IL-17 and TNF-α sustain neutrophil recruitment during inflammation through synergistic effects on endothelial activation

Abstract

IL-17A (IL-17) is the signature cytokine produced by Th17 cells and has been implicated in host defense against infection and the pathophysiology of autoimmunity and cardiovascular disease. Little is known, however, about the influence of IL-17 on endothelial activation and leukocyte influx to sites of inflammation. We hypothesized that IL-17 would induce a distinct pattern of endothelial activation and leukocyte recruitment when compared with the Th1 cytokine IFN-γ. We found that IL-17 alone had minimal activating effects on cultured endothelium, whereas the combination of TNF-α and IL-17 produced a synergistic increase in the expression of both P-selectin and E-selectin. Using intravital microscopy of the mouse cremaster muscle, we found that TNF-α and IL-17 also led to a synergistic increase in E-selectin-dependent leukocyte rolling on microvascular endothelium in vivo. In addition, TNF-α and IL-17 enhanced endothelial expression of the neutrophilic chemokines CXCL1, CXCL2, and CXCL5 and led to a functional increase in leukocyte transmigration in vivo and CXCR2-dependent neutrophil but not T cell transmigration in a parallel-plate flow chamber system. By contrast, endothelial activation with TNF-α and IFN-γ preferentially induced the expression of the integrin ligands ICAM-1 and VCAM-1, as well as the T cell chemokines CXCL9, CXCL10, and CCL5. These effects were further associated with a functional increase in T cell but not neutrophil transmigration under laminar shear flow. Overall, these data show that IL-17 and TNF-α act in a synergistic manner to induce a distinct pattern of endothelial activation that sustains and enhances neutrophil influx to sites of inflammation.

Figure 1

IL-17A and TNFα synergistically increase endothelial selectin expression in vitro. A and B, Time course (0.5 – 16h) of selectin surface protein expression by MHEC in response to IL-17A (100ng/ml) and/or TNFα (50ng/ml), as determined by immunofluorescent flow cytometry. Data are represented as the fold change in the geometric mean of the fluorescence intensity (GMFI) relative to treatment with vehicle alone. Error bars show the 95% confidence interval for each time point. C and D, qRT-PCR of selectin mRNA expression after 8h of stimulation with IL-17A (100ng/ml) and/or TNFα (50ng/ml). Data are normalized to the level of β-actin (Actb) expression and represented as the mean fold change in %Actb expression +/− SEM relative to treatment with TNFα alone. E and F, Flow cytometry of selectin expression after 16h of treatment with IL-17A (100ng/ml), IFNγ (100U/ml), and/or TNFα (50ng/ml). Data are represented as the mean fold change in GMFI +/− SEM relative to treatment with TNFα alone. All data are from at least 3 independent experiments. *, p < 0.05. **, p < 0.01.

Figure 2

IL-17A and TNFα synergistically enhance rolling interactions of leukocytes with microvascular endothelium in vivo. Leukocyte rolling behavior in the mouse cremaster muscle was quantified by intravital microscopy at three time points (2, 6, 16h) following intrascrotal injection with IL-17A (1000ng/mouse) and/or TNFα (100ng/mouse). A – C, Leukocyte rolling flux versus average leukocyte rolling velocity for each treatment condition. Data are represented as mean +/− 95% confidence interval. D and E, Total number of rolling leukocytes per 100 µm vessel segment (D) and expressed as a ratio (E) normalized for differences in vessel diameter, volume, and systemic leukocyte count. Data are represented as mean +/− SEM. F – I, Representative still images of total rolling leukocytes for each treatment condition after 6h of stimulation. Sizes of experimental groups and hemodynamic parameters for all data are summarized in Sup Table 1. *, p < 0.05. **, p < 0.01.

Figure 3

IL-17A and TNFα enhance leukocyte slow-rolling on microvascular endothelium in vivo. Leukocyte rolling velocities in the mouse cremaster muscle were quantified by intravital microscopy following intrascrotal injection with IL-17A (1000ng/mouse) and/or TNFα (100ng/mouse). A – C, Cumulative frequency distribution of leukocyte rolling velocity after 2, 6, and 16h. D and E, Proportion of slow-rolling leukocytes (< 10 µm/s) in each vessel (D) and the average leukocyte rolling velocity (E) after 2, 6, and 16h of cytokine activation. F and G, Effects of E-selectin (9A9) blocking antibody injection (50 µg/mouse) on the proportion of slow-rolling leukocytes (F) and the average leukocyte rolling velocity (G) at 16h after stimulation with TNFα alone or in combination with IL-17A. Data (D – G) are represented as mean +/− SEM. Sizes of experimental groups and hemodynamic parameters for all data are summarized in Sup Table 1. E-selectin blocking studies were performed with n = 3 mice per group. *, p < 0.05. **, p < 0.01.

Figure 4

IL-17A does not enhance endothelial expression of integrin-ligands in vitro. A – F, Immunofluorescent flow cytometry of integrin-ligand expression after 16h of treatment with IL-17A (100ng/ml), IFNγ (100U/ml), and/or TNFα (50ng/ml). Data are represented as the mean fold change in the geometric mean of the fluorescence intensity (GMFI) +/− SEM relative to treatment with TNFα alone. All data are from at least 3 independent experiments. *, p < 0.05. **, p < 0.01.

Figure 5

IL-17A and TNFα synergistically increase endothelial chemokine expression in vitro. A – I, qRT-PCR of chemokine mRNA expression after 8h of stimulation with IL-17A (100ng/ml), IFNγ (100U/ml), and/or TNFα (50ng/ml). Data are normalized to the level of β-actin (Actb) gene expression and represented as the mean fold change in %Actb expression +/− SEM relative to treatment with TNFα alone. All data are from at least 3 independent experiments. *, p < 0.05. **, p < 0.01.

Figure 6

IL-17A and TNFα synergistically increase leukocyte transendothelial migration in vivo. A – F, Transmigrated leukocytes in the perivascular space (50 × 100 µm area) were quantified at three time points (2, 6, 16h) following intrascrotal injection with IL-17A (1000ng/mouse) and/or TNFα (100ng/mouse). Data are represented as the mean +/− SEM of the total number of transmigrated cells per field of view (A) and as a ratio normalized for differences in microvessel volume and systemic leukocyte count (B). C – F, Representative still images of transmigrated leukocytes for each treatment condition after 16h of stimulation. Sizes of experimental groups and hemodynamic parameters for all data are summarized in Sup Table 1. *, p < 0.05. **, p < 0.01.

Figure 7

IL-17A and TNFα synergistically increase neutrophil but not effector CD4+ T cell transmigration through endothelium under laminar shear flow. A and B, Bone-marrow derived neutrophils (PMN) were perfused at a defined shear stress (0.8 dynes/cm²) across MHEC monolayers that had been pre-activated for 16h with IL-17A (100ng/ml), IFNγ (100U/ml), and/or TNFα (50ng/ml). Accumulated (A) and transmigrated (B) cells were then quantified by video microscopy. C and D, MHEC were pre-activated for 16h with both IL-17A (100ng/ml) and TNFα (50ng/ml) and incubated with blocking antibodies (20 µg/ml) against E-selectin (9A9), P-selectin (RB40.34), ICAM1 (YN1), and ICAM2 (3C4). Neutrophils were then perfused and accumulated (C) and transmigrated (D) cells were quantified. E and F, Neutrophils from Cxcr2^−/− and Cxcr2^+/+ mice were perfused over MHEC that had been activated for 16h with both IL-17A (100ng/ml) and TNFα (50ng/ml), and accumulated (E) and transmigrated (F) cells were quantified. G and H, Effector CD4+ T cells (TEFF) were differentiated in vitro from naive CD4+ cells isolated from the spleens of WT mice. Cells were then perfused over MHEC that had been activated for 16h with IL-17A (100ng/ml), IFNγ (100U/ml), and/or TNFα (50ng/ml), and accumulated (G) and transmigrated (H) cells were quantified. All data are expressed as mean +/− SEM. All data are from at least 3 independent experiments, except for E and F, which were from 2 independent experiments. *, p < 0.05. **, p < 0.01.