A central role for CD68(+) macrophages in hepatopulmonary syndrome. Reversal by macrophage depletion

Abstract

Rationale: The etiology of hepatopulmonary syndrome (HPS), a common complication of cirrhosis, is unknown. Inflammation and macrophage accumulation occur in HPS; however, their importance is unclear. Common bile duct ligation (CBDL) creates an accepted model of HPS, allowing us to investigate the cause of HPS.

Objectives: We hypothesized that macrophages are central to HPS and investigated the therapeutic potential of macrophage depletion.

Methods: Hemodynamics, alveolar-arterial gradient, vascular reactivity, and histology were assessed in CBDL versus sham rats (n = 21 per group). The effects of plasma on smooth muscle cell proliferation and endothelial tube formation were measured. Macrophage depletion was used to prevent (gadolinium) or regress (clodronate) HPS. CD68(+) macrophages and capillary density were measured in the lungs of patients with cirrhosis versus control patients (n = 10 per group).

Measurements and main results: CBDL increased cardiac output and alveolar-arterial gradient by causing capillary dilatation and arteriovenous malformations. Activated CD68(+)macrophages (nuclear factor-κB+) accumulated in HPS pulmonary arteries, drawn by elevated levels of plasma endotoxin and lung monocyte chemoattractant protein-1. These macrophages expressed inducible nitric oxide synthase, vascular endothelial growth factor, and platelet-derived growth factor. HPS plasma increased endothelial tube formation and pulmonary artery smooth muscle cell proliferation. Macrophage depletion prevented and reversed the histological and hemodynamic features of HPS. CBDL lungs demonstrated increased medial thickness and obstruction of small pulmonary arteries. Nitric oxide synthase inhibition unmasked exaggerated pulmonary vasoconstrictor responses in HPS. Patients with cirrhosis had increased pulmonary intravascular macrophage accumulation and capillary density.

Conclusions: HPS results from intravascular accumulation of CD68(+)macrophages. An occult proliferative vasculopathy may explain the occasional transition to portopulmonary hypertension. Macrophage depletion may have therapeutic potential in HPS.

Figure 1.

Common bile duct ligation (CBDL) recapitulates human hepatopulmonary syndrome (HPS). (A) Masson trichrome staining demonstrating cirrhosis of CBDL liver. Note increased collagen (blue) and dilated bile ducts (×40). (B) Three-way scatter plot noting parallel increase in cardiac output and common bile duct (CBD) diameter (measured by echocardiography). (C) Top panel: Hematoxylin and eosin (H&E) staining of the lungs demonstrating dilated pulmonary capillaries (arrows) in CBDL. Bottom panel: Representative images and quantitative assessment of the increased microvessel count based on von Willebrand factor (vWF) immunostaining (red) in CBDL versus sham lungs (×40; n = 4 per group). Blue staining is 4′,6-diamidino-2-phenylindole (DAPI).

Figure 2.

Proliferative, obstructive vasculopathy in hepatopulmonary syndrome (HPS) affects small pulmonary arteries (PA). (A) Representative micrographs of hematoxylin and eosin (H&E) staining of the resistance PA in common bile duct ligation (CBDL) and sham animals. (B, C) Representative and mean data showing increased % medial thickness of small PA in HPS, as measured using immunofluorescent images costained for smooth muscle actin (red) and von Willebrand factor (vWF, green) (×40; n = 4 in each group). (D) Representative computed tomography (CT) angiogram (left panel). Note that CBDL lungs have significantly fewer resistance PAs (0.1–0.2 mm, yellow) than sham but have more pulmonary alveolar capillaries (red). Right panel: quantification of vessel count, binned by diameter (bin width: 0.1 mm).

Figure 3.

Vasoconstrictor diathesis in hepatopulmonary syndrome (HPS) is revealed by nitric oxide synthase (NOS) inhibition. (A) There is enhanced pulmonary vasoconstriction in response to angiotensin II in common bile duct ligation (CBDL) versus sham animals. (B) Hypoxic pulmonary vasoconstriction is augmented in HPS lungs after inhibition of NOS by N^G-nitro-arginine methyl ester (L-NAME) (n = 3 per group). (C) Representative and quantitative data showing increased proliferative activity in CBDL versus sham rats. Images obtained using double-label immunofluorescent stains for proliferating cell nuclear antigen (red) and smooth muscle actin (green) (×40; n = 4 per group). hpf = high-power field; PCNA = proliferating cell nuclear antigen.

Figure 4.

Common bile duct ligation (CBDL) induces accumulation of CD68(+) macrophages in the lungs. (A) Note greater accumulation of CD68 (red) and nuclear factor (NF)-κB (green) positive intravascular macrophages in CBDL versus sham animals (×40). (B) Representative immunofluorescent image demonstrating nuclear translocation of NF-κB (green) in CBDL. Note colocalization with the nuclear marker, 4′,6-diamidino-2-phenylindole (DAPI, blue), which indicates macrophage activation. (C) Plasma endotoxin level is increased in CBDL (n = 8 per group). (D) Monocyte chemoattractant protein (MCP)-1 mRNA is increased in CBDL versus sham lungs (n = 7 per group). PA = pulmonary arteries.

Figure 5.

Increased inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) in pulmonary intravascular macrophages in hepatopulmonary syndrome (HPS). (A–C) Representative and quantitative data showing greater accumulation of CD68(+) (red) macrophages and increased expression of (A) iNOS (green), (B) VEGF (green), and (C) PDGF (green) in common bile duct ligation (CBDL) versus sham animals (×40) (n = 3 per group; 10 high-power fields [hpf] per slide). Note iNOS, VEGF, and PDGF are largely expressed within the macrophages. (D) Representative immunoblots and mean expression shows greater expression of phosphorylated extracellular signal–regulated kinase (p-ERK1) in CBDL lungs. This is reduced in the gadolinium chloride (GdCl)3-treated CBDL group (n = 5 per group).

Figure 6.

Hepatopulmonary syndrome (HPS) plasma increases endothelial tube formation and pulmonary artery smooth muscle cells (PASMC) proliferation. (A) Representative and mean data from vascular endothelial growth factor (VEGF) dot blot assay showing increased plasma VEGF level in common bile duct ligation (CBDL) and sham animals (n = 6 per group). (B) Representative images and mean data from matrigel assay showing a VEGF-dependent increase in endothelial tube formation in response to CBDL versus sham plasma (n = 3 per group). (C) Representative and mean data from platelet-derived growth factor (PDGF) dot blot assay showing increased plasma PDGF level in CBDL and sham animals (n = 9 per group). (D) The in vitro BrdU assay shows increased PDGF-dependent PASMC proliferation in response to CBDL versus sham plasma (n = 3 per group). hpf = high-power field.

Figure 7.

Depletion of macrophages prevents and reverses hepatopulmonary syndrome (HPS). (A–D) Representative and mean data showing that macrophage depletion strategies reduce accumulation of CD68 (red) and nuclear factor (NF)-κB (green) intravascular macrophages in common bile duct ligation (CBDL) and normalize capillary density (CD31, red), cell proliferation (proliferating cell nuclear antigen [PCNA], green), and percent medial thickness of small pulmonary arteries (PAs) (smooth muscle actin, green; ×10; n = 4 in each group). For CD68, CD31, and PCNA staining, 10 high-power fields (hpf) per animal at ×40 were studied. Scale bar = 20 μm. For % medial thickness, 120 blood vessels less than 150 μm were studied. Scale bar = 200 μm. (D) Comparison of alveolar–arterial (A–a) O₂ gradient and hemodynamics in the four study groups (sham, n = 8; CBDL, n = 8; CBDL + gadolinium chloride [GdCl]3, n = 6; and CBDL+clodronate, n = 3).

Figure 8.

(A) Increased capillary density based on CD31 staining in patients who died with cirrhosis versus age- and sex-matched control patients (×20). (B) Increased CD68(+) macrophages within the pulmonary vasculature in patients who died with cirrhosis versus age- and sex-matched control patients (×40). (C) Representative images showing plugging of small pulmonary arteries with pulmonary intravascular macrophages in a patient with cirrhosis versus age- and sex-matched control patient (×40). PIMS = pulmonary intravascular macrophages.

Figure 9.

Schematic illustrating the proposed central role of macrophages in the pathogenesis of hepatopulmonary syndrome. A–a = alveolar–arterial; CBDL = common bile duct ligation; iNOS = inducible nitric oxide synthase; NO = nitric oxide; PASMC = pulmonary artery smooth muscle cells; PDGF = platelet-derived growth factor; PVR = pulmonary vascular resistance; VEGF = vascular endothelial growth factor.