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Antibiotic-induced apoptosis in human activated peripheral lymphocytes
International Journal of Antimicrobial Agents 25 (2005) 216–220
Antibiotic-induced apoptosis in human activated peripheral lymphocytes Jun-Ichi Kadota∗ , Syunji Mizunoe, Kenji Kishi, Issei Tokimatsu, Hiroyuki Nagai, Masaru Nasu Division of Pathogenesis and Disease Control, Department of Infectious Diseases, Oita University Faculty of Medicine, 1-1 Hasama, Oita 879-5593, Japan Received 6 July 2004; accepted 6 October 2004
Abstract Long-term administration of macrolide antibiotics reduced the number of lymphocytes in bronchoalveolar lavage fluid in patients with chronic airway inflammatory disease. To evaluate the inflammatory activity of macrolides, their effect on apoptosis of activated lymphocytes isolated from human peripheral blood was compared with that of other antibiotics. Macrolides, including clarithromycin and azithromycin, at a final concentration of 100 g/ml accelerated apoptosis of activated lymphocytes, while other antibiotics such as fosfomycin sodium, -lactams — ceftazidime, piperacillin sodium and biapenem, and a quinolone, ofloxacin, did not cause significant induction of apoptosis. Our results suggest that 14- or 15-membered ring macrolides are specifically involved in the augmentation of apoptosis of activated lymphocytes, and this may be of value therapeutically for chronic airway diseases. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Macrolide antibiotics; Clarithromycin; Azithromycin; Apoptosis; Lymphocytes
1. Introduction The anti-inflammatory action of macrolide antibiotics has been an area of intensive investigation since Kudoh et al. first reported the effectiveness of low-dose, long-term treatment with erythromycin in chronic lower respiratory tract infections, including diffuse panbronchiolitis (DPB) [1]. The antiinflammatory activity of the drugs have been reported to be the action on lymphocytes [2,3], promoting monocytes to macrophage differentiation [4], modulating IL-8 expression/production [5,6] and inhibiting Cl and glycoconjugate secretion [7,8]; this may account for the clinical effectiveness of erythromycin in the treatment of the disease [1,9]. We have previously described how long-term macrolide antibiotics reduced the number of lymphocytes in bronchoalveolar lavage fluid (BALF) of the patients with DPB [10,11], and that lymphocyte elimination by apoptosis is im∗
Corresponding author. Tel.: +81 97 586 5804; fax: +81 97 549 4245. E-mail address: [email protected] (J.-I. Kadota).
portant to terminate the chronic immune response of the murine lung to intratracheal particulate antigen exposure [12]. In this context, it has been reported that roxithromycin (RXM) augumented apoptosis in Dermatophagoides farinaestimulated peripheral blood lymphocytes (PBLs) from patients with bronchial asthma, while other antibiotics, including cefazolin and ampicillin, did not cause significant induction of apoptosis [13]. Jun et al. also found that ciprofloxacin (CPFX) or RXM induced apoptosis of anti-CD3-activated Jurkat T lymphocytes [14]. Additionally, we demonstrated that macrolides including clarithromycin (CLR), azithromycin (AZM) and josamycin (JM), at higher concentration than 200 g/ml, induced apoptosis on unstimulated PBLs from normal subjects, while other antibiotics such as -lactams, a carbapenem and a quinolone did not (Ishimatsu et al. Abstract: The 2000 International Conference of the American Thoracic Society. Am J Respir Crit Care Med 161;A888:2000). In this study, therefore, we investigated whether macrolides and other antibiotics induce apoptosis of PBLs
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J.-I. Kadota et al. / International Journal of Antimicrobial Agents 25 (2005) 216–220
activated by anti-CD3/CD28 antibody obtained from normal subjects.
2. Materials and methods 2.1. Agents The antimicrobial agents were gifts from the manufacturers. They included JM (Yamanouchi Pharmaceutical Co., Tokyo, Japan), CLR (Taisho-Toyama Pharmaceuticals Co., Tokyo), AZM (Pfizer Pharmaceuticals Co., Tokyo), fosfomycin sodium (FOM) (Meiji Pharmaceutical Co., Tokyo), the -lactams, ceftazidime (CAZ, Tanabe Pharmaceutical Co., Tokyo), piperacillin sodium (PIPC) (Taisho-Toyama Pharmaceuticals Co., Tokyo) and biapenem (BIPM) (Meiji Pharmaceutical Co., Tokyo), and a new quinolone, ofloxacin (OFLX) (Daiichi Pharmaceutical Co., Tokyo). 2.2. In vitro culture and stimulation of lymphocytes Venous blood was obtained from healthy adult volunteers and was collected in heparinized tubes. Lymphocytes were isolated by Ficoll-Hypaque and Percoll gradient centrifugation under sterile procedures. Lymphocytes (5 × 105 /well) in culture medium (RPMI 1640) supplemented with 10% foetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM l-glutamine (all from Sigma, St. Louis, MO, USA), were cultured in a 96-well flat bottom microtitre plate (Nunc, Roskilde, Denmark) in the presence of anti-CD3 monoclonal antibody (mAb) at 100 ng/ml (Immunotech, Marseilles Cedex, France) and anti-CD28 mAb at 100 ng/ml (Immunotech) with IL-2 at 50 U/ml (Sigma) for 24 h at 37 ◦ C in a humidified 5% CO2 atmosphere. Thereafter, the drugs were added at a concentration of 10 or 100 g/ml at 37 ◦ C for 48 h. Macrolide antibiotics were dissolved in dimethyl sulfoxide (DMSO: final concentration, < 0.1%), and other antibiotics were prepared in sterilized water. Culture medium with DMSO (0.1%) was used as a control.
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2.4. Statistical analysis All data are expressed as mean ± standard error of the mean (S.E.M.). Statistical comparisons among groups were made using analysis of variance with Tukey–Kramer post hoc analysis by the StatView 5.0 statistical package. A P-value of <0.05 was considered significant.
3. Results Fig. 1 shows representative results of flow cytometry for the apoptosis of anti-CD3 and anti-CD28 antibody-activated lymphocytes after incubation in the presence or absence of various antibiotics at 100 g/ml for 48 h. The lower left quadrant of the histogram shows the viable cells, which exclude PI and are negative for FITC-Annexin-V binding. The lower right compartment represent the early apoptotic cells, which are PI negative and Annexin-V positive, indicating the translocation of phophatidylserine to the external cell surface but the integrity of the cytoplasmic membrane [15]. The upper right quadrant represents the nonviable necrotic and late-stage apoptotic cells, which are positive for Annexin-V binding and PI uptake. In this example, the percentages of Annexin-V-positive cells were 14.6% (control), 10.5% (FOM), 10.5% (BIPM), 15.4% (OFLX), 13.3% (PIPC), 12.6% (CAZ), 7.1% (JM), 23.8% (CLR) and 38.8% (AZM). The same experiments were performed 10 times for the control group, three times for non-macrolide antibiotics or four times for macrolide antibiotics, and the same results were obtained. Neither of the antibiotics at a concentration of 10 g/ml induced apoptosis (Fig. 2), while CLR and AZM at 100 g/ml, but not JM, significantly enhanced apoptosis compared with the control group or other antibiotic-treated groups (31.7 ± 1.3% and 46.4 ± 2.9% versus 21.1 ± 1.2% for control, 17.1 ± 0.5% for FOM, 17.7 ± 1.1% for BIPM, 21.3 ± 3.3% for OFLX, 19.3 ± 1.3% for PIPC, 17.8 ± 1.3% for CAZ, and 23.8 ± 1.0% for JM, respectively; P < 0.05, Fig. 2). In addition, the apoptosis of activated lymphocytes was more dramatically increased in the AZM-treated cells than in CLR-treated cells (P < 0.05).
2.3. Measurement of apoptosis Induction of apoptosis was measured after 48 h using an apoptosis detection kit (Trevigen Inc., Gaithersburg, MD) with fluorescein isothiocyanate (FITC)-conjugated AnnexinV, which is an early apoptosis marker on flow cytometry (FACS Calibur version 1.0 cell sorter, Becton Dickinson, San Jose, CA). Briefly, cells (5 × 105 /well) were treated with FITC-conjugated Annexin-V mAb and propidium iodide (PI) in Annexin-V binding buffer (10 mM Hepes, 0.15 M NaCl, 5 mM KCl, 1 mM MgCl2 , and 1.8 mM CaCl2 , pH 7.4) for 15 min in the dark. Cells were then analyzed by flow cytometry using the FL1 channel for detecting Annexin-VFITC staining and the FL-2 channel for detecting PI staining. Annexin-V-positive cells were defined as apoptotic cells.
4. Discussion Apoptosis is critical for normal development and tissue homeostasis, including that of the immune system [16]. There is increasing evidence that dysregulation of apoptotic pathways are associated with airway disease, including bronchial asthma. Most of the T-lymphocytes infiltrating the airway of asthmatics are not apoptotic, suggesting that the persistence of airway inflammation may depend upon their continuing proliferation and their increased survival in the bronchial mucosa [17]. It was previously reported that the anti-inflammatory effects of macrolide antibiotics against lymphocytes included inhibition of the mitogen-mediated
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Fig. 1. Analysis of apoptosis induced by various antibiotics in stimulated lymphocytes. After pretreatment with anti-CD3/CD28 mAb and IL-2 for 24 h, cells were incubated in the absence (A) or in the presence of fosfomycin sodium (FOM) (B), biapenem (BIPM) (C), ofloxacin (OFLX) (D), piperacillin sodium (PIPC) (E), ceftazidime (CAZ) (F), josamycin (JM) (G), clarithromycin (CLR) (H) or azithromycin (AZM) (I) at 100 g/ml. After 48 h, induction of apoptosis was analyzed by flow cytometry using Annexin-V/PI staining. The percentage in the lower right compartment and upper right quadrant shows the percentage of Annexin-V-positive cells. Representative data from several separate experiments are shown.
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Fig. 2. Effects of various antibiotics on apoptosis of stimulated lymphocytes in vitro. After pretreatment with anti-CD3/CD28 mAb and IL-2 for 24 h, stimulated cells were incubated with or without antibiotics at 10 (open column) or 100 (closed column) g/ml. After 48 h, induction of apoptosis was measured using flow cytometry. Data are mean ± S.E.M. of proportions of cells positive for Annexin-V determined in three or four independent experiments. C: control; FOM: fosfomycin sodium; BIPM: biapenem; OFLX: ofloxacin; PIPC: piperacillin sodium; CAZ: ceftazidime; JM: josamycin; CLR: clarithromycin; AZM: azithromycin.
lymphocyte proliferative response and reduction of activated lymphocytes in BAL fluid of DPB patients after treatment with macrolide antibiotics [2,10,11]. In addition, RXM augumented apoptosis in D. farinae-stimulated PBLs from asthma patients [13] and CPFX or RXM induced apoptosis of antiCD3-activated Jurkat T lymphocytes [14]. Thus, in this study, the immunomodulatory effect of macrolides and other antibiotics was focused on lymphocyte apoptosis so as to compare our results with past published data. Our present study showed that CLR and AZM at 100 g/ml induced apoptosis in anti-CD3/CD28-activated PBLs obtained from normal subjects but not at 10 g/ml. This increase of apoptosis was not induced by other antibiotics, including -lactams, the quinolone, FOM, and 16-membered ring macrolide, JM. Additionally, we previously reported that macrolide antibiotics at a higher concentration of 200 g/ml, but not lower concentrations, induced apoptosis in unstimulated PBLs from normal subjects (Ishimatsu et al. Abstract: The 2000 International Conference of the American Thoracic Society. Am J Respir Crit Care Med 161;A888:2000). These results partially coincided with the findings that RXM at a concentration of 10 g/ml did not significantly affect apoptosis in PBLs stimulated with D. farinae or phytohaemagglutinin from normal subjects or in unstimulated cells [13]. In contrast, RXM at low concentrations (1–500 ng/ml) or 1 g/ml augumented the early phase or both the early and late phases of apoptosis, respectively in D. farinae-stimulated PBLs from asthma patients, while other antibiotics, including cefazolin and ampicillin, did not induce apoptosis [13]. Collectively, these results suggest that 14- or 15-membered ring macrolides are specifically involved in the augmentation of apoptosis and that concentrations of macrolides less than 1 g/ml are sufficient to induce apoptosis when PBLs are activated by a relevant antigen in patients, whereas a higher concentration at 100 or 200 g/ml is necessary to augment apoptosis in PBLs obtained from normal subjects.
By contrast, the enhanced effect of CPFX at 2.5-10 g/ml or RXM at 10–50 g/ml on the apoptosis of anti-CD3activated Jurkat T cells has been demonstrated [14]. This difference regarding the new quinolone between ours and the previous study might be caused by experimental conditions. Jurkat T cells are different in some ways from normal peripheral T cells. Additionally, we used anti-CD3 and -CD28 antibodies to activate PBLs from normal subjects, while Jun et al. used anti-CD3 antibody to activate Jurkat T cells. AntiCD3 antibody is known as an activator and inducer of Fas/Fas ligand-mediated apoptosis of T cells [18], and the interaction of Fas-ligand with Fas induces Fas-mediated killing of lymphocytes [19]. On the other hand, the CD3 and CD28 accessory receptors act synergistically in the activation of Tlymphocytes, including stimulation of cellular proliferation, prevention of apoptosis and maintenance of antigen responsiveness. Their combination is responsible for the integrated delivery of two signals, also known as signals 1 and 2, which induce IL-2 transcription, Bcl-xL expression and suppression of energy-inducing factors. One of the major functional effects of CD28 is Bcl-xL expression [20–23]. The Bcl-2 protein family regulates one of the steps in the conserved apoptosis pathway. Among the members of this family, Bcl2 and Bcl-xL act as inhibitors of apoptosis, whereas Bax and Bad act as promoters [24,25]. In this regard, we speculate that CLR and AZM, but not OFLX, may induce apoptosis of anti CD3/CD28-activated PBLs by influencing the Bcl-2 protein family, although presently this is not clear. In contrast, Jun et al. demonstrated that CPFX- or RXM-induced apoptosis of Jurkat T cells was increased through up-regulation of Fas ligand expression, and that the Bcl-2/Bax ratios were not different between antibiotic-treated and control Jurkat T cells [14]. Furthermore, some differences of mechanism inducing apoptosis of activated Jurkat T cells between CPFX and RXM could exist since the activities of caspase-3 and -8 were increased in the CPFX-treated cells, but only caspase-3
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activity was increased in the RXM-treated cells, which was less than that of CPFX [14]. Considering these facts together, it is certain that macrolide antibiotics could induce apoptosis of lymphocytes, and it could be done by a different intracellular pathway from that of the quinolone. This may indicate the therapeutic importance for chronic airway diseases of asthma and DPB. In conclusion, our preliminary experiments revealed that 14- and 15-membered macrolide antibiotics have the potential to accelerate apoptosis of activated PBLs from normal subjects in vitro. This suggests that patients with chronic airway diseases such as asthma and DPB, may improve clinically through such action by which macrolides induce apoptosis of lymphocytes localized in the affected bronchioles. Further investigations regarding detailed and precise regulatory mechanisms in the cells and clinical relevance at the inflammatory sites are warranted.
Acknowledgment This study was supported in part by a Grant-in-Aid for Scientific Research (C) (13670607) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
References [1] Kudoh S, Azuma A, Yamamoto M, Izumi T, Ando M. Improvement of survival in patients with diffuse panbronchiolitis treated with lowdose erythromycin. Am J Respir Crit Care Med 1998;157:1829–32. [2] Keicho N, Kudoh S, Yotsumoto H, Akagawa KS. Antilymphocytic activity of erythromycin distinct from that of FK506 or cyclosporin A. J Antibiot 1993;46:1406–13. [3] Morikawa K, Oseko F, Morikawa S, Iwamoto K. Immunomodulatory effect of three macrolides, midekamycin acetate, josamycin, clarithromycin, on human T-lymphocyte function in vitro. Antimicrob Agents Chemother 1994;38:2643–7. [4] Keicho N, Kudoh S, Yotsumoto H, Akagawa KS. Erythromycin promotes monocytes to macrophage differentiation. J Antibiot 1994;46:80–9. [5] Takizawa H, Desaki M, Ohtoshi T, et al. Erythromycin modulates IL-8 expression in human bronchial epithelial cells: studies with normal and inflamed airway epithelium. Am J Respir Crit Care Med 1997;156:266–71. [6] Fujii T, Kadota J, Morikawa T, et al. Inhibitory effect of erythromycin on interleukin 8 production by 1 alpha 25hydroxyvitamin D3-stimulated THP-1 cells. Antimicrob Agents Chemother 1996;40:1548–51. [7] Tamaoki J, Isono K, Sakai N, Kanemura T, Konno K. Erythromycin inhibits Cl secretion across canine tracheal epithelial cells. Eur Respir J 1992;5:234–8.
[8] Goswami SK, Kivity S, Marom Z. Erythromycin inhibits respiratory glycoconjugate secretion from human airway in vitro. Am Rev Respir Dis 1990;141:72–8. [9] Kadota J, Sakito O, Kohno S, et al. A mechanism of erythromycin treatment in patients with diffuse panbronchiolitis. Am Rev Respir Dis 1993;147:153–9. [10] Mukae H, Kadota J, Kohno S, et al. Increase in activated CD8+ cells in bronchoalveolar lavage fluid in patients with diffuse panbronchiolitis. Am J Respir Crit Care Med 1995;152:613– 8. [11] Kawakami K, Kadota J, Iida K, et al. Phenotypic characterization of T cells in bronchoalveolar lavage fluid (BALF) and peripheral blood of patients with diffuse panbronchiolitis; the importance of cytotoxic T cells. Clin Exp Immunol 1997;107:410–6. [12] Milik AM, Buechner-Maxwell VA, Sonstein J, et al. Lung lymphocyte elimination by apoptosis in the murine response to intratracheal particulate antigen. J Clin Invest 1997;99:1082–91. [13] Ogawa N, Sugawara Y, Fujiwara Y, Noma T. Roxithromycin promotes lymphocyte apoptosis in Dermatophagoides-sensitive asthma patients. Eur J Pharmacol 2003;474:273–81. [14] Jun YT, Kim HJ, Song MJ, et al. In vitro effects of ciprofloxacin and roxithromycin on apoptosis of Jurkat T lymphocytes. Antimicrob Agents Chemother 2003;47:1161–4. [15] Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis flow cytometric detection of phosphatidyl-serine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995;184:39– 51. [16] Krammer PH. CD95’s deadly mission in the immune system. Nature 2000;407:789–95. [17] Vignola AM, Chanez P, Chiappara G, et al. Evaluation of apoptosis of eosinophils, macrophages, and T lymphocytes in mucosal biopsy specimens of patients with asthma and chronic bronchitis. J Allergy Clin Immunol 1999;103:563–73. [18] Ju ST, Panka DJ, Cui H, et al. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature 1995;373:444–8. [19] Tanaka M, Suda T, Takahashi T, Nagata S. Expression of the functional soluble form of human fas ligand in activated lymphocytes. EMBO J 1995;14:1129–35. [20] Boise LH, Minn AJ, Noel PJ, et al. CD28 costimulation can promote T cell survival by enhancing the expression of bcl-xL. Immunity 1995;3:87–98. [21] Fraser JD, Irving BA, Crabtree GR, Weiss A. Regulation of interleukin-2 gene enhancer activity by the T cell accessory molecule CD28. Science 1991;251:313–6. [22] Noel PJ, Boise LH, Green JM, Thompson CB. CD28 costimulation prevents cell death during primary T cell activation. J Immunol 1996;157:636–42. [23] Schwartz RH. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell 1992;71:1065–8. [24] Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609–19. [25] Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 1996;88:386–401.
Antibiotic-induced apoptosis in human activated peripheral lymphocytes Jun-Ichi Kadota∗ , Syunji Mizunoe, Kenji Kishi, Issei Tokimatsu, Hiroyuki Nagai, Masaru Nasu Division of Pathogenesis and Disease Control, Department of Infectious Diseases, Oita University Faculty of Medicine, 1-1 Hasama, Oita 879-5593, Japan Received 6 July 2004; accepted 6 October 2004
Abstract Long-term administration of macrolide antibiotics reduced the number of lymphocytes in bronchoalveolar lavage fluid in patients with chronic airway inflammatory disease. To evaluate the inflammatory activity of macrolides, their effect on apoptosis of activated lymphocytes isolated from human peripheral blood was compared with that of other antibiotics. Macrolides, including clarithromycin and azithromycin, at a final concentration of 100 g/ml accelerated apoptosis of activated lymphocytes, while other antibiotics such as fosfomycin sodium, -lactams — ceftazidime, piperacillin sodium and biapenem, and a quinolone, ofloxacin, did not cause significant induction of apoptosis. Our results suggest that 14- or 15-membered ring macrolides are specifically involved in the augmentation of apoptosis of activated lymphocytes, and this may be of value therapeutically for chronic airway diseases. © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Macrolide antibiotics; Clarithromycin; Azithromycin; Apoptosis; Lymphocytes
1. Introduction The anti-inflammatory action of macrolide antibiotics has been an area of intensive investigation since Kudoh et al. first reported the effectiveness of low-dose, long-term treatment with erythromycin in chronic lower respiratory tract infections, including diffuse panbronchiolitis (DPB) [1]. The antiinflammatory activity of the drugs have been reported to be the action on lymphocytes [2,3], promoting monocytes to macrophage differentiation [4], modulating IL-8 expression/production [5,6] and inhibiting Cl and glycoconjugate secretion [7,8]; this may account for the clinical effectiveness of erythromycin in the treatment of the disease [1,9]. We have previously described how long-term macrolide antibiotics reduced the number of lymphocytes in bronchoalveolar lavage fluid (BALF) of the patients with DPB [10,11], and that lymphocyte elimination by apoptosis is im∗
Corresponding author. Tel.: +81 97 586 5804; fax: +81 97 549 4245. E-mail address: [email protected] (J.-I. Kadota).
portant to terminate the chronic immune response of the murine lung to intratracheal particulate antigen exposure [12]. In this context, it has been reported that roxithromycin (RXM) augumented apoptosis in Dermatophagoides farinaestimulated peripheral blood lymphocytes (PBLs) from patients with bronchial asthma, while other antibiotics, including cefazolin and ampicillin, did not cause significant induction of apoptosis [13]. Jun et al. also found that ciprofloxacin (CPFX) or RXM induced apoptosis of anti-CD3-activated Jurkat T lymphocytes [14]. Additionally, we demonstrated that macrolides including clarithromycin (CLR), azithromycin (AZM) and josamycin (JM), at higher concentration than 200 g/ml, induced apoptosis on unstimulated PBLs from normal subjects, while other antibiotics such as -lactams, a carbapenem and a quinolone did not (Ishimatsu et al. Abstract: The 2000 International Conference of the American Thoracic Society. Am J Respir Crit Care Med 161;A888:2000). In this study, therefore, we investigated whether macrolides and other antibiotics induce apoptosis of PBLs
0924-8579/$ – see front matter © 2004 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2004.10.009
J.-I. Kadota et al. / International Journal of Antimicrobial Agents 25 (2005) 216–220
activated by anti-CD3/CD28 antibody obtained from normal subjects.
2. Materials and methods 2.1. Agents The antimicrobial agents were gifts from the manufacturers. They included JM (Yamanouchi Pharmaceutical Co., Tokyo, Japan), CLR (Taisho-Toyama Pharmaceuticals Co., Tokyo), AZM (Pfizer Pharmaceuticals Co., Tokyo), fosfomycin sodium (FOM) (Meiji Pharmaceutical Co., Tokyo), the -lactams, ceftazidime (CAZ, Tanabe Pharmaceutical Co., Tokyo), piperacillin sodium (PIPC) (Taisho-Toyama Pharmaceuticals Co., Tokyo) and biapenem (BIPM) (Meiji Pharmaceutical Co., Tokyo), and a new quinolone, ofloxacin (OFLX) (Daiichi Pharmaceutical Co., Tokyo). 2.2. In vitro culture and stimulation of lymphocytes Venous blood was obtained from healthy adult volunteers and was collected in heparinized tubes. Lymphocytes were isolated by Ficoll-Hypaque and Percoll gradient centrifugation under sterile procedures. Lymphocytes (5 × 105 /well) in culture medium (RPMI 1640) supplemented with 10% foetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM l-glutamine (all from Sigma, St. Louis, MO, USA), were cultured in a 96-well flat bottom microtitre plate (Nunc, Roskilde, Denmark) in the presence of anti-CD3 monoclonal antibody (mAb) at 100 ng/ml (Immunotech, Marseilles Cedex, France) and anti-CD28 mAb at 100 ng/ml (Immunotech) with IL-2 at 50 U/ml (Sigma) for 24 h at 37 ◦ C in a humidified 5% CO2 atmosphere. Thereafter, the drugs were added at a concentration of 10 or 100 g/ml at 37 ◦ C for 48 h. Macrolide antibiotics were dissolved in dimethyl sulfoxide (DMSO: final concentration, < 0.1%), and other antibiotics were prepared in sterilized water. Culture medium with DMSO (0.1%) was used as a control.
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2.4. Statistical analysis All data are expressed as mean ± standard error of the mean (S.E.M.). Statistical comparisons among groups were made using analysis of variance with Tukey–Kramer post hoc analysis by the StatView 5.0 statistical package. A P-value of <0.05 was considered significant.
3. Results Fig. 1 shows representative results of flow cytometry for the apoptosis of anti-CD3 and anti-CD28 antibody-activated lymphocytes after incubation in the presence or absence of various antibiotics at 100 g/ml for 48 h. The lower left quadrant of the histogram shows the viable cells, which exclude PI and are negative for FITC-Annexin-V binding. The lower right compartment represent the early apoptotic cells, which are PI negative and Annexin-V positive, indicating the translocation of phophatidylserine to the external cell surface but the integrity of the cytoplasmic membrane [15]. The upper right quadrant represents the nonviable necrotic and late-stage apoptotic cells, which are positive for Annexin-V binding and PI uptake. In this example, the percentages of Annexin-V-positive cells were 14.6% (control), 10.5% (FOM), 10.5% (BIPM), 15.4% (OFLX), 13.3% (PIPC), 12.6% (CAZ), 7.1% (JM), 23.8% (CLR) and 38.8% (AZM). The same experiments were performed 10 times for the control group, three times for non-macrolide antibiotics or four times for macrolide antibiotics, and the same results were obtained. Neither of the antibiotics at a concentration of 10 g/ml induced apoptosis (Fig. 2), while CLR and AZM at 100 g/ml, but not JM, significantly enhanced apoptosis compared with the control group or other antibiotic-treated groups (31.7 ± 1.3% and 46.4 ± 2.9% versus 21.1 ± 1.2% for control, 17.1 ± 0.5% for FOM, 17.7 ± 1.1% for BIPM, 21.3 ± 3.3% for OFLX, 19.3 ± 1.3% for PIPC, 17.8 ± 1.3% for CAZ, and 23.8 ± 1.0% for JM, respectively; P < 0.05, Fig. 2). In addition, the apoptosis of activated lymphocytes was more dramatically increased in the AZM-treated cells than in CLR-treated cells (P < 0.05).
2.3. Measurement of apoptosis Induction of apoptosis was measured after 48 h using an apoptosis detection kit (Trevigen Inc., Gaithersburg, MD) with fluorescein isothiocyanate (FITC)-conjugated AnnexinV, which is an early apoptosis marker on flow cytometry (FACS Calibur version 1.0 cell sorter, Becton Dickinson, San Jose, CA). Briefly, cells (5 × 105 /well) were treated with FITC-conjugated Annexin-V mAb and propidium iodide (PI) in Annexin-V binding buffer (10 mM Hepes, 0.15 M NaCl, 5 mM KCl, 1 mM MgCl2 , and 1.8 mM CaCl2 , pH 7.4) for 15 min in the dark. Cells were then analyzed by flow cytometry using the FL1 channel for detecting Annexin-VFITC staining and the FL-2 channel for detecting PI staining. Annexin-V-positive cells were defined as apoptotic cells.
4. Discussion Apoptosis is critical for normal development and tissue homeostasis, including that of the immune system [16]. There is increasing evidence that dysregulation of apoptotic pathways are associated with airway disease, including bronchial asthma. Most of the T-lymphocytes infiltrating the airway of asthmatics are not apoptotic, suggesting that the persistence of airway inflammation may depend upon their continuing proliferation and their increased survival in the bronchial mucosa [17]. It was previously reported that the anti-inflammatory effects of macrolide antibiotics against lymphocytes included inhibition of the mitogen-mediated
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Fig. 1. Analysis of apoptosis induced by various antibiotics in stimulated lymphocytes. After pretreatment with anti-CD3/CD28 mAb and IL-2 for 24 h, cells were incubated in the absence (A) or in the presence of fosfomycin sodium (FOM) (B), biapenem (BIPM) (C), ofloxacin (OFLX) (D), piperacillin sodium (PIPC) (E), ceftazidime (CAZ) (F), josamycin (JM) (G), clarithromycin (CLR) (H) or azithromycin (AZM) (I) at 100 g/ml. After 48 h, induction of apoptosis was analyzed by flow cytometry using Annexin-V/PI staining. The percentage in the lower right compartment and upper right quadrant shows the percentage of Annexin-V-positive cells. Representative data from several separate experiments are shown.
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Fig. 2. Effects of various antibiotics on apoptosis of stimulated lymphocytes in vitro. After pretreatment with anti-CD3/CD28 mAb and IL-2 for 24 h, stimulated cells were incubated with or without antibiotics at 10 (open column) or 100 (closed column) g/ml. After 48 h, induction of apoptosis was measured using flow cytometry. Data are mean ± S.E.M. of proportions of cells positive for Annexin-V determined in three or four independent experiments. C: control; FOM: fosfomycin sodium; BIPM: biapenem; OFLX: ofloxacin; PIPC: piperacillin sodium; CAZ: ceftazidime; JM: josamycin; CLR: clarithromycin; AZM: azithromycin.
lymphocyte proliferative response and reduction of activated lymphocytes in BAL fluid of DPB patients after treatment with macrolide antibiotics [2,10,11]. In addition, RXM augumented apoptosis in D. farinae-stimulated PBLs from asthma patients [13] and CPFX or RXM induced apoptosis of antiCD3-activated Jurkat T lymphocytes [14]. Thus, in this study, the immunomodulatory effect of macrolides and other antibiotics was focused on lymphocyte apoptosis so as to compare our results with past published data. Our present study showed that CLR and AZM at 100 g/ml induced apoptosis in anti-CD3/CD28-activated PBLs obtained from normal subjects but not at 10 g/ml. This increase of apoptosis was not induced by other antibiotics, including -lactams, the quinolone, FOM, and 16-membered ring macrolide, JM. Additionally, we previously reported that macrolide antibiotics at a higher concentration of 200 g/ml, but not lower concentrations, induced apoptosis in unstimulated PBLs from normal subjects (Ishimatsu et al. Abstract: The 2000 International Conference of the American Thoracic Society. Am J Respir Crit Care Med 161;A888:2000). These results partially coincided with the findings that RXM at a concentration of 10 g/ml did not significantly affect apoptosis in PBLs stimulated with D. farinae or phytohaemagglutinin from normal subjects or in unstimulated cells [13]. In contrast, RXM at low concentrations (1–500 ng/ml) or 1 g/ml augumented the early phase or both the early and late phases of apoptosis, respectively in D. farinae-stimulated PBLs from asthma patients, while other antibiotics, including cefazolin and ampicillin, did not induce apoptosis [13]. Collectively, these results suggest that 14- or 15-membered ring macrolides are specifically involved in the augmentation of apoptosis and that concentrations of macrolides less than 1 g/ml are sufficient to induce apoptosis when PBLs are activated by a relevant antigen in patients, whereas a higher concentration at 100 or 200 g/ml is necessary to augment apoptosis in PBLs obtained from normal subjects.
By contrast, the enhanced effect of CPFX at 2.5-10 g/ml or RXM at 10–50 g/ml on the apoptosis of anti-CD3activated Jurkat T cells has been demonstrated [14]. This difference regarding the new quinolone between ours and the previous study might be caused by experimental conditions. Jurkat T cells are different in some ways from normal peripheral T cells. Additionally, we used anti-CD3 and -CD28 antibodies to activate PBLs from normal subjects, while Jun et al. used anti-CD3 antibody to activate Jurkat T cells. AntiCD3 antibody is known as an activator and inducer of Fas/Fas ligand-mediated apoptosis of T cells [18], and the interaction of Fas-ligand with Fas induces Fas-mediated killing of lymphocytes [19]. On the other hand, the CD3 and CD28 accessory receptors act synergistically in the activation of Tlymphocytes, including stimulation of cellular proliferation, prevention of apoptosis and maintenance of antigen responsiveness. Their combination is responsible for the integrated delivery of two signals, also known as signals 1 and 2, which induce IL-2 transcription, Bcl-xL expression and suppression of energy-inducing factors. One of the major functional effects of CD28 is Bcl-xL expression [20–23]. The Bcl-2 protein family regulates one of the steps in the conserved apoptosis pathway. Among the members of this family, Bcl2 and Bcl-xL act as inhibitors of apoptosis, whereas Bax and Bad act as promoters [24,25]. In this regard, we speculate that CLR and AZM, but not OFLX, may induce apoptosis of anti CD3/CD28-activated PBLs by influencing the Bcl-2 protein family, although presently this is not clear. In contrast, Jun et al. demonstrated that CPFX- or RXM-induced apoptosis of Jurkat T cells was increased through up-regulation of Fas ligand expression, and that the Bcl-2/Bax ratios were not different between antibiotic-treated and control Jurkat T cells [14]. Furthermore, some differences of mechanism inducing apoptosis of activated Jurkat T cells between CPFX and RXM could exist since the activities of caspase-3 and -8 were increased in the CPFX-treated cells, but only caspase-3
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activity was increased in the RXM-treated cells, which was less than that of CPFX [14]. Considering these facts together, it is certain that macrolide antibiotics could induce apoptosis of lymphocytes, and it could be done by a different intracellular pathway from that of the quinolone. This may indicate the therapeutic importance for chronic airway diseases of asthma and DPB. In conclusion, our preliminary experiments revealed that 14- and 15-membered macrolide antibiotics have the potential to accelerate apoptosis of activated PBLs from normal subjects in vitro. This suggests that patients with chronic airway diseases such as asthma and DPB, may improve clinically through such action by which macrolides induce apoptosis of lymphocytes localized in the affected bronchioles. Further investigations regarding detailed and precise regulatory mechanisms in the cells and clinical relevance at the inflammatory sites are warranted.
Acknowledgment This study was supported in part by a Grant-in-Aid for Scientific Research (C) (13670607) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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