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The Journal of Immunology, 1999, 163: 2909-2915.
Copyright © 1999 by The American Association of Immunologists

Macrolide Antibiotics Protect Against Immune Complex-Induced Lung Injury in Rats: Role of Nitric Oxide from Alveolar Macrophages1

Jun Tamaoki2, Mitsuko Kondo, Kazuhiro Kohri, Kazutetsu Aoshiba, Etsuko Tagaya and Atsushi Nagai

First Department of Medicine, Tokyo Women’s Medical University School of Medicine, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Macrolide antibiotics have unique immunomodulatory actions apart from antimicrobial properties. We studied the effects of macrolides on IgG immune complex (IgG-ICx)-induced lung injury in rats in vivo and in vitro. Intrapulmonary deposition of IgG-ICx produced a time-dependent increase in the concentration of NO in exhaled air. There were corresponding increases in the number of neutrophils accumulated into alveolar spaces, and lung wet-to-dry weight ratio. All of these changes were inhibited by pretreatment with erythromycin or josamycin, but not by amoxicillin or cephaclor. Incubation of cultured pulmonary alveolar macrophages caused up-regulation of NO production and expression of inducible NO synthase mRNA, an effect that was dose dependently inhibited by erythromycin, roxithromycin, or josamycin. The macrolides also reduced IgG-ICx-induced release of IL-1ß and TNF-{alpha}, but did not alter the release of NO induced by exogenously added IL-1ß and TNF-{alpha}. These results suggest that macrolide antibiotics specifically inhibit immune complex-induced lung injury presumably by inhibiting cytokine release and the resultant down-regulation of inducible NO synthase gene expression and NO production by rat pulmonary alveolar macrophages.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide is generated from the amino acid L-arginine by NO synthase (NOS),3 which exists in both constitutive and inducible isoforms. In the respiratory tract, endogenous NO plays a role in the regulation of airway and vascular smooth muscle tone, microvascular permeability, neurotransmission, and host defense (1). Apart from these regulatory actions, NO also exerts deleterious effects when it is inappropriately generated or overproduced, and excessive amounts of NO and its metabolites such as peroxynitrite anion and hydroxyl radical may contribute to the pathophysiology of inflammation and the resultant tissue damage (1, 2, 3). Constitutive form of NOS is localized to several cell types, including vascular endothelial cells, nerve cells, and airway epithelial cells (4), indicating that these cells are constitutively generating NO. On the other hand, the production of NO is up-regulated in pulmonary inflammatory diseases (5, 6, 7), in which the cell sources of NO may be pulmonary alveolar macrophages (PAM) and airway epithelial cells that contain inducible NOS (iNOS).

There is increasing evidence that macrolide antibiotics have a variety of biological actions in addition to their antimicrobial properties. For instance, erythromycin and roxithromycin inhibit cytokine production and chloride secretion from airway epithelial cells (8, 9), expression of adhesion molecules on neutrophils (10), and neurotransmission in vagal nerve fibers (11). However, it is unknown whether macrolides affect NO generating system. Because lung injury induced by immune complex may depend on L-arginine (12), we hypothesized that, if macrolides could inhibit the release of NO, then the drugs might protect against lung injury in this model. Therefore, objective of the present study was to determine the effects of macrolide antibiotics on immune complex-induced lung injury and, if so, to elucidate whether the effect is associated with alterations in NO generation. To accomplish this goal, we studied lung histopathology and exhaled NO concentration in the rats following intrapulmonary deposition of IgG immune complex (IgG-ICx) in vivo, and the release of NO and expression of iNOS mRNA by the rat PAM incubated with IgG-ICx in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Except where noted, all reagents were purchased from Sigma (St. Louis, MO). Rabbit polyclonal IgG rich in anti-BSA was obtained from Organon Teknika (Westchester, PA). Anti-iNOS mAb was obtained from Transduction Laboratories (Lexington, KY). All antibiotics were generous gifts from pharmaceutical companies, as follows: erythromycin (DAINABOT, Tokyo, Japan); josamycin (Yamonouchi Pharmaceutical, Tokyo, Japan); roxithromycin (Hoechst Marion Roussel Pharmaceutical, Tokyo, Japan); amoxicillin (Takeda Pharmaceutical Industries, Osaka, Japan); and cefaclor (Shionogi Pharmaceutical, Osaka, Japan).

Animal model

All experimental procedures were approved by the Animal Research Committee of Tokyo Women’s Medical University. Pathogen-free male Sprague Dawley rats, weighing 200–220 g, were obtained from SLC Japan (Hamamatsu, Japan) and used in this study. The rats were given a macrolide antibiotic, erythromycin, or josamycin at a dose of 50 mg/kg, or its vehicle (0.9% sterile saline) 3 h before induction of lung injury. Freshly prepared diluted macrolide or vehicle was administered in 0.5 ml vol by oral gavage with a 21-gauge feeding tube fitted to a 1-ml syringe. To test whether other antibiotics can also affect lung injury, the rats were given amoxicillin, a penicillin derivative, or cephaclor, a cephalosporin derivative, at a dose of 50 mg/kg in a similar manner.

The rats were anesthetized i.p. with sodium pentobarbital (40 mg/kg) and were given supplemental doses as needed. The blood samples were taken from right femoral vein, and the plasma concentration of erythromycin was measured by a HPLC (13). The larynx and upper trachea were exposed, and the trachea just below the larynx was incised. To induce IgG-ICx-induced lung injury, the rats were given intratracheal instillation of rabbit polyclonal IgG rich in anti-BSA (2.5 mg in 0.3 ml saline) and the subsequent i.v. injection of BSA (10 mg in 0.5 ml saline), as has been described previously (12). In the control group, the same volumes of saline alone were consecutively given. An intubation tube was then inserted into the trachea, and the rats were artificially ventilated (frequency, 60 breaths/min; tidal volume, 10 ml/kg) with a volume-cycled ventilator (model SN-480-7; Shinano, Tokyo, Japan).

Measurement of exhaled NO concentration

At 1, 3, 6, 12, 24, and 36 h after the application of IgG-ICx, exhaled air was collected over a 10-min period by connecting the expiratory port of the ventilator to a 3-L nondiffusing gas collection bag (Sasaki Medical, Tokyo, Japan). To measure the concentration of NO in the exhaled air, 50-ml aliquots of the collected gas were introduced into a chemiluminescence analyzer (model 200-A; Riken Keiki, Tokyo, Japan) through a modified purging chamber, which was continuously flushed with nitrogen at a flow rate of 50 ml/min. The electrical signals were amplified and integrated to measure the area under the curve with an integration time of 2 s (14). The NO analyzer was calibrated by injecting different volumes of a certified NO gas (10 ppm balanced nitrogen; Yamato Sanki, Tokyo, Japan) with establishment of a linear calibrated scale (range, 2–100 pmol of NO; r2 = 0.99) of NO amount (pmol) vs analyzer output. The NO concentration in the exhaled air was expressed as ppb and calculated as the quotient of the NO content in the sample and the sample volume (50 ml).

Lung inflammation and injury

To assess lung inflammation, 24 h after administration of IgG-ICx, the rats were anesthetized with sodium pentobarbital, and the right cranial lobe was fixed by inflating with 10% Formalin. Sagittal sections were cut for embedding in paraffin. Sections 5 µm thick were taken from the blocks and stained with hematoxylin-eosin, and the number of neutrophils was counted in 10 randomly selected fields at a magnification of x400. The fields containing large vessels and bronchi were excluded, and the number of neutrophils was divided by the number of alveoli to compensate for variable lung inflation (15).

To evaluate lung injury, vascular leakage in the lung was assessed. Both caudal lobes were dissected, stored in a tared container, and weighed for the measurement of wet lung weight. They were then dried in a vacuum oven at 70°C and 4 mmHg for 24 h, weighed for the measurement of dry weight, and the lung wet-to-dry (W/D) weight ratio was calculated (16).

Culture of PAM

Previous study has shown that immune complex can stimulate both PAM and type II alveolar epithelial cells to produce NO, in which the former cells may be a predominant source of NO (17). We thus focused on PAM for in vitro experiments. Bronchoalveolar lavage cells were collected by lavaging whole lungs by five instillations of 10 ml of saline via tracheal canula and withdrawing slowly while gently massaging thorax. The lavage cells were pelleted (200 x g, 10 min), washed twice with PBS, and resuspended in RPMI 1640 medium. More than 95% of the cells in this preparation were PAM, as determined morphologically by May-Giemsa staining. The viability was more than 95%, as determined by trypan blue exclusion. The PAM were adjusted to 106 cells/ml with RPMI 1640 medium containing 10% heat-inactivated FBS, penicillin (50 U/ml), and streptomycin (50 µg/ml), and seeded on 60-mm-diameter plastic culture dishes. After the cells were incubated for 120 min at 37°C in 5% CO2-95% air at 98% relative humidity, nonadherent cells were removed by aspiration. The adherent cells were cultured with 4 ml serum-free RPMI 1640 medium, and stimulated for 24 h with IgG-ICx (3 µg/ml) in the absence or presence of various concentrations of a macrolide. Our preliminary experiments showed that the final concentration of PAM in the culture medium was 6.7 ± 1.1 x 105 cells/ml (n = 24). In the control experiment, the cells were incubated in the culture medium containing the vehicle alone.

Cytokine production and NO generation by PAM

The cytokines IL-1ß and TNF-{alpha} may play a role in immune complex-induced NO generation by PAM (17). Therefore, to assess possible involvement of IL-1ß and TNF-{alpha} in the effect of macrolides, we measured the cytokine concentration in the medium bathing cultured PAM. The concentration of IL-1ß was determined using a murine IL-1ß ELISA kit (BioSource, Camarillo, CA), and TNF-{alpha} activity was determined using a standard LM cell cytotoxicity assay, as previously described (18).

To assess NO release, the concentration of NO in the medium was determined by an NO meter (Model NO-501; Inter Medical, Tokyo, Japan) by measuring a redox current between the following two electrodes (19). A counter electrode was made of carbon fiber, and an NO-selective working electrode was made of Pt/Ir alloy (0.2 mm in diameter; Pt 90%, Ir 10%) coated with a three-layered membrane that consisted of KCl, NO-selective resin, and normal silicone membranes. The KCl membrane was electrochemically deposited on the Pt/Ir wire to suppress overvoltage in the NO discharge. The NO-selective resin was made of puroxyline lacquer and coated by immersion of the wire in 0.8% nitrocellulose solution and dried under air for 12 h. The silicon membrane was affixed as the outermost membrane to avoid a nonspecific ionic effect and electrochemical reactions. The working electrode was supplied with 0.4–0.8 V for the electrochemical oxidization of NO. The NO diffusing through the membrane was detected as an electrical current based on the following reaction: NO + 2H2O -> NO3- + 4H+ + 3e-. The current flow was proportional to the rate of diffusing through the membrane, which was, in turn, proportional to the concentration of NO at the outer surface of the membrane.

After a 24-h incubation of PAM with IgG-ICx (3 µg/ml), the working electrode and the counter electrode were placed 5 mm apart in the medium, and L-arginine (10-3 M) was added to the medium. The response of polarographic current was detected by a current-voltage converter circuit and continuously recorded for 1 h on a pen recorder (Model SR-6355; Graphtec, Tokyo, Japan). In addition, to test whether macrolides can also inhibit NO generation induced by exogenously applied cytokines, PAM were stimulated for 24 h with a mixture of IL-1ß (1500 pg/ml) and TNF-{alpha} (3000 pg/ml) in the presence of erythromycin (10-4 M) or roxithromycin (10-4 M), and the release of NO was determined in a similar manner.

Calibration of the electrode was performed daily before the experiments. Briefly, using the nitrosothiol NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; Inter Medical) as a standard (20), the relationship between the magnitude of electrical current and the concentration of SNAP in the medium was determined. The current increased linearly as SNAP concentration increased, so that the concentration of NO could be determined from the electrical current recorded. Addition of heat-decomposed SNAP produced only 2% of the original current, and oxyhemoglobin (4 x 10-6 M) and 2-phenyl-4,4,5,5-tetramethylimidazole-3-oxide-1-oxyl (PTIO, 3 x 10-5 M; Tokyo Kasei Kogyo, Tokyo, Japan) suppressed the current produced by 5 x 10-5 M SNAP. Thus, there was no significant direct effect of SNAP itself on the electrical current. Moreover, immersion of the electrode in the medium containing L-arginine (10-3 M) and each macrolide (10-4 M), but not PAM, was without effect on the current, indicating that these drugs per se had no effect on the electrode.

Expression of iNOS mRNA

Total RNA was extracted from cultured PAM 24 h after IgG-ICx stimulation by guanidinium-phenol-chloroform method. Total RNA (2.5 µg) was fractionated by 1% formaldehyde-agarose gel electrophoresis, transferred to nylon membranes (Hybond N+; Amersham, Bucks, U.K.), and immobilized with UV cross-linking. Rat iNOS partial cDNA probe (685 bp) was prepared by RT-PCR. RNA prepared from PAM was reverse transcribed into cDNA, and PCR was performed to amplify the 685-bp cDNA fragment. The sequences of the forward (5'-GAGATCAATGCAGCTGTG-3') and reverse (5'-GCTTCTGGTCGATGTCATGAGCAA-3') primers were based on the published iNOS cDNA sequence (21, 22). Authenticity of the PCR products was confirmed by sequencing by dideoxy sequencing methods. The cDNA probe for human ß-actin was purchased from Clontech (Palo Alto, CA).

The probes for rat iNOS and human ß-actin were labeled with [{alpha}-32P]dCTP (Amersham) by a random prime labeling method, and hybridization and autoradiography were performed. In all experiments, the values obtained by densitometric scanning of the blots hybridized with the probe for iNOS were corrected by the values obtained with hybridization with the ß-actin probe.

Statistics

All values are expressed as means ± SEM and analyzed on a Macintosh computer using the Statview 4.5 statistical software package (Abacus Concepts, Berkeley, CA). Statistical analysis was performed by ANOVA with Scheffe’s test for multiple comparison. Student’s t test was used for comparison between two groups. A p value of less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma concentration of erythromycin

Immediately before intrapulmonary deposition of IgG-ICx, the concentration of erythromycin in the rat plasma was 4.4 ± 0.8 x 10-6 M (n = 8).

Exhaled NO concentration

The changes in NO concentration in the exhaled air of six groups of rats are shown in Fig. 1Go. In the control group, exhaled NO concentration remained unchanged at least for 36 h from the baseline value of 9.1 ± 0.5 ppb (n = 12). In the animals subjected to immune complex, exhaled NO concentration increased 3-fold after 6 h of IgG-ICx deposition, which reached a plateau level of 43.3 ± 4.9 ppb (p < 0.001 vs control, n = 12) after 12 h and remained elevated thereafter. Oral administration of erythromycin (50 mg/kg) reduced the IgG-ICx-induced increase in exhaled NO concentration, the maximal level of NO concentration being only 16.9 ± 2.1 ppb (p < 0.001 vs IgG-ICx alone, n = 12). Josamycin at a same dose likewise attenuated the effect of IgG-ICx on exhaled NO, whereas amoxicillin and cephaclor were without effect.



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  		FIGURE 1. Time course of exhaled NO concentration in rats. The animals were treated with 50 mg/kg erythromycin ({blacksquare}), josamycin ({blacktriangleup}), amoxicillin ({triangleup}), cephaclor ({square}), or vehicle alone (0.9% sterile saline, •). After intrapulmonary deposition of IgG-ICx at time 0, concentration of NO in exhaled air was measured by a chemiluminescence analyzer. In the control group, the animals were not subjected to IgG-ICx ({circ}). Data are means ± SEM; n = 12 for each point.

 
Lung inflammation and injury

Lung histopathology and W/D weight ratio were assessed 24 h after IgG-ICx. As shown in Fig. 2Go, the typical features of IgG-ICx-induced lung reaction were marginated neutrophils in blood vessels and the presence of neutrophils, red cells, macrophages, and strands of fibrin in alveolar spaces. The number of neutrophils present in alveoli was increased in the IgG-ICx-treated rats. The rats pretreated with erythromycin or josamycin had fewer neutrophils in the alveoli than the IgG-ICx alone animals did, but pretreatment with amoxicillin or cephaclor did not significantly alter the IgG-ICx-induced neutrophil accumulation (Fig. 3Go, upper panel). The lung W/D weight ratio was increased by IgG-ICx from 5.31 ± 0.19 to 6.78 ± 0.23 (p < 0.01, n = 8). Administration of erythromycin and josamycin each reduced the IgG-ICx-induced increase in W/D weight ratio to 5.52 ± 0.30 and 5.44 ± 0.19, respectively (p < 0.05, n = 8 vs IgG-ICx alone), but amoxicillin and cephaclor did not (Fig. 3Go, lower panel).



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  		FIGURE 2. Photomicrographs showing the effect of erythromycin on morphologic features of lung injury in rats induced by intrapulmonary deposition of IgG-ICx. A, The animals were not subjected to IgG-ICx. B, The animals were given IgG-ICx without pretreatment. C, The animals were teated with erythromycin (50 mg/kg) and then given IgG-ICx. Hematoxylin-eosin staining. Scale bar, 50 µm.

 


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  		FIGURE 3. Effects of antibiotics on the accumulation of neutrophils into alveolar compartment (upper panel) and the increase in lung W/D weight ratio (lower panel) induced by intrapulmonary deposition of IgG-ICx in rats. The animals were given 50 mg/kg erythromycin (EM), josamycin (JM), amoxicillin (AMPC), or cephaclor (CCL), and then IgG-ICx. In the control experiment, the animals received nothing. Data are means ± SEM; n = 8 for each column. **, p < 0.01; ***, p < 0.001, significantly different from control values. {dagger}, p < 0.05, {dagger}{dagger}, p < 0.01, significantly different from the values for IgG-ICx alone.

 
Release of cytokines from cultured PAM

As shown in Fig. 4Go, incubation of PAM for 24 h with IgG-ICx (3 µg/ml) caused increases in the concentrations of IL-1ß and TNF-{alpha} in the medium. Coincubation of cells with erythromycin (10-4 M) inhibited the IgG-ICx-induced release of IL-1ß by 71 ± 9% and TNF-{alpha} by 64 ± 5% (p < 0.01, n = 8 for each), whereas amoxicillin (10-4 M) had no effect.



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  		FIGURE 4. Effects of antibiotics on the release of IL-1ß (upper panel) and TNF-{alpha} (lower panel) from rat PAM stimulated with IgG-ICx (3 µg/ml). The PAM were incubated for 24 h in the absence and presence of erythromycin (EM, 10-4 M) or amoxicillin (AMPC, 10-4 M). Values are expressed as the concentrations of cytokines in the culture medium. Data are means ± SEM; n = 8 for each column. **, p < 0.01; ***, p < 0.001, significantly different from values for unstimulated PAM (Control). {dagger}{dagger}, p < 0.01, significantly different from values for IgG-ICx alone.

 
Release of NO from cultured PAM

The output current of the NO-selective electrode in the medium bathing rat-cultured PAM is demonstrated in Fig. 5Go. Immersion of the electrode in the medium did not produce electrical current in PAM that had been incubated for 24 h with the vehicle (saline) alone even in the presence of L-arginine (10-3 M) (Fig. 5GoA). When the cells were incubated with IgG-ICx (3 µg/ml), the baseline current was not detected, but application of L-arginine caused an increase in the current within 10 min. Between 15 and 30 min after the addition of L-arginine, the response showed the maximal value of 116 ± 23 pA (p < 0.001, n = 11), which corresponds to the medium NO concentration of 92 ± 18 nM, and the subsequent electrical current remained elevated for at least next 1 h (Fig. 5GoB). To assess whether the observed response was associated with iNOS-mediated release of NO, after the response of electrical current reached a plateau, aminoguanidine (10-5 M), a specific inhibitor of NO synthesis through iNOS (23), was added to the medium. As shown in Fig. 5GoC, aminoguanidine rapidly decreased the current by 94 ± 5% (p < 0.001, n = 8) in IgG-ICx-treated PAM.



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  		FIGURE 5. Representative tracing of the current detected by an NO-selective electrode in RPMI medium containing rat PAM. After equilibration, L-arginine (L-Arg, 10-3 M) was added to the medium (arrows). A, Response of electrical current in the medium containing unstimulated PAM. B and C, PAM were incubated for 24 h with IgG-ICx (3 µg/ml). When the response reached a plateau, aminoguanidine (AG, 10-5 M) was added. D and E, PAM were incubated for 24 h with IgG-ICx (3 µg/ml) in the presence of erythromycin (EM, 10-4 M) or amoxicillin (AMPC, 10-4 M).

 
Then, the effects of antibiotics on the IgG-ICx-induced NO release were determined. As shown in Fig. 5Go, D and E, the NO-selective electrical current induced by IgG-ICx (3 µg/ml, 24 h) was greatly reduced by erythromycin (10-4 M), but not by amoxicillin (10-4 M). The inhibitory effects of macrolide antibiotics on the NO release were dose dependent, the concentration of the drug required to reduce 50% of NO contents being 9.1 ± 1 x 10-6 M for erythromycin, 8.7 ± 0.6 x 10-6 M for roxithromycin, and 1.2 ± 1.1 x 10-5 M for josamycin (n = 8 for each) (Fig. 6Go).



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  		FIGURE 6. Dose-dependent effects of antibiotics on the release of NO from rat PAM stimulated with IgG-ICx (3 µg/ml). The PAM were incubated for 24 h either with IgG-ICx alone ({circ}) or in combination with various concentrations of erythromycin (•), roxithromycin ({blacksquare}), josamycin ({blacktriangleup}), amoxicillin ({triangleup}), or cefaclor ({square}). Values are expressed as the concentration of NO in the culture medium after addition of L-arginine (10-3 M). Data are means ± SEM; n = 8 for each point.

 
Incubation of PAM for 24 h with a mixture of IL-1ß (1500 pg/ml) and TNF-{alpha} (3000 pg/ml) increased the release of NO to the same degree as did IgG-ICx. However, this effect was not inhibited by 10-4 M erythromycin or roxithromycin (Fig. 7Go).



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  		FIGURE 7. Representative tracing of the current detected by an NO-selective electrode in RPMI medium containing rat PAM. After equilibration, L-arginine (L-Arg, 10-3 M) was added to the medium (arrows). A, Response of electrical current in the medium containing unstimulated PAM. B, PAM were incubated for 24 h with a mixture (Cytomix) of IL-1ß (1500 pg/ml) and TNF-{alpha} (3000 pg/ml). C and D, PAM were incubated for 24 h with Cytomix in the presence of erythromycin (EM, 10-4 M) or roxithromycin (RXM, 10-4 M).

 
iNOS gene expression

To assess the expression of iNOS gene, PAM were incubated for 24 h with IgG-ICx (3 µg/ml) in the absence or presence of each antibiotic agent or dexamethasone, and total RNA was then extracted and analyzed by Northern blotting with iNOS and ß-actin cDNA probes. Three distinct bands were observed in the hybridization for iNOS, as reported previously (24, 25). The most prominent 4.4-kb transcript is shown in Fig. 8Go. When the cells were treated with IgG-ICx, a marked increase in the steady state level of iNOS mRNA was seen. This induction of iNOS mRNA expression was inhibited by coincubation with 10-4 M erythromycin (percentage of inhibition: 68%), 10-4 M roxithromycin (77%), 10-4 M josamycin (75%), and 10-7 M dexamethasone (97%) (Fig. 8GoA), and the inhibitory effect of erythromycin was concentration dependent (Fig. 8GoB). Coincubation of PAM with amoxicillin or cefaclor at 10-4 M did not alter IgG-ICx-induced iNOS mRNA expression (Fig. 8GoC).



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  		FIGURE 8. Effects of antibiotics and steroid on iNOS mRNA expression by rat PAM stimulated with IgG-ICx (3 µg/ml). In the control experiment, PAM were treated with saline alone. Total RNA was isolated after a 24-h incubation period, and analyzed by Northern blotting with iNOS and ß-actin cDNA probes. A, Effects of erythromycin (EM), roxithromycin (RXM), and josamycin (JM) at 10-4 M, and dexamethasone (DEX) at 10-7 M on IgG-ICx-induced iNOS mRNA expression. B, Effects of various concentrations of EM on IgG-ICx-induced iNOS mRNA expression. C, Effects of erythromycin (EM), amoxicillin (AMPC), and cefaclor (CCL) at 10-4 M on IgG-ICx-induced iNOS mRNA expression.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main findings of the present in vivo and in vitro studies are: 1) intrapulmonary deposition of IgG-ICx in the rats elicited lung injury and increased NO concentration in the exhaled air; 2) incubation of cultured rat PAM with IgG-ICx stimulated the release of NO and induced iNOS mRNA expression; and 3) all of these effects of IgG-ICx were inhibited by macrolide antibiotics. Our results indicate for the first time that macrolides protect against immune complex-induced lung injury presumably by inhibiting PAM to generate iNOS-mediated NO.

In the in vivo model of lung injury, intratracheal instillation of anti-BSA Ab followed by i.v. injection of BSA increased the number of neutrophils in the alveolar compartment and the lung W/D weight ratio, indicating the presence of neutrophilic lung inflammation and vascular hyperpermeability induced by IgG-ICx. There is indirect evidence that acute lung injury occurring after immune complex deposition may be related to NO generated by PAM. Previous studies have shown that pretreatment of rats with NG-monomethyl-L-arginine, a NOS inhibitor, reduces IgG-ICx-induced lung hemorrhage (12) and that IgG-ICx stimulates the production of nitrite and nitrate, which are metabolites of NO, from PAM (17). In the present study, involvement of NO was confirmed by a direct measurement of exhaled NO in mechanically ventilated rats. In our experimental condition, basal NO concentration was low, because the animals were ventilated through tracheal cannulae to exclude contribution of large amounts of NO released from nasal mucosa (26). The rise in exhaled NO concentration was observed at 6 h after the deposition of IgG-ICx and thereafter. This delayed response is consistent with the finding in endotoxin-induced lung injury (27), and may reflect induction of iNOS within the cells of lower respiratory tract.

The concentration of NO in exhaled air is increased in patients with asthma (5, 6) and bronchiectasis (7), and it has been reported that long-term administration of erythromycin, roxithromycin, or clarithromycin is effective in the treatment of these airway diseases (28, 29). Although the mechanism for the efficacy is uncertain, it may not be accounted for by antimicrobial properties of macrolides, but rather by immunomodulatory and/or anti-inflammatory actions (8, 9, 10). In our study, pretreatment of rats with erythromycin, a 14-membered ring macrolide, and josamycin, a 16-membered ring macrolide, each inhibited the accumulation of neutrophils into alveoli and reduced the increase in lung W/D weight ratio induced by IgG-ICx, and these effects were accompanied by the corresponding reductions in exhaled NO concentration. We thus speculate that the suppressive action of antibiotics on lung inflammation and injury is specific for macrolides, and that the effect might be associated with the inhibition of NO generation. Additionally, the potency of the inhibition of IgG-ICx-induced increase in exhaled NO concentration was not different between erythromycin and josamycin, implying that macrolide antibiotics may possess inhibitory actions against NO production regardless of their structures of the lactone ring.

It is known that NO and its metabolite peroxinitrite cause injury to endothelial cells, leading to vascular leakage in the lung. On the other hand, NO does not stimulate, but rather inhibits neutrophil chemotaxis in vitro (30). Thus, the observed inhibition of lung injury in the macrolide-treated rats, as assessed by the lung W/D weight ratio, is most likely attributable to the reduced NO production, but the inhibition of neutrophil accumulation into alveoli may not be related to NO. One possible mechanism for the inhibition of neutrophil accumulation would be macrolides’ inhibition of the expression of adhesion molecule for neutrophil (10) and/or the release of neutrophil chemoattractant cytokines such as IL-8 (8).

Warner et al. (17) have shown that, in the rat model of IgG-ICx-induced lung injury, the percentage of iNOS-positive cells was much greater in PAM than type II alveolar epithelial cells. Therefore, to characterize the effects of macrolides on NO generation, we studied the response of isolated PAM to IgG-ICx in vitro. Direct measurement of NO using an amperometric NO sensor showed that incubation with IgG-ICx caused a release of NO into the culture medium. We also found that the subsequent addition of aminoguanidine, a specific inhibitor of iNOS (23), abolished the NO-selective electrical current. This implies that the observed production of NO was most likely mediated by iNOS, and this notion was further supported by the corresponding induction of iNOS mRNA after stimulation with IgG-ICx.

The release of NO from PAM was inhibited by macrolides in a dose-dependent manner, supporting our in vivo findings that macrolides decreased NO generation in inflamed lung. In this regard, a discrepancy seems to exist in the concentrations of erythromycin required to exert its in vivo and in vitro effects. The plasma concentration of erythromycin at the time of IgG-ICx deposition was 4.4 x 10-6 M, whereas in the in vitro experiments a significant inhibition of NO release was observed at concentrations of 10-5 M and higher. However, plasma concentrations of macrolide antibiotics do not accurately reflect the local concentrations because these drugs most likely concentrate intracellularly (31), and Patel et al. (13) recently showed that the cellular concentration of clarithromycin in human PAM was between 10-4 and 10-3 M, which was 35 times greater than the plasma concentration.

It has been shown that tracheal instillation of IgG-ICx stimulates the release of IL-1ß and TNF-{alpha} into bronchoalveolar lavage fluid (18), and that these cytokines are necessary for IgG-ICx-induced expression of PAM iNOS in the rats (17). In our in vitro experiments, IgG-ICx increased the release of IL-1ß and TNF-{alpha} from PAM, and incubation of the cells with a mixture of these cytokines caused NO production. Pretreatment with erythromycin inhibited the cytokine release, but had little effect on the cytokine-induced NO release. These findings suggest that macrolides’ inhibition of immune complex-mediated NO production may be derived from the inhibition of cytokine generation. Indeed, macrolides are capable of down-regulating the release of IL-6 and IL-8 from human bronchial epithelium (8) and TNF-{alpha} from rat PAM (32). However, further studies are required to elucidate subcellular mechanisms of macrolide action.

In conclusion, we assessed the expression of iNOS mRNA and made a direct measurement of NO release from rat PAM, and found that macrolide antibiotics inhibited iNOS gene expression and the consequent production of NO induced by IgG-ICx. Therefore, it is possible that macrolides could favorably affect immune complex-mediated airway inflammation and lung injury.


   Acknowledgments
 
We thank Masayuki Shino and Yoshimi Sugimura for their technical assistance. We also thank Hoechst Marion Roussel Pharmaceutical and Yamanouchi Pharmaceutical for providing us with roxithromycin and josamycin, respectively.


   Footnotes
 
1 This work was supported in part by Grants-in-Aid 06670243 and 09770429 from the Ministry of Education, Science, and Culture, Japan. Back

2 Address correspondence and reprint requests to Dr. Jun Tamaoki, First Department of Medicine, Tokyo Women’s Medical University School of Medicine, 8-1 Kawada-Cho, Shinjuku, Tokyo 162-8666, Japan. E-mail address: Back

3 Abbreviations used in this paper: NOS, NO synthase; IgG-ICx, IgG immune complex; iNOS, inducible NOS; PAM, pulmonary alveolar macrophage; SNAP, S-nitroso-N-acetyl-DL-penicillamine; W/D, wet-to-dry. Back

Received for publication March 5, 1999. Accepted for publication June 28, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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