Cigarette smoke impairs clearance of apoptotic cells through oxidant-dependent activation of RhoA

Abstract

Rationale: Cigarette smoke (CS) is the primary cause of chronic obstructive pulmonary disease (COPD), an effect that is, in part, due to intense oxidant stress. Clearance of apoptotic cells (efferocytosis) is a critical regulator of lung homeostasis, which is defective in smokers and in patients with COPD, suggesting a role in disease pathogenesis.

Objectives: We hypothesized that CS would impair efferocytosis through oxidant-dependent activation of RhoA, a known inhibitor of this process.

Methods: We investigated the effect of CS on efferocytosis in vivo and ex vivo, using acute, subacute, and long-term mouse exposure models.

Measurements and main results: Acute and subacute CS exposure suppressed efferocytosis by alveolar macrophages in a dose-dependent, reversible, and cell type-independent manner, whereas more intense CS exposure had an irreversible effect. In contrast, CS did not alter ingestion through the Fc gamma receptor. The inhibitory effect of CS on apoptotic cell clearance depended on oxidants, because the effect was blunted in oxidant-resistant ICR mice, and was prevented by either genetic or pharmacologic antioxidant strategies in vivo and ex vivo. CS inhibited efferocytosis through oxidant-dependent activation of the RhoA-Rho kinase pathway because (1) CS activated RhoA, (2) antioxidants prevented RhoA activation by CS, and (3) inhibitors of the RhoA-Rho kinase pathway reversed the suppressive effect of CS on apoptotic cell clearance in vivo and ex vivo.

Conclusions: These findings advance the hypothesis that impaired efferocytosis may contribute to the pathogenesis of COPD and suggest the therapeutic potential of drugs targeting the RhoA-Rho kinase pathway.

Figure 1.

Cigarette smoke (CS) specifically impairs efferocytosis in vivo. To determine the effect of CS on efferocytosis in vivo, C57BL/6J mice were exposed to air or CS at either 25 mg/m³ total particulate matter (TPM) or 100 mg/m³ TPM for 5 hours and were then examined for their ability to remove apoptotic cells, using our in vivo clearance assay (42). At time points ranging from 0 to 5 days after CS exposure, 10 million apoptotic murine thymocytes were instilled intratracheally into anesthetized mice. Sixty minutes later, bronchoalveolar lavage was performed, cytospins were stained, and alveolar macrophage ingestions of apoptotic cells were quantified by calculating a phagocytic index (PI). (A) Photomicrographs (original magnification, ×40) show alveolar macrophages that have ingested instilled apoptotic thymocytes (arrows). (B) Low-dose CS (25 mg/m³ TPM) decreased efferocytosis immediately postexposure (Day 0) and trended toward a decrease at 1 day postexposure, compared with air control. *Significantly different from Day 0 control (P = 0.008); ^†different from Day 1 control (P = 0.07). (C) Moderate-dose CS (100 mg/m³ TPM) decreased efferocytosis at 0, 1, and 2 days postexposure. *Significantly different from Day 0 control (P = 0.02), Day 1 control (P = 0.03), and Day 2 control (P = 0.03).

Figure 2.

Subacute and long-term cigarette smoke (CS) impairs efferocytosis in vivo. (A) C57BL/6J mice were exposed to air or CS at 100 mg/m³ total particulate matter (TPM) for 5 days at 5 hours/day in a subacute exposure model, and were then examined for their ability to ingest apoptotic cells in vivo. Subacute CS exposure decreased efferocytosis at 1 week, but not at 4 weeks postexposure. *Significantly different from Week 1 air control (P = 0.02). (B) In a long-term CS exposure model, FVB/N mice were exposed to air or CS at 100 mg/m³ TPM for the first week, and then increased to 250 mg/m³ TPM for a total exposure period of 22 weeks. Mice were then returned to conventional caging for 20 weeks before being examined for their ability to clear apoptotic human neutrophils. *Significantly different from control (P = 0.02; Mann-Whitney test). (C) Collections of pigmented macrophages (arrows) were diffusely present within the lungs of FVB/N mice exposed to CS (left: original magnification, ×10; right: original magnification, ×20). (D) Patches of macrophages containing 10 or more macrophages were significantly more common in the lungs of CS-exposed mice, compared with air-exposed mice. *Significantly different from control (P = 02).

Figure 3.

Cigarette smoke (CS) selectively suppresses efferocytosis ex vivo. (A) To determine whether CS suppressed efferocytosis ex vivo, C57BL/6J mice were exposed to air or moderate-dose CS (100 mg/m³ total particulate matter [TPM]) for 5 hours. Whole lung lavage was then performed and alveolar macrophages were cultured ex vivo overnight. Alveolar macrophages were then examined for their ability to ingest apoptotic Jurkat T cells during a 60-minute coculture experiment. *Significantly different from Day 0 air control (P = 0.002). PI = phagocytic index. (B) To examine whether the suppressive effect of CS was specific for efferocytosis, C57BL/6J mice were exposed to CS and processed as in (A), and were then tested for their ability to ingest various targets. CS specifically decreased ingestion of apoptotic Jurkat T cells ex vivo, but had no significant effect on ingestion of viable or IgG-opsonized Jurkat T cells. *Significantly different from air control (P = 0.003).

Figure 4.

Cigarette smoke (CS) suppresses efferocytosis in vivo and ex vivo through an oxidant-dependent mechanism. To determine the effect of CS on efferocytosis in vivo, oxidant-resistant ICR mice were exposed to air or CS at 100 mg/m³ total particulate matter (TPM) for 5 hours, and were then examined for their ability to remove apoptotic cells at time points ranging from 0 to 2 days postexposure (as in Figure 1C). (A) Moderate-dose CS (100 mg/m³ TPM) trended toward a decrease in efferocytosis only immediately postexposure (Day 0), but had no effect at 1 and 2 days postexposure. *Nonsignificantly different from Day 0 control (P = 0.07). The role of oxidant stress was explored further in (B) mice treated with a superoxide dismutase (SOD) mimetic, manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP), and in (C) extracellular SOD–overexpressing (ecSOD OE) mice. (B) MnTBAP (5 mg/kg) or vehicle control was administered intraperitoneally to C57BL/6J mice three times: (1) immediately before and (2) after exposure to air or CS at 100 mg/m³ TPM for 5 hours, and (3) again the next morning before experimentation. CS did not inhibit efferocytosis in mice pretreated with MnTBAP, as it was in mice that received vehicle control. *Significantly different from vehicle control (P ≤ 0.05). (C) ecSOD OE mice were exposed to air or CS at 100 mg/m³ TPM for 5 hours, and were then examined for their ability to remove apoptotic cells at 1 day postexposure. CS inhibited efferocytosis in wild-type mice, but not in ecSOD OE mice. *Significantly different from vehicle control (P ≤ 0.05). To determine whether CS suppresses efferocytosis ex vivo through an oxidant-dependent mechanism. C57BL/6J mice were exposed to air or moderate-dose CS (100 mg/m³ TPM) for 5 hours. Whole lung lavage was then performed and alveolar macrophages were cultured ex vivo overnight in the presence of (D) increasing concentrations of N-acetylcysteine (NAC) or phosphate-buffered saline (PBS) control, or (E) with increasing concentrations of MnTBAP (or PBS control) for 4 hours before experimentation. Alveolar macrophages were then examined for their ability to ingest apoptotic Jurkat T cells during a 60-minute coculture experiment. In both experiments, CS suppressed efferocytosis, but not in the presence of (D) NAC or (E) MnTBAP at all concentrations tested. (D) *Nonsignificantly different from phosphate buffered saline (PBS) control (P = 0.08). (E) *Significantly different from PBS control (P ≤ 0.05). PI = phagocytic index.

Figure 5.

Cigarette smoke (CS) activates RhoA via an oxidant-dependent mechanism. We examined the hypothesis that CS suppresses efferocytosis through oxidant-dependent activation of RhoA, by (A) investigating the effect of CS on RhoA activation, and then by (B) determining whether RhoA activation could be prevented by the superoxide dismutase (SOD) mimetic manganese(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP). Alveolar macrophages were collected 0 and 24 hours after exposure to air or CS (100 mg/m³ total particulate matter [TPM] for 5 h) and then assayed for RhoA activity. (A) CS increased RhoA activity at 24 hours, but had no effect immediately postexposure. *Significantly different from air control (P = 0.01). (B) C57BL/J mice were pretreated with MnTBAP and exposed to CS as described in Fig. 4B, after which alveolar macrophages were collected by bronchoalveolar lavage 24 hours postexposure and assayed for RhoA activity. CS activated RhoA in the absence, but not the presence, of MnTBAP. *Significantly different from air control (P ≤ 0.02). To determine whether acute exposure to CS impairs efferocytosis through activation of the RhoA–Rho kinase pathway, we first used the ex vivo system described in Fig. 3. Alveolar macrophages taken from air- or CS-exposed C57BL/6 mice were tested for their ability to ingest apoptotic cells in the presence or absence of (C) C3 transferase (a direct RhoA blocker) or (D) Y-27632 (a Rho kinase inhibitor). CS decreased alveolar macrophage efferocytosis, but not in the presence of (C) C3 transferase or (D) Y-27632. *Significantly different from air control (P ≤ 0.05). (E) To determine the role of the RhoA–Rho kinase pathway in the ability of CS to impair efferocytosis in vivo, C57BL/6J mice were exposed to CS at 100 mg/m³ TPM for 5 hours, and then assessed for their ability to clear instilled apoptotic cells the next day, after pretreatment with phosphate-buffered saline or the Rho kinase inhibitor Y-27632. At both doses used, Y-27632 prevented the suppressive effect of CS on efferocytosis, confirming that acute CS impairs efferocytosis through a RhoA–Rho kinase-dependent mechanism. *Significantly different from air control (P ≤ 0.05). PBS = phosphate-buffered saline; PI = phagocytic index.

Figure 6.

Acute cigarette smoke (CS) exposure impairs efferocytosis in vivo through a tumor necrosis factor (TNF)-α–dependent mechanism. To determine whether CS suppressed efferocytosis in vivo through a TNF-α–dependent mechanism, wild-type and TNF-α receptor double knockouts (mice deficient in TNF receptors 1 and 2; TNFa RI/RII KO) were exposed to air or CS, and then tested their ability to clear apoptotic cells (A) immediately or (B) 24 hours after exposure. *Significantly different from air control (P ≤ 0.05).