Lactoferrin regulates an axis involving CD11b and CD49d integrins and the chemokines MIP-1α and MCP-1 in GM-CSF-treated human primary eosinophils

Abstract

Eosinophils are multifunctional immune cells that contribute to innate and adaptive immune/repair responses. Lactoferrin (LF) is an iron-binding protein indicated to alter cell adhesion and immune function by receptor-mediated interactions or by participating in redox mechanisms. The eosinophil adhesion molecules, αMβ2 and α4β1, are differentially expressed following exposure to the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) and various redox agents. We hypothesized that LF can alter the function and production of proteins involved in adhesion/migration. Utilizing eosinophil peroxidase activity or fluorescent labeling adhesion assays, LF reduced GM-CSF-induced eosinophil adhesion in the presence of fibronectin or vascular adhesion molecule-1 compared with GM-CSF treatment alone. Flow cytometric analysis of eosinophil αM (CD11b) and α4 (CD49d) integrins revealed that cotreatments (24 h) with LF plus GM-CSF induced a significant increase in CD11b compared with control and GM-CSF treatments but a significant decrease in CD49d compared with control and GM-CSF treatments. These changes in CD11b and CD49d levels were significantly correlated with the increased production of chemokines (macrophage inflammatory Protein-1α, monocyte chemotactic protein-1) and an identified increase in S100A9 production. Thus, LF release at sites of inflammation may alter eosinophil recruitment/activation and possibly the progression of diseases such as cancer and asthma where significant eosinophil influx has been described.

FIG. 1.

LF-modulated adherence to fibronectin compared to BSA. Purified human blood eosinophils (1×10⁵cells/mL) were added to plates coated with 10 μg/mL BSA or 10 μg/mL FN. Cells were treated with 100 pM GM-CSF or increasing concentrations of bovine LF for 1 h. EPO activity was assessed at 490 nm as described under Materials and Methods. The average number of eosinophils adhered relative to a standard curve is displayed±SEM, n=5, *P<0.02 versus buffer control, ^†P<0.04 versus GM-CSF alone. GM-CSF, granulocyte-macrophage colony-stimulating factor; EPO, eosinophil peroxidase; BSA, bovine serum albumin; LF, lactoferrin; FN, fibronectin.

FIG. 2.

LF-modulated adherence to FN and VCAM-1. Purified human blood eosinophils were cultured in plates coated with 10 μg/mL FN or 10 μg/mL VCAM-1. Cells were treated with 100 pM GM-CSF and 10 μg/mL bovine or human (H) LF for 1 h and examined for eosinophil adherence via EPO activity or CFSE labeling. (A) The average number of eosinophils adhered relative to an EPO activity standard curve is displayed±SEM, n=5, *P<0.02 versus buffer control, ^†P<0.03 versus GM-CSF. (B) The average number of eosinophils adhered relative to a CFSE fluorescence standard curve is displayed±SEM, n=8, *P<0.03 versus buffer control, ^†P<0.04 versus GM-CSF. (C) Bovine versus human (H) LF: The average number of eosinophils adhered relative to a CFSE fluorescence standard curve is displayed±SEM, n=4, *P<0.04 versus buffer control, ^†P<0.05 versus GM-CSF; VCAM-1, vascular adhesion molecule-1; CFSE, carboxyfluorescein diacetate succinimidyl ester.

FIG. 3.

LF-modulated CD49d and CD11b expression. Purified human blood eosinophils (2×10⁶cells/mL) were cultured in plates coated with 10 μg/mL fibronectin (FN). Cells were treated with buffer control, 100 pM GM-CSF, and/or 10 μg/mL bovine LF for 24 h. Live cells were gated (A, C) and charted (B, D) for integrin expression. CD11b and CD49d are identified via flow cytometry and reported as the gMFI. Charts represent treatment with LF alone (B) (±SEM, n=4, *P=0.01 versus CD11b buffer control) or cotreatment with LF and GM-CSF (D) (±SEM, n=23 *P<0.02 versus CD49d buffer control, ^§P<0.0001 versus respective buffer control, ^†P=0.0002 versus CD49d GM-CSF, ^‡P=0.0008 versus CD11b GM-CSF) compared to controls. The values in parenthesis indicate the ratio of CD11b gMFI/CD49d gMFI. gMFI, geometric mean fluorescent intensity.

FIG. 4.

Donor to donor variability in LF-modulated CD49d and CD11b expression. Purified human blood eosinophils from each of the 23 donors charted in Figure 3D are plotted. Identified markers represent the CD49d or CD11b gMFI of eosinophils treated with 100 pM GM-CSF±10 μg/mL bovine LF normalized to the GM-CSF treatment.

FIG. 5.

LF-modulated CD49d and CD11b expression compared to NAC. Purified human blood eosinophils (2×10⁶cells/mL) were cultured in plates coated with 10 μg/mL FN. Cells were treated with buffer control, 100 pM GM-CSF, 10 mM NAC, and/or 10 μg/mL bovine LF for 24 h. Live cells were gated (A) and charted (B) for integrin expression. CD11b and CD49d are identified using flow cytometry and reported as the gMFI,±SEM, n=9, *P<0.02 versus respective buffer control, ^†P<0.03 versus GM-CSF, ^‡P=0.03 versus GM-CSF+LF, ^§P=0.002 versus GM-CSF+NAC, ^¶P<0.008 versus GM-CSF+NAC. The values in parenthesis indicate the ratio of CD11b gMFI/CD49d gMFI. NAC, N-acetyl-l-cysteine; FN, fibronectin.

FIG. 6.

LF-modulated MIP-1α and MCP-1 production. Purified human blood eosinophils (2×10⁶cells/mL) were cultured in plates coated with 10 μg/mL FN. Cells were treated with buffer control, 100 pM GM-CSF, 10 mM NAC, and/or 10 μg/mL bovine LF for 24 h. Cell-free conditioned media was measured in triplicate per experiment for the specified protein. Repeat experiments were averaged and are displayed as the mean concentration (pg/mL). Charted concentrations represent the chemokines MIP-1α (A) (mean±SEM, n=16, *P<0.003 versus control, ^†P<0.05 versus GM-CSF, ^‡P<0.004 versus GM-CSF+NAC) and MCP-1 (B) (mean±SEM, n=16, *P<0.01 versus control, ^†p=0.001 versus GM-CSF, ^‡P<0.004 versus GM-CSF+NAC). MIP-1α, macrophage inflammatory protein-1alpha; MCP-1, mononocyte chemotactic protein-1; FN, fibronectin.

FIG. 7.

Immunoblot detection of intracellular and extracellular eosinophil S100A9 in response to LF. Purified human blood eosinophils (5×10⁶/mL) were cultured in plates coated with 10 μg/mL FN. Cells were suspended in 0.1% human serum albumin RPMI and treated with buffer control, 100 pM GM-CSF or 10 μg/mL bovine LF for 24 h. Total eosinophil cell lysates from 5×10⁵ cells and the total corresponding conditioned media were loaded onto a 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel. A representative immunoblot is displayed in (A). Data were also quantified with ImageJ software, expressed as S100A9 mean band densitometry relative to actin controls and normalized to the respective media control, n=4, *P<0.04 versus conditioned media buffer control, ^†P<0.05 versus conditioned media GM-CSF control.