Journal of Otology & RhinologyISSN: 2324-8785

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Research Article, J Otol Rhinol S Vol: 0 Issue: 1

Effects of Erythromycin on Neutrophil Chemotaxis in Rats

Fuyuki Enomoto1, Ryutaku Kin2, Takeshi Kataoka2, Yoko Sakai1, Hidenori Yokoi1, Masato Fujimori1, Ginichiro Ichikawa1 and Katsuhisa Ikeda1*
1Department of Otorhinolaryngology, School of Medicine, Juntendo University, Tokyo, Japan
2Department of Otorhinolaryngology, Juntendo University Urayasu Hospital, Tokyo Japan
Corresponding author : Katsuhisa Ikeda, MD
2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
Tel: +81-3-5802-1094, Fax: +81-3-5689-0547
E-mail: [email protected]
Received: November 17, 2014 Accepted: March 19, 2015 Published: March 27, 2015
Citation: Enomoto F, Kin R, Kataoka T, Sakai Y, Yokoi H, et al. (2015) Effects of Erythromycin on Neutrophil Chemotaxis in Rats. J Otol Rhinol S1:1. doi:10.4172/2324-8785.S1-018

Abstract

Background: Erythromycin showed an inhibitory effect on neutrophil exudation to the middle ear cavity in a rat model of otitis media.

Methods: Erythromycin (EM) was administered intraperitoneally into male Splague-Dawley rats at 10 mg/kg once daily for 7 or 14 days. A purified neutrophil suspension was employed in the neutrophil chemotaxis experiment using the Boyden chamber method.

Results: A significant increase in migration was observed at 50 and 100 ng/ml growth-related gene product (GRO) as compared to the assay with the addition of serum alone. Strong migration activity was also seen in the presence of zymosanactivated C5a in the assay medium. Treatment with EM for both 7 and 14 days suppressed the enhanced migration of neutrophils induced by 50 and 100 ng/ml GRO. In contrast, C5a significantly enhanced neutrophil migration under the treatment of EM for both 7 and 14 days.

Conclusion: The present study first revealed that EM administration to animals selectively inhibited the GRO-induced migration of neutrophils, which may contribute to its antiinflammatory effect on the upper respiratory mucosa.

Keywords: Neutrophil; Chemotaxis; Growth-related gene product; C5a

Keywords

Neutrophil; Chemotaxis; Growth-related gene product; C5a

Abbreviations:

EM: Erythromycin; GRO: Growth-related Gene Product; ZAP: Zymosan-activated Plasma; PBS: Phosphate Buffered Saline

Background

Macrolide antibiotics have potent immunomodulatory activity. The spectrum of action of macrolides extends to the regulation of leukocyte function and production of inflammatory mediators, control of mucus hypersecretion, resolution of inflammation, and modulation of host defense mechanisms [1]. Intense research of the upper and lower respiratory tract is ongoing to further elucidate the targets and mechanisms of action of macrolides in leukocytes.
To investigate the mechanism of the anti-inflammatory action of erythromycin, we previously reported that administration of the 14- membered macrolides for 2 weeks demonstrated an inhibitory effect on neutrophils exuded to the middle ear cavity in a rat experimental otitis media model [2]. In the present study, we measured neutrophil chemotaxis to examine the effect of erythromycin on neutrophil function.

Methods

Fifteen male Splague-Dawley rats weighing 200 g were used. Erythromycin (Erythromycin Lactobionate : Abbot Pharma. Illinois. USA) was administered intraperitoneally at 10 mg/kg once daily for 7 or 14 days. Blood was collected from the heart under intraperitoneal pentobarbital anesthesia using a heparinized syringe. Neutrophils were isolated from blood treated with sodium citrate anticoagulant using PolymorphoperpTM (Nycomed Pharma AS, Oslo, Norway) centrifugation as described previously [3]. The purity of neutrophils obtained was greater than 98%. More than 97% of the cells were viable as determined by trypan blue dye exclusion. In accordance with the institutional guidelines, rats received proper care and maintenance and did not suffer unnecessary discomfort. The study was approved by the ethics committee of the Juntendo University Faculty of Medicine.
A purified neutrophil suspension was employed in the neutrophil chemotaxis experimentusing the Boyden chamber method [4]. For the neutrophil chemotaxis assay, the cell suspension and normal rat serum were injected into the upper chamber. Serum containing graded concentrations of zymosan-activated complement component C5a and/or those of the rat chemokine GRO (growth-relted gene product) (Rat recombinant GRO. Endogen. INC) was placed in the lower chamber.
To prepare zymosan-activated plasma (ZAP) as a source of activated complement fragment C5a, blood was collected from rat into sodium heparin. Platelet-poor plasma was separated by centrifugation at 1000 x g for 5 min. The plasma was then incubated with zymosan (8 mg/ml) for 30 min at 37°C, followed by centrifugation at 10,000 x g for 5 min and filtration through a sterile 0.2-μm pore size filter. ZAP was used at 1% in PBS to activate neutrophils [5]. For the rat neutrophil chemotaxis inhibitory test with GRO, neutrophils were preincubated for 1 hour with a mixture of GRO with anti-N-terminal antibody (Anti-Rat GRO/CINC-1 Rabbit IgG N-terminal specific, Immuno- Biological Laboratories, Fuzioka, Japan) and anti-C-terminal antibody (Anti-Rat GRO/CINC-1 Rabbit IgG C-terminal specific, Immuno- Biological Laboratories, Fuzioka, Japan), which are anti-rat GRO polyclonal antibodies [5]. Cellulose filters were used for cell trapping. The chemotactic chambers were incubated for 30 minutes. After the incubation period, the filter was removed and stained with Meyer’s hematoxylin. The migrated neutrophils were counted microscopically under 400× magnification.
Data were statistically compared using Wilcoxon’s Test; values of P<0.05 were considered significant.

Results

Rat peripheral blood neutrophils from the control group without EM treatment exhibited increased migration in the presence of GRO in a concentration (1 to 100 ng/ml)-dependent manner. A significant increase in migration was observed at 50 and 100 ng/ml GRO as compared to the assay with the addition of serum alone. A strong migration activity was seen in the presence of zymosan-activated C5a in assay medium (Figure 1).
Figure 1: Chemotactic effect of GRO and C5a on neutrophils.GROinduced enhancement of neutrophil migration was dosedependent. Chemotactic activity was significantly increased in the presence of GRO at 50 and 100 ng/ml, and C5a in the assay medium, compared to the assay with the addition of serum alone. *:p<0.05
To ascertain whether the enhanced neutrophil migration observed in the presence of GRO could be attributable to GRO itself, GRO was preincubated with anti-rat GRO/CINC-1 polyclonal antibodies. The enhancement of migration by GRO was neutralized by the addition of its antibodies. However, anti-GRO antibodies failed to inhibit the enhanced migration induced by zymosan-activated C5a (Figure 2).
Figure 2: Effects of anti-GRO on chemotactic activity of GRO (100 ng/ml) and C5a on neutrophils. The addition of anti-GRO antibody significantly suppresses the enhancement of neutrophil migration induced by GRO, but not that by C5a. *: p<0.05
Neutrophils from rats dosed with erythromycin for 7 days and those from rats dosed with erythromycin for 14 days showed no appreciable difference from control rat neutrophils in terms of migration in the presence of serum alone in the assay medium. Neutrophils from the control rat showed a significant increase in migration in the presence of 50 and 100 ng/ml GRO. Treatment with EM for both 7 and 14 days suppressed the enhanced migration of neutrophils induced by 50 and 100 ng/ml GRO. In contrast, during treatment with EM for both 7 and 14 days, C5a significantly enhanced neutrophil migration (Figure 3).
Figure 3: Effects of anti-GRO on chemotactic activity of GRO (100 ng/ml) and C5a on neutrophils. The addition of anti-GRO antibody significantly suppresses the enhancement of neutrophil migration induced by GRO, but not that by C5a. *: p<0.05
Erythromycin added to the upper chamber or lower chamber at concentrations ranging from 1 to 100 μg/ml had no effect on the migration of the neutrophils derived from the control rat (Figure 4).
Figure 4: Effect of erythromycin (EM) on neutrophil migration. EM added to the upper chamber or lower chamber at concentrations ranging from 1 to 100 mg/ml had no effect on neutrophil migration.

Discussion

Neutrophils are known to infiltrate into tissues in response to cytokines such as GRO) and interleukin-8 (IL-8) [6,7]. It has been suggested that neutrophils that produce cytokines after infiltration are involved in the development of chronic inflammation and immune reaction. Several studies have shown the relevance of neutrophils and its related mediators in purulent otitis media with effusion [8,9]. Cytokines and immune reactions are known to be involved in the mechanism of development of exudative otitis media and chronic sinusitis [10]. Therefore, inhibiting the infiltration of neutrophils would seem likely to contribute to preventing the transition to chronic inflammation. Our previous report demonstrated a significant suppression of exudative cells in the middle ear by EM or CAM in experimental otitis media of rats [4]. The suppressive effect consisted primarily of suppressing neutrophil migration [5]. On the basis of this hypothesis, we directed our attention to neutrophil chemotaxis and examined how neutrophil chemotaxis was affected by EM [11-14].
The present study was conducted to examine whether and how neutrophil chemotactic activity might be affected by EM and the neutrophil-activating factor GRO. As a result, neutrophil migration was found to be increased by GRO in a concentration-dependent manner and also to be enhanced in the presence of zymosan-activated C5a. The GRO-induced increase in neutrophil migration was reversed by the addition of GRO polyclonal antibodies to the assay medium, indicating that GRO-induced neutrophil chemotaxis is specifically mediated by GRO-receptor. No such inhibitory effect of the antibodies on increased migration was observed in the presence of zymosanactivated C5a, which suggests that the neutrophil migration induced by C5a is mediated by a pathway that does not involve the GROreceptor. C5a binds to at least two seven-transmembrane domain receptors, C5aR (C5R1, CD88) and C5L2 (gpr77), expressed ubiquitously on a wide variety of cells but particularly on the surface of immune cells like macrophages, neutrophils and T cells [15].
The administration of EM for both 7 and 14 days significantly inhibited the neutrophil migration induced by GRO, but not C5a. The effect of EM on neutrophil migration may be related to the GROreceptor, such as inhibition of its expression on neutrophil, since EM inhibits the expression of beta2-integrins (Mac 1) adhesion molecules on neutrophils [16]. EM added directly to the assay system in vitro had no effect on neutrophil migration [17,18]. Thus, the inhibitory effect of GRO-receptor is speculated to be brought about by derivatives or metabolites of EM, but not EM itself. Further studies are required to clarify the underlying mechanisms involved in the the inhibition of neutrophil migration.

Conclusion

The present study first revealed that EM administration to animals selectively inhibited GRO-induced migration of neutrophils, which may contribute to its anti-inflammatory effect on the upper respiratory mucosa.

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