TNF-related apoptosis-inducing ligand (TRAIL) regulates inflammatory neutrophil apoptosis and enhances resolution of inflammation

Abstract

Novel therapeutics targeting neutrophilic inflammation are a major unmet clinical need in acute and chronic inflammation. The timely induction of neutrophil apoptosis is critical for inflammation resolution, and it is thought that acceleration of apoptosis may facilitate resolution at inflammatory sites. We previously demonstrated that a death receptor ligand, TRAIL, accelerates neutrophil apoptosis in vitro. We examined the role of TRAIL in neutrophil-dominant inflammation in WT and TRAIL-deficient mice. TRAIL deficiency did not alter constitutive neutrophil apoptosis, whereas exogenous TRAIL accelerated apoptosis of murine peripheral blood neutrophils. We compared TRAIL-deficient and WT mice in two independent models of neutrophilic inflammation: bacterial LPS-induced acute lung injury and zymosan-induced peritonitis. In both models, TRAIL-deficient mice had an enhanced inflammatory response with increased neutrophil numbers and reduced neutrophil apoptosis. Correction of TRAIL deficiency and supraphysiological TRAIL signaling using exogenous protein enhanced neutrophil apoptosis and reduced neutrophil numbers in both inflammatory models with no evidence of effects on other cell types. These data indicate the potential therapeutic benefit of TRAIL in neutrophilic inflammation.

Figure 1.. Effects of TRAIL on apoptosis of murine peripheral blood neutrophils.

(A) Cytocentrifuge preparation of purified peripheral blood neutrophils from TRAIL-deficient mice shows apoptotic neutrophils (arrows) readily distinguishable from neutrophils of nonapoptotic morphology. (B) Neutrophils were purified from peripheral blood and sampled after 6, 12, and 18 h in culture. Apoptosis was assessed by cytospin morphology of neutrophils from control (open bars) and TRAIL-deficient (filled bars) and plotted against time (h); n=4. At t = 0 h, apoptosis was <1.0%. There were no statistically significant differences between WT and TRAIL-deficient in constitutive neutrophil apoptosis. (C) Addition of rTRAIL accelerated apoptosis of WT neutrophils. Freshly isolated peripheral blood neutrophils were cultured with or without TRAIL (100 ng/ml). Mean ± sem of the percentage of apoptotic cells is shown for control (open bars) and TRAIL-treated cells (filled bars); n = 4. In both populations at both time-points, TRAIL treatment caused a significant increase in levels of neutrophil apoptosis.

Figure 2.. Inflammatory cell numbers and apoptosis following i.t. administration of LPS in WT and TRAIL-deficient mice.

Cell counts were obtained from cytocentrifuge preparations and hemocytometer counts of BALF lavaged from the lungs at time-points up to 144 h; n = 10 animals in each group. In all panels, WT mice are depicted as a solid line and TRAIL-deficient mice as a dashed line. (A) Total leukocyte counts. At 48 h, there were significantly more leukocytes in the TRAIL-deficient when compared with WT BAL. (B) Total neutrophil counts. At 48 h, there were significantly more neutrophils in the TRAIL-deficient when compared with WT BAL. (C) The percentage of neutrophils in TRAIL-deficient mice was significantly increased after LPS instillation when compared with WT mice. (D) The percentage of macrophages rose in WT and TRAIL-deficient mice over time as inflammation resolved, and TRAIL-deficient mice had a significantly lower percentage. (E) The percentage of BALF neutrophils with morphological appearances of apoptosis was assessed. The percentage of apoptotic neutrophils increased in both populations, but at 48, 72, and 96 h, there was a significantly lower proportion of apoptotic neutrophils in the TRAIL-deficient mice. Neutrophil apoptosis was also assessed by flow cytometry. (F) The percentage of apoptotic neutrophils (IA8+/Annexin V+) was significantly reduced in TRAIL-deficient mice at 48 h compared with WT mice (P<0.05). Histologic sections of mice 24 h post-LPS treatment were TUNEL-stained for detection of apoptotic events. (G) Numbers of TUNEL-positive (+ve) cells were significantly higher in WT (H) than in TRAIL-deficient mice (I). HPF, per high power field.

Figure 3.. IgM levels in BALF following i.t. administration of LPS in WT and TRAIL-deficient mice.

Total IgM levels measured by ELISA in BAL at 48 h and 72 h were significantly higher in TRAIL-deficient (filled bars) compared with WT mice (open bars; P<0.05; n=5).

Figure 4.. TRAIL expression following i.t. LPS.

Lung sections 48 h after i.t. instillation of saline (control) or LPS were immunostained for TRAIL. (A) Lung section demonstrating lack of positive TRAIL staining in the airways of a WT mouse following saline administration. TRAIL staining is identified only where associated with small pulmonary arteries. (B) Lung section demonstrating TRAIL expression principally in the bronchial epithelium following LPS challenge of WT mice, with occasional staining of alveolar macrophages. (C) Lung section demonstrating absence of specific TRAIL staining in a TRAIL-deficient lung (negative control). These sections are representative of multiple sections cut in a minimum of three experiments. All images, 200× original magnification; scale bar represents 100 μm.

Figure 5.. Neutrophil counts and apoptosis in peritoneal lavage fluid following i.p. administration of zymosan in WT and TRAIL-deficient mice.

(A) Total neutrophil counts were obtained from cytocentrifuge preparations of lavage fluid from the peritoneum at 24 h and 48 h (n=5 experiments), multiplying the differential count by the total leukocyte number obtained from hemocytometer counts. (B) The percentage of lavage neutrophils with morphological appearances of apoptosis was calculated. At 24 h and 48 h, the percentage of apoptotic neutrophils was increased in both populations, but there was a significantly lower proportion of apoptotic neutrophils in the TRAIL-deficient (filled bars) compared with WT (open bars) mice.

Figure 6.. i.t. administration of rTRAIL induces neutrophil apoptosis and reduces neutrophil numbers in vivo.

TRAIL (5 μg) was administered i.t. 24 h after nebulized administration of 3 mg LPS; n = 6 animals in each group. Total neutrophil counts at 48 h from WT mice are shown as open bars and from TRAIL-deficient mice as filled bars. There were significantly more (A) cells and (B) neutrophils in the TRAIL-deficient mice lavages when compared with WT, which significantly reduced in response to TRAIL therapy. (C) At 48 h, the percentage of apoptotic neutrophils was reduced in TRAIL-deficient mice compared with WT mice but significantly increased in response to TRAIL treatment. Histologic sections of WT mice 24 h post-TRAIL treatment were TUNEL-stained for detection of apoptotic events. (D) Numbers of TUNEL-positive cells were significantly higher in WT (E and F) mice following TRAIL treatment (tx). Neb, Nebulized.

Figure 7.. i.p. administration of rTRAIL induces neutrophil apoptosis and restores neutrophil numbers in vivo.

TRAIL (5 μg) was injected into the peritoneal cavity 6 h after zymosan administration; n = 6 animals in each group. Total neutrophil counts from WT mice are shown as open bars and from TRAIL-deficient mice, as filled bars. There were significantly more (A) cells and (B) neutrophils in the TRAIL-deficient mice lavage when compared with WT, which significantly reduced in response to TRAIL therapy. (C) At 24 h, the percentage of apoptotic neutrophils was reduced in TRAIL-deficient mice compared with WT mice but significantly increased in response to TRAIL treatment.

Figure 8.. TRAIL regulates inflammatory neutrophil apoptosis.

A proposed model for TRAIL regulating inflammation resolution by modulation of neutrophil lifespan. In WT mice, TRAIL expression by tissue cells, e.g., the respiratory epithelium, can induce apoptosis of recruited neutrophils. In TRAIL-deficient mice, inflammatory cell survival is prolonged until engagement of a constitutive program of neutrophil apoptosis. Exogenous TRAIL accelerates the apoptotic death of neutrophils in TRAIL-deficient and WT mice.