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Ann Thorac Surg 1998;66:313-317
© 1998 The Society of Thoracic Surgeons

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Original articles: general thoracic

Nitric oxide downregulates lung macrophage inflammatory cytokine production

^a Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado, USA

Address reprint requests to Dr Meldrum, Department of Surgery, University of Colorado Health Sciences Center, C-306, 4200 E Ninth Ave, Denver, CO 80262

Presented at the Thirty-fourth Annual Meeting of The Society of Thoracic Surgeons, New Orleans, LA, Jan 26–28, 1998.

	Abstract

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Background. Inflammatory cytokine production contributes to lung injury after lung ischemia reperfusion and during lung transplant rejection. Although nitric oxide has been demonstrated to reduce lung injury associated with the adult respiratory distress syndrome, it remains unknown whether the mechanism of nitric oxide’s beneficial effects involves reducing lung macrophage inflammatory cytokine production. The purpose of this study was to determine whether nitric oxide downregulates lung macrophage inflammatory cytokine production.

Methods. Lung macrophages were harvested by bronchoalveolar lavage (10⁶ macrophage per milliliter from normal Sprague-Dawley rats, 6 animals per group) and treated under ex vivo tissue culture conditions with the nitric oxide releasing compound S-nitoso-N-acetyl-D, L-penicillamine (0, 10^-5, 10^-4, 10^-3, 10^-2 mol/L) before induction of inflammatory cytokines with endotoxin, (50 ng/mL for 24 hours). Supernatants were assayed for inflammatory cytokine production (tumor necrosis factor , interleukin-1ß) by enzyme-linked immunosorbent assay.

Results. Continuous nitric oxide release by S-nitoso-N-acetyl-D, L-penicillamine decreased lung macrophage tumor necrosis factor- and interleukin-1ß production in a dose-dependent fashion (6 rats per group; data were analyzed for significance [p < 0.05] using two-way analysis of variance with Tukey’s post-hoc correction).

Conclusions. Nitric oxide decreases inflammatory cytokine production by lung macrophage. The mechanism of nitric oxide’s beneficial effects may be partially attributable to decreased production of inflammatory cytokines. Nitric oxide may serve an expanded role for reducing inflammatory cytokine production during acute lung injury, ischemia-reperfusion–induced inflammation, or lung transplant rejection.

	Introduction

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Nitric oxide (NO), originally known as endothelial-derived relaxing factor, is important in the regulation of vascular tone, white blood cell adhesion to vascular endothelium, host defense against infection, and platelet aggregation [1–3]. Inhalational use of NO avoids systemic vasodilation and the ensuing hypotension, thus permitting selective therapeutic delivery to the lungs [1]. In experimental settings, NO decreased pulmonary parenchymal damage, lung neutrophil sequestration, and pulmonary hypertension during acute lung injury [3–5]. Clinically, inhaled NO reduces the pulmonary hypertension [3] as well as the lung inflammation [6] that are characteristic of patients with adult respiratory distress syndrome (ARDS) [7]. Although the effect of NO on vascular reactivity and neutrophil function has been the focus of much investigation, relatively little is known regarding the effects of NO on lung macrophage. Lung macrophage proinflammatory monokine production may be an important contributor to the lung parenchymal damage that occurs during ARDS [6]. We hypothesized that NO downregulates lung macrophage inflammatory cytokine production.

	Material and methods

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Animals
Male Sprague-Dawley rats (weight, 250 to 350 g; Sasco Inc, Omaha, NE) were fed a standard diet and acclimated in a quiet quarantine room for 2 weeks before the experiments. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985).

	Materials

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Enzyme-linked immunosorbent assays for tumor necrosis factor- (TNF) and interleukin-1ß (IL-1ß) were obtained from Genzyme (Cambridge, MA). Dulbecco’s Modified Eagle Medium (DMEM/F-12) with glutamine and 15 mM HEPES buffer was obtained from Life Technologies, Inc (Grand Island, NY). Fetal bovine serum was obtained from Summit Biotechnology (Ft. Collins, CO). All other chemicals and reagents were obtained from Sigma Chemical Co (St. Louis, MO).

Harvest of lung macrophage
Lung macrophages were harvested by bronchoalveolar lavage [8]. After median sternotomy, the right and left bronchi were cannulated by using aseptic technique. Each lung was lavaged with 3 mL of cold (4°C) DMEM a total of 3 times. Greater than 90% of the lavage fluid was retrieved and collected in 15-mL polypropylene centrifuge tubes and kept on ice. The cells were centrifuged at 300 g for 10 minutes at 4°C, the supernatant was decanted, and the pellets were washed twice with DMEM (4°C) and resuspended in 1 mL DMEM (4°C). Leukocytes were counted by hemocytometer and viability was assessed by Trypan blue exclusion. Greater than 98% of cells were viable by Trypan blue exclusion. Leukocytes were centrifuged again (300 g for 10 minutes at 4°C) and resuspended in the appropriate volume of DMEM (with 10% fetal bovine serum) to achieve a final cell concentration of 1 x 10⁶ viable cells per milliliter. Cell suspensions were plated in 24-well plastic culture plates (Corning Glass, Corning, NY) and incubated for 2 hours at 37°C, 5% CO₂, 90% humidity to allow macrophage adherence. Nonadherent cells were removed by vigorous washing with DMEM and adherent cells were counted. This technique yields greater than 95% macrophage by nonspecific esterase staining and by rabbit anti-rat macrophage antibody stain [9–11].

Macrophage culture and measurement of tumor necrosis factor- and interleukin-1ß
Dulbecco’s Modified Eagle Medium (10% fetal bovine serum) was added to each well to achieve a final alveolar macrophage concentration of 10⁶ macrophage per mL per well. Macrophages were incubated with and without the NO releasing compound S-nitoso-N-acetyl-D, L-penicillamine (SNAP; 0, 10^-5, 10^-4, 10^-3, 10^-2 M) before induction of inflammatory cytokines with endotoxin (50 ng/mL for 24 hours). Cultures were incubated for 24 hours at 37°C, 5% CO₂, and 90% humidity. After 24-hour incubation, the supernatants were removed, filtered, aliquoted, and stored at -70°C until assayed for TNF and IL-1ß concentrations [12–17]. Tumor necrosis factor- and IL-1ß concentrations in culture supernatants were measured by enzyme-linked immunosorbent assay according to the manufacturer’s instructions.

Presentation of data and statistical analysis
All reported values are mean ± standard error of the mean (6 rats per group). Differences at the 95% confidence level were considered significant at a p value less than 0.05. Groups were compared using a one-way analysis of variance with post-hoc Bonferroni/Dunn adjustment (SuperANOVA; Abacus Concepts, Berkeley, CA).

	Results

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Tumor necrosis factor
Lung macrophage TNF production is shown in Figure 1. After endotoxin was administered, macrophage TNF production increased compared with unstimulated macrophage (49 ± 21 pg/mL) (p < 0.05). Continuous NO release by SNAP decreased lung macrophage TNF production in a dose-dependent fashion, such that at 10^-2 M SNAP, TNF production was downregulated to 312 ± 162 pg/mL (p < 0.05 versus endotoxin).

View larger version (23K): [in this window] [in a new window]   Fig 1. The effect of nitric oxide on lung macrophage tumor necrosis factor (TNF) production. (ETX = endotoxin; NS = not stimulated; SNAP = S-nitoso-N-acetyl-D, L-penicillamine).

View larger version (24K): [in this window] [in a new window]   Fig 2. The effect of nitric oxide on lung macrophage interleukin-1ß production (ETX = endotoxin; NS = not stimulated; SNAP = S-nitoso-N-acetyl-D, L-penicillamine).

	Comment

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View larger version (32K): [in this window] [in a new window]   Fig 3. Representation of the initiation of lung injury after an insult and the potential mechanisms by which nitric oxide downregulates the inflammatory response. Resident lung macrophage is the first inflammatory cell type to experience an inflammatory challenge (ischemia and reperfusion, endotoxin, etc). Inciting stimuli induce lung macrophage to release the inflammatory cytokine tumor necrosis factor (TNF) and interleukin-1ß (IL-1). Tumor necrosis factor and interleukin-1ß promote lung parenchymal damage in two ways, by direct parenchymal damage through the cellular calcium dyshomeostasis or apoptosis induction and by recruitment of neutrophils that release injurious reactive oxygen metabolites. Nitric oxide (NO) may circumvent this vicious cycle by decreasing inflammatory cytokine (TNF and IL-1) production by the resident lung macrophage. Nitric oxide may inhibit inflammatory cytokine production by affecting the transcription signal (nuclear factor kappa B, NFB) required for their production (inset). (CD14 = LPS receptor; IB = inhibitory B; LBP = LPS binding protein; LPS = lipopolysaccharide; mRNA = messenger ribonucleic acid.)

Chollet-Martin and colleagues [6] have reported an early (day 0) increase in IL-6 and IL-8 concentrations in bronchoalveolar lavage fluid from patients with ARDS. They also observed enhanced spontaneous H₂O₂ production and ß₂ integrin CD11b and CD18 expression by neutrophils isolated from the bronchoalveolar lavage fluid [6]. In addition, Sutter and associates [23] observed increased TNF in the bronchoalveolar lavage supernatants of patients with ARDS. Because macrophage are the major source of these proinflammatory monokines, these clinical studies suggest that lung macrophage proinflammatory monokine production may be a clinically relevant factor in the pathogenesis of ARDS [6]. Furthermore, because we previously reported that proinflammatory monokines activate neutrophils [24, 25], it is possible that lung macrophages extend neutrophil-mediated tissue injury in a positive feedback fashion (see Fig 3).

It has been reported that endotoxin induces an increase in L-arginine metabolism, which is further increased by the provision of exogenous L-arginine [26]. This finding suggests that there is a relative cellular deficiency of L-arginine during endotoxemia. If NO indeed acts as a negative feedback regulator of proinflammatory monokine production, as proposed by Persoons and coworkers [27], then a relative deficiency during acute lung injury may contribute to uncontrolled inflammation. This deficiency would theoretically result in the deficiency of NO as a negative feedback regulator, which may contribute to uncontrolled inflammation. Indeed, L-arginine’s effects appear to be caused by enhanced local production of endogenous NO, as Palmer and colleagues [26] demonstrated that L-arginine is the exclusive amino acid precursor of endogenous NO production. In the present study, exogenous supplementation of NO (through the continuous NO donor, SNAP) resulted in decreased TNF and IL-1ß production by lung macrophage. This finding suggests that, when supplied in ample amounts from an exogenous source, the potentially limited endogenous L-arginine metabolism (NO formation) can be circumvented, and inflammatory cytokine production can be therapeutically downregulated (Fig 3).

The ultimate mechanism by which NO decreases lung macrophage proinflammatory monokine production remains unknown. Recent experimental evidence suggests that NO may inhibit inflammatory cytokine production at the pretranscriptional level [28, 29]. In this regard, nuclear factor kappa B (NFB) serves as the transcription factor for TNF and IL-1ß production [14]. Schwartz and colleagues [30] have demonstrated that the transcription factor NFB is activated in alveolar macrophages of patients with ARDS. Nitric oxide may prevent lung macrophage TNF and IL-1ß production by inhibiting NFB activation (Fig 3). Chen and coworkers [29] have reported that NO inhibits endotoxin-induced NFB activation in the murine macrophage cell line RAW 264.7. Additionally, Peng and associates [28] have demonstrated that NO inhibits NFB in TNF-activated endothelial cells. However, it remains to be determined whether L-arginine and NO decrease endotoxin-induced NFB activation in alveolar macrophage.

	Acknowledgments

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Supported by National Institutes of Health grants HL-43696, HL-44186, and GM-08315 (A.H.H.), an American College of Surgeons Faculty Research Grant (R.C.M.), and a National Institutes of Health National Research Service Award (D.R.M.).

	References

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