immunostaining (green channel). C: Co-localization of CD3 and CX3CR1 in cell with eccentric nucleus (merged channels). D: Absence of immunostaining in studies using Armenian hamster IgG and rabbit IgG control antibodies. E: Gr-1⁺ immunostaining (red channel). F: CX3CR1 immunostaining (green channel). G: White arrow indicates polymorphonuclear cell is Gr-1⁺ but CX3CR1^−, and yellow arrow indicates mononuclear cell is CX3CR1⁺ but Gr-1⁻ (merged channels). H: Absence of immunostaining in studies using rat IgG and rabbit IgG control antibodies. I: Pro-SPC immunostaining (red channel) in unstimulated B6.129P-cx3cr1^tm1Litt/J mouse lung. J: GFP signal (yellow arrow) indicating endogenous CX3CR1 expression (green channel). K: Yellow arrow indicates cell that is CX3CR1⁺ but pro-SPC⁻, and white arrows indicate cells that are pro-SPC⁺ but CX3CR1⁻ (merged channels). L and M: Serial sections from AKR/J mouse lungs after 12 weeks of cigarette smoke exposure confirm that pro-SPC⁺ cells are discrete from CX3CR1⁺ cells. White arrows indicate pro-SPC⁺ cells (L, green channel). Yellow arrows indicate CX3CR1⁺ cells (M, red channel). N: Absence of immunostaining in studies using rabbit IgG control antibody. Original magnifications, ×400.

Figure 6

Relative gene expression in lung tissue homogenates and BAL cells of mice after chronic air or cigarette smoke exposure. A: Relative gene expression in lung tissue homogenates from air-exposed mice (gray bar) compared to cigarette smoke-exposed mice (black bar) shows up-regulation of TNF-α, IL-6, and CX3CL1 after 12 weeks of cigarette smoke exposure. *P ≤ 0.03. B: Relative gene expression in lung tissue homogenates after 24 weeks of cigarette smoke exposure shows CX3CL1 up-regulated compared to air-exposed controls. *P = 0.03. C: The relative gene expression in BAL cells from air-exposed mice (gray bar) compared to cigarette smoke-exposed mice (black bar) demonstrates increase in TNF-α, IL-6, CX3CL1, CX3CR1, and CCL2 after 12 weeks of cigarette smoke exposure. *P ≤ 0.008. D: Relative gene expression of metalloproteinase (MMP)-7, -9, and -12 in lung tissue homogenates after 24 weeks of cigarette smoke exposure. MMP-12 gene expression was up-regulated with cigarette smoke exposure when compared to air-exposed controls. *P = 0.006. Gene expression was analyzed by the ΔΔ-threshold cycle (ΔΔ-Ct) method, with 18S rRNA as the endogenous control. The average ΔCt of age-matched, gender-matched, air-exposed mouse samples served as the calibrator. Gray bars represent air-exposed lungs. Black bars represent cigarette smoke-exposed lungs. n = 3 to 5 mice in each group for 12-week lung homogenate data; n = 5 to 7 mice for 12-week BAL and 24-week lung homogenate data.

Figure 7

LPS induces CX3CR1 expression in RAW264.7 cells in vitro and in alveolar mononuclear phagocytes in vivo. A: Absence of immunostaining in RAW264.7 cells using rabbit IgG control antibody. B: CX3CR1 expression in unstimulated RAW264.7 cells. C: CX3CR1 expression in RAW264.7 cells after 24 hours of stimulation with LPS (10 ng/ml). D: CX3CR1 expression in RAW264.7 cells after 24 hours of stimulation with LPS (100 ng/ml). E–H: CX3CR1 expression in mononuclear phagocytes from BAL 24 hours after intratracheal instillation of stimulus. E: Absence of immunostaining with rabbit IgG control antibody. F: CX3CR1 expression after intratracheal instillation of PBS. G: CX3CR1 expression, after intratracheal instillation of CX3CL1 (1 μmol/L). H: CX3CR1 expression, after intratracheal instillation of CX3CL1 (1 μmol/L) + LPS (0.5 mg/ml). N = 3 to 7 mice in each group, two independent experiments. Original magnifications, ×100.