Apolipoproteins and apolipoprotein mimetic peptides modulate phagocyte trafficking through chemotactic activity

Abstract

The plasma lipoprotein-associated apolipoproteins (apo) A-I and apoE have well described anti-inflammatory actions in the cardiovascular system, and mimetic peptides that retain these properties have been designed as therapeutics. The anti-inflammatory mechanisms of apolipoprotein mimetics, however, are incompletely defined. Whether circulating apolipoproteins and their mimetics regulate innate immune responses at mucosal surfaces, sites where transvascular emigration of leukocytes is required during inflammation, remains unclear. Herein, we report that Apoai(-/-) and Apoe(-/-) mice display enhanced recruitment of neutrophils to the airspace in response to both inhaled lipopolysaccharide and direct airway inoculation with CXCL1. Conversely, treatment with apoA-I (L-4F) or apoE (COG1410) mimetic peptides reduces airway neutrophilia. We identify suppression of CXCR2-directed chemotaxis as a mechanism underlying the apolipoprotein effect. Pursuing the possibility that L-4F might suppress chemotaxis through heterologous desensitization, we confirmed that L-4F itself induces chemotaxis of human PMNs and monocytes. L-4F, however, fails to induce a calcium flux. Further exploring structure-function relationships, we studied the alternate apoA-I mimetic L-37pA, a bihelical analog of L-4F with two Leu-Phe substitutions. We find that L-37pA induces calcium and chemotaxis through formyl peptide receptor (FPR)2/ALX, whereas its D-stereoisomer (i.e. D-37pA) blocks L-37pA signaling and induces chemotaxis but not calcium flux through an unidentified receptor. Taken together, apolipoprotein mimetic peptides are novel chemotactic agents that possess complex structure-activity relationships to multiple receptors, displaying anti-inflammatory efficacy against innate immune responses in the airway.

FIGURE 1.

ApoE deficiency and supplementation with peptide have reciprocal effects on leukocyte trafficking to inflamed lung. A, C, and E, Apoe^+/+ and Apoe^−/− mice were exposed to inhaled LPS, and then bronchoalveolar lavage (BAL) total leukocytes (WBC) (A), neutrophils (PMNs) (C), and macrophages (MΦ) (E) were quantified at the indicated post-exposure time points. B, D, and F, Apoe^+/+ (C57BL/6) mice were pretreated intravenously with 1.2 mg/kg of COG1410 or control (CTL) peptide 264 2 h before LPS inhalation. BAL WBCs (B), PMNs (D), and MΦ (F) were then quantified at the indicated post-LPS time points. Data are mean ± S.E. n = 4–5 mice (A, B, and D); n = 5–10 mice (E and F); and n = 9–10 mice (C) per condition/time point (*, p < 0.01; **, p < 0.001; ***, p < 0.0001; Φ, p = 0.058).

FIGURE 2.

ApoA-I deficiency and supplementation with peptide have reciprocal effects on leukocyte trafficking to inflamed lung. A, C, and E, Apoai^+/+ and Apoai^−/− mice were exposed to inhaled LPS, and then bronchoalveolar lavage (BAL) total leukocytes (WBC) (A), neutrophils (PMNs) (C), and macrophages (MΦ) (E) were quantified at the indicated post-exposure time points. B, D, and F, Apoai^+/+ (C57BL/6) mice were pretreated intravenously with 20 mg/kg of L-4F or scrambled control peptide (sc-4F) 2 h before LPS inhalation. BAL WBCs (B), PMNs (D), and MΦ (F) were then quantified at the indicated post-LPS time points. Data are mean ± S.E. n = 9–10 mice per condition/time point (B, D, and F) (*, p < 0.01; **, p < 0.001; ***, p < 0.0001).

FIGURE 3.

Apolipoprotein deficiency and supplementation with peptide have reciprocal effects on PMN migration to the chemokine-inoculated airspace. Apoe^−/− (A) or Apoai^−/− (C) mice and corresponding WT controls received 0.5 μg of CXCL1 intrapulmonary, and the BAL PMNs were quantified 4 h later. Wild type (C57BL/6) mice pretreated intravenously at −2 h with either 1.2 mg/kg of COG1410 versus control peptide (B), or with 20 mg/kg of L-4F versus sc-4F (D) had BAL PMNs quantified 4 h after intrapulmonary administration of CXCL1. Data are mean ± S.E. and represent n = 6–10 mice per condition (*, p < 0.05; **, p < 0.01).

FIGURE 4.

Apolipoprotein mimetics suppress CXCR2-directed PMN chemotaxis. A-C, wild type murine bone marrow-derived PMNs were pretreated for 1 h with apoA-I (20 μg/ml) or media control (A), L-4F or sc-4F (10 μg/ml) (B), or COG1410 or control (CTL) peptide 264 (8.5 μg/ml) (C), and then assayed for chemotaxis (represented as % of total input PMNs) up a MIP-2 gradient. Data are mean ± S.E. and represent 2 or more independent experiments (**, p < 0.01). D, cell surface display of CXCR2 by murine PMNs was quantified by flow cytometric measurement of CXCR2 mean fluorescence intensity (MFI) after cell treatment (1 h) ex vivo with the indicated concentrations of sc-4F or L-4F. Data are mean ± S.E. and represent 2 independent experiments; **, p = 0.001. A representative flow cytometry histogram is shown at right.

FIGURE 5.

ApoA-I mimetic L-4F is a chemoattractant for human PMNs and monocytes. A, human PMNs purified from peripheral blood of normal, healthy donors were assayed for chemotaxis up a concentration range of gradients of IL-8 (control), L-4F, or sc-4F. Data are mean ± S.D. of chemotaxis index, which represents the fold-increase in the number of migrated cells counted in three high-power fields in response to chemoattractants over spontaneous cell migration to medium control (0)(*, p < 0.05 compared with medium control). B, chemotaxis of human peripheral blood monocytes in response to either fMLF or L-4F was assayed as in A. C and D, human peripheral blood monocytes (C) and neutrophils (D) were tested for chemotaxis up gradients of COG1410. fMLF and IL-8 were used as positive controls. *, p < 0.05 compared with medium control (− for monocytes; 0 for neutrophils).

FIGURE 6.

ApoA-I mimetic L-37pA induces chemotaxis and calcium flux by human monocytes and cross-desensitizes with a defined FPR2/ALX ligand. A, human monocytes were assayed, as described in the legend to Fig. 5, for chemotaxis to a concentration range of D-37pA, L-37pA, or fMLF (*, p < 0.05 compared with medium control (chemotaxis index set to 1)). B-D, calcium flux in Fura-2-loaded monocytes under different stimulation conditions was calculated as the ratio of fluorescence at 340 and 380 nm wavelengths using the FLWinLab program. B, calcium flux in response to a concentration range of L-37pA. C, reduction of L-37pA-induced calcium flux by pre-exposure of monocytes to D-37pA, which itself does not induce a calcium flux. D, cross-desensitization of L-37pA with MMK-1, a defined FPR2/ALX agonist. Pre-exposure of monocytes to MMK-1 and L-37pA reciprocally densensitizes calcium fluxes induced by the other.

FIGURE 7.

ApoA-I mimetic L-37pA induces FPR2/ALX-dependent calcium flux and chemotaxis. A-C, calcium flux in FPR2/ALX-transfected HEK293 cells was assayed as described in the legend to Fig. 6. A, calcium flux was quantified in response to a concentration range of L-37pA. B, W peptide, a defined FPR2/ALX ligand desensitizes subsequent calcium flux in response to L-37pA. C, D-37pA does not induce a calcium flux, but does reduce subsequent L-37pA-induced calcium flux. D, chemotaxis by FPR2/ALX-HEK293 cells was quantified, as described in the legend to Fig. 6, up a concentration range gradient of L-37pA and MMK-1 (*, p < 0.05 compared with medium control (0)). E, C57BL/6 mice were instilled intratracheally with saline, 0.25 μg of KC, 100 μg of L-37pA, or 100 μg of scrambled L-37pA (sc-37pA) and bronchoalveolar lavage neutrophils (PMNs) and macrophages (MΦ) were counted 6 h later. Results are n = 5/condition and are representative of 2 independent experiments.