Modulation of oxidative burst of neutrophils by doxycycline in patients with acute myocardial infarction

Satoshi Takeshitaa,*, Yasuo Onob, Ken Kozumaa, Masatoshi Suzukia, Yutaka Kawamuraa, Naoyuki Yokoyamaa, Taiji Furukawaa and Takaaki Isshikia

a Departments of Medicine and b Microbiology and Immunology, Teikyo University School of Medicine, Tokyo, Japan


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
We investigated to what extent doxycycline hydrochloride (DOXY) can modify polymorphonuclear leucocyte (PMN) activity in patients with acute myocardial infarction (AMI). PMN activity was measured in blood samples obtained at 0, 15, 30 and 60 min after DOXY administration. In patients receiving saline, PMN activity did not change over 60 min. In contrast, patients receiving DOXY showed a >=10% reduction in PMN activity at 30 and 60 min. These data support the use of DOXY to reduce myocardial cell damage by PMNs in patients with AMI.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In acute myocardial infarction (AMI), activated polymorphonuclear leucocytes (PMNs) generate reactive oxygen species (ROS) in the coronary circulation, which result in myocardial cell damage.1 We reported previously that certain classes of antibiotic agents such as tetracyclines and trimethoprim–sulfamethoxazole suppress PMN activity in normal subjects.2 Whether these agents similarly reduce PMN activity in patients with AMI has not yet been determined. We investigated to what extent PMN activity in AMI patients could be modified by a single intravenous administration of doxycycline hydrochloride (DOXY).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The study protocol was approved by the institutional ethics committee. Written informed consent was obtained from all patients before enrolment.

Study patients

The in vitro study was undertaken to determine the optimal dose of DOXY administration to suppress PMN activity in the ex vivo study (see below). Owing to the limited availability of blood samples from AMI patients for the in vitro study, we used blood samples obtained from 10 patients with coronary artery disease including angina pectoris and previous myocardial infarction (seven males; mean age 67 ± 8 years, range 57–80 years). The freshly drawn sample (total volume 0.1 mL) containing 10 U/mL of heparin was diluted with 0.8 mL of Hank':s balanced salt solution (Gibco-BRL, Gaithersburg, MD, USA). The sample was then mixed with 0.1 mL of 0.9% saline containing various concentrations of DOXY, and assayed for PMN activity as described previously.3,4

For the ex vivo study, blood samples were obtained from 22 patients with AMI. In all 22 patients, direct percutaneous coronary intervention (PCI) was carried out, and procedural success, defined as residual stenosis of <50% without precipitating a major cardiac event, was achieved. Within 30 min after the PCI, intravenous administration of DOXY (100 mg) dissolved in 0.9% saline was carried out for 5 min (total volume of 20 mL; n = 10). The dose of 100 mg of DOXY was chosen because this is the recommended dose for clinical usage, and when 100 mg of DOXY is administered, a maximum blood concentration of 15–20 mg/L can be expected, which is within the range that achieves significant reduction of PMN activity in vitro (see Results). Blood samples were obtained at 0, 15, 30 and 60 min after DOXY administration, heparinized, and then diluted as described above. In the control patients (n = 12), saline was administered instead of DOXY.

Measurements of PMN activity

The generation of ROS by PMNs is accompanied by a chemiluminescence (CL) response. Such a CL response can be used as a marker of PMN activation in a variety of pathological conditions including coronary artery diseases.3,4 The CL assay was carried out as described previously.3,4 The CL was monitored for 20 min, and the integrated CL count was calculated. PMN activity was expressed as the percentage of integrated CL count at each concentration (in vitro study) or time point (ex vivo study) divided by the integrated CL count at baseline.

Statistical analyses

All measurements were carried out in duplicate. Continuous variables were expressed as mean ± s.e. Statistical significance was assessed by the unpaired Student':s t-test for comparison of two means, and by ANOVA followed by Scheffe':s procedure for repeated measurements, using the StatView application (SAS Institute Inc., Cary, NC, USA). A value of P < 0.05 was considered to be statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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In vitro study

At a concentration of >=10 mg/L, DOXY induced a significant reduction of PMN activity compared with the baseline level (10 mg/L: 89.2 ± 2.8%, P < 0.005; 15 mg/L: 82.4 ± 2.3%, P < 0.0001; 20 mg/L: 66.0 ± 2.5%, P < 0.0001). Higher concentrations of DOXY were not tested since such range is beyond clinical usage (Figure, aGo).



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Figure. Effect of DOXY on PMN activity. (a) In the in vitro study, DOXY induced a dose-dependent reduction in the activity of PMNs that had been obtained from patients with ischaemic heart disease. *P < 0.005, **P < 0.0001 versus baseline (n = 10 at each point). (b) DOXY (100 mg) or saline was intravenously administered to patients with acute myocardial infarction intravenously. In the control group who received saline (n = 12), PMN activity did not change over 60 min. In the DOXY-treated group (n = 10), significant reductions in PMN activity were observed at 30 and 60 min after DOXY administration (n = 10). *P < 0.05 versus baseline. •, DOXY; {circ}, control.

 
Ex vivo study

The clinical characteristics of the 22 patients with AMI are summarized in the TableGo. In the control group, PMN activity did not significantly change after saline administration compared with the baseline level (15 min: 106.8 ± 7.4%; 30 min: 112.7 ± 8.7%; 60 min: 113.1 ± 8.3%). In contrast, the patients treated with DOXY showed significant reduction in PMN activity at 30 and 60 min (15 min: 91.8 ± 2.9%, P = NS; 30 min: 87.4 ± 2.7%, P < 0.005; 60 min: 90.0 ± 3.8%, P < 0.05). The PMN activity of the control and DOXY groups differed significantly at 30 and 60 min (P < 0.05 for both) (Figure, bGo).


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Table. Characteristics of the AMI patients
 

    Discussion
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 Abstract
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 Materials and methods
 Results
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The in vitro study demonstrated that at concentrations of 10–20 mg/L, DOXY suppressed PMN activity by 11–34%. This is consistent with our previous observations that DOXY inhibited PMN activity in vitro in blood samples from healthy volunteers at a concentration of 12.5–25 mg/L.2 The in vitro effect of this range of concentration is proportional to that achieved when therapeutic doses of DOXY are administered to patients. In fact, the ex vivo study confirmed that administration of 100 mg DOXY resulted in >=10% reduction of PMN activity. Although the precise mechanism of the inhibitory effect of DOXY on PMN activity was not clarified in the present study, other investigators have suggested that it may be based on the intracellular penetration ability and the divalent cation (Ca2+, Mg2+) chelating ability of tetracyclines.5

The routine use of tetracyclines such as DOXY and minocycline hydrochloride in patients with coronary artery disease may also have an advantage over other classes of antibiotics in terms of management of Chlamydia pneumoniae infection.6 For example, Gupta et al.7 reported that a short course of azithromycin (3 or 6 days) significantly lowered the incidence of adverse cardiovascular events following AMI. Similarly, Gurfinkel et al.8 reported that 30 day treatment with roxithromycin (RXM) improved the clinical outcome of patients with acute non-Q-wave coronary syndrome. It must be noted that RXM directly inhibited oxidant production by PMNs in vitro.9 Whether similar beneficial effects on clinical outcome can be expected with DOXY remains to be determined.

In summary, the present study demonstrated that suppression of PMN activity could be achieved by a single intravenous administration of DOXY in patients with AMI. DOXY may thus serve as the first-line antibiotic regimen for patients with AMI. Future studies need to clarify whether the use of DOXY improves the clinical outcome of these patients.


    Notes
 
* Correspondence address. Department of Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. Tel: +81-3-3964-1211; Fax: +81-3-5375-1308; E-mail: stake{at}blue.ocn.ne.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Vandeplassche, G., Bernier, M., Thone, F., Borgers, M., Kusama, Y. & Hearse, D. J. (1990). Singlet oxygen and myocardial injury: ultrastructural, cytochemical and electrocardiographic consequences of photoactivation of rose bengal. Journal of Molecular and Cellular Cardiology 22, 287–301.[ISI][Medline]

2 . Ono, Y. & Kunii, O. (1989). Influence of forty-two antimicrobial agents on the chemiluminescence response of human phagocytic cells. Chemotherapy 37, 583–9.

3 . Takeshita, S., Isshiki, T., Ochiai, M., Ishikawa, T., Nishiyma, Y., Fusano, T. et al. (1997). Systemic inflammatory responses in acute coronary syndrome: increased activity observed in polymorphonuclear leukocytes but not T lymphocytes. Atherosclerosis 135, 187–92.[ISI][Medline]

4 . Takeshita, S., Hashimoto, H., Ono, Y., Ochiai, M., Yokoyama, N., Terakura, M. et al. (2001). Increased leukocyte activity as a predictor for flow-limiting coronary lesions in patients with angina pectoris. Atherosclerosis 158, 477–81.[ISI][Medline]

5 . Siegel, J. P. & Remington, J. S. (1982). Effect of antimicrobial agents on chemiluminescence of human polymorphonuclear leukocytes in response to phagocytosis. Journal of Antimicrobial Chemotherapy 10, 505–15.[ISI][Medline]

6 . Danesh, J., Collins, R. & Peto, R. (1997). Chronic infections and coronary heart disease: is there a link? Lancet 350, 430–6.[ISI][Medline]

7 . Gupta, S., Leatham, E. W., Carrington, D., Mendall, M. A., Kaski, J. C. & Camm, A. J. (1997). Elevated Chlamydia pneumoniae antibodies, cardiovascular events, and azithromycin in male survivors of myocardial infarction. Circulation 96, 404–7.[Abstract/Free Full Text]

8 . Gurfinkel, E., Bozovich, G., Beck, E., Testa, E., Livellara, B. & Maunter, B. (1999). Treatment with antibiotic roxithromycin in patients with acute non-Q-wave coronary syndromes: the final report of ROXIS study. European Heart Journal 20, 121–7.[Abstract/Free Full Text]

9 . Labro, M. T., el Benna, J. & Babin-Chevaye, C. (1989). Comparison of the in-vitro effect of several macrolides on the oxidative burst of human neutrophils. Journal of Antimicrobial Chemotherapy 24, 561–72.[Abstract]

Received 28 August 2001; returned 18 October 2001; revised 20 November 2001; accepted 22 November 2001





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