Upregulation of phagocyte-derived catecholamines augments the acute inflammatory response

Abstract

Following our recent report that phagocytic cells (neutrophils, PMNs, and macrophages) are newly discovered sources of catecholamines, we now show that both epinephrine and norepinephrine directly activate NFkappaB in macrophages, causing enhanced release of proinflammatory cytokines (TNFalpha, IL-1beta, IL-6). Both adrenal-intact (AD+) and adrenalectomized (ADX) rodents were used, because ADX animals had greatly enhanced catecholamine release from phagocytes, facilitating our efforts to understand the role of catecholamines released from phagocytes. Phagocytes isolated from adrenalectomized rats displayed enhanced expression of tyrosine-hydroxylase and dopamine-beta-hydroxylase, two key enzymes for catecholamine production and exhibited higher baseline secretion of norepinephrine and epinephrine. The effects of upregulation of phagocyte-derived catecholamines were investigated in two models of acute lung injury (ALI). Increased levels of phagocyte-derived catecholamines were associated with intensification of the acute inflammatory response, as assessed by increased plasma leak of albumin, enhanced myeloperoxidase content in lungs, augmented levels of proinflammatory mediators in bronchoalveolar lavage fluids, and elevated expression of pulmonary ICAM-1 and VCAM-1. In adrenalectomized rats, development of ALI was enhanced and related to alpha(2)-adrenoceptors engagement but not to involvement of mineralocorticoid or glucocorticoid receptors. Collectively, these data demonstrate that catecholamines are potent inflammatory activators of macrophages, upregulating NFkappaB and further downstream cytokine production of these cells. In adrenalectomized animals, which have been used to further assess the role of catecholamines, there appears to be a compensatory increase in catecholamine generating enzymes and catecholamines in macrophages, resulting in amplification of the acute inflammatory response via engagement of alpha(2)-adrenoceptors.

Figure 1. Isolated peritoneal mouse macrophages (A, B) were exposed to various concentrations of norepinephrine or epinephrine for 30 min at 37°C.

Then, nuclear proteins were extracted from 10×10⁶ cells protein concentrations adjusted and NFκB p65 activation assessed. Each bar represents n = 4. In a second set of experiments, following incubation with various concentrations of norepinephrine or epinephrine (30 min, 37°C), peritoneal mouse macrophages (5×10⁶/ml; C, D) were lysed and cytosol subjected to Western blotting analysis for IκBα. Depicted blots are representative of 3 independent experiments. Neg ctrl, incubation with HBSS.

Figure 2. Isolated peritoneal mouse macrophages were exposed to various concentrations of norepinephrine (10⁻¹¹–10⁻⁸ M) or LPS (20 ng/ml, positive control) for 30 min at 37°C.

Then, supernatant fluids were obtained and subjected to ELISA analysis for TNF-α (A), IL-1β (B), IL-6 (C) and MIP-2 (D). Each bar represents n = 4–7. Neg ctrl, incubation with HBSS.

Figure 3. Following isolation, peritoneal mouse macrophages incubated to HBSS (negative conrol), LPS (20 ng/ml, positive control) or 10⁻¹¹–10⁻⁸ M epinephrine (30 min, 37°C).

Obtained supernatants were analyzed for TNF-α (A), IL-1β (B), IL-6 (C) and MIP-2 (D) using ELISA measurements. n = 4–7 per experimental group.

Figure 4. Rat blood neutrophils and rat alveolar macrophages were obtained from AD+ and ADX animals and mRNA isolated and subjected to real-time PCR analysis for both key-enzymes of catecholamine synthesis, tyrosine-hydroxylase (rate limiting step) and dopamine-β-hydroxylase (final conversion step) (A, B).

Following 15 min culture of unstimulated phagocytes derived from AD+ and ADX animals, supernatant fluids were analyzed for norepinephrine (C) and epinephrine (D) by ELISA. Each bar n = 5.

Figure 5. IgG immune complex (IC)-induced lung injury was induced in adrenal-intact (AD+) and adrenalectomized (ADX) rats.

Vascular leakage (A), content of myeloperoxidase in lung extracts (B) and total white cell content in BAL fluids (C) were assessed. In negative controls, intratracheal administration of anti-BSA was replaced by PBS. Lungs were surgically removed 4 hr after intrapulmonary deposition of IgG immune complexes, tissues fixed in buffered 5% formaldehyde, and paraffin-embedded lung sections were stained with hematoxylin and eosin (D–I). Frames D–F, histology of lungs from adrenal-intact animals; frames G–I represent lungs from adrenalectomized littermates. Sections are representative for at least three rats per group. For each bar n>5 rats.

Figure 6. BAL fluids 4 hr after immune complex-induced lung injury, showing content of interleukin-6 (IL-6) (A), tumor necrosis factor α (TNFα) (B), interleukin-1β (IL-1β) (C) and norepinephrine (D).

Four hours after induction of injury, whole lungs were flushed, surgically removed, homogenized and subjected to Western blot analysis for the adhesion molecules ICAM-1 (E) and VCAM-1 (F). For Western blot studies, experiments were repeated, using 5 different samples per group. Blots shown are of representative bands. For each bar n≥5 rats. Abbreviations: neg ctrl, negative control; AD+, adrenal-intact animals; ADX, adrenalectomized animals; IC-ALI, immune complex-induced acute lung injury.

Figure 7. ALI was induced by airway instillation of LPS (300 µg).

In parallel to immune complex-induced ALI, vascular leakage (A) and myeloperoxidase (MPO) activity in lung extracts (B) 6 hr after initiation of injury were measured. Bronchioalveolar lavage fluids obtained 6 hr after introduction of LPS-induced lung injury were assessed for content of IL-6 (C), TNFα (D), and IL-1β (E). Following surgical removal 6 hr after intrapulmonary LPS deposition, lungs were formalin-fixed, paraffin-embedded and stained with hematoxylin and eosin (F–I). Insets F and G show lungs of adrenal-intact animals, while lungs of rats lacking their adrenal glands are presented in insets H and I. For each bar n>5 rats. Abbreviations used: neg ctrl, negative control; AD+, adrenal-intact animals; ADX, adrenalectomized animals; LPS-ALI, LPS-induced acute lung injury.

Figure 8. Severity of IgG-IC ALI following pharmacologic blockade of mineralocorticoid and glucocorticoid receptors (by spironolactone and RU 28362, respectively) in adrenal-intact and ADX animals, as assessed by permeability index.

The α₂-adrenergic antagonist RX 821002 was employed in ADX rats. Each bar represents n≥5 rats. Neg ctrl, negative control with intratracheal instillation of PBS.