a First Department of Internal Medicine, Gunma University School of Medicine, 3-39-22, Showa, Maebashi, Gunma, 371-8511; b Department of Endocrinology, Dokkyo University School of Medicine, Mibu, Japan
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Abstract |
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Introduction |
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The bactericidal action of neutrophils consists of oxygen-dependent and -independent processes. In the oxygen-dependent bactericidal process, the importance of several oxygen-derived free radicals (e.g. superoxide anion [O2·-], hydrogen peroxide [H2O2], and hypochlorite [OCL-]) has been identified.4 These oxygen-derived free radicals are produced during oxidative metabolism.
There has been increased interest in recent years in the relationship between antibiotics and the immune response. Several studies have shown that various antibacterial agents (e.g. benzylpenicillin, cephalothin, cefuroxime, cefotaxime, gentamicin, erythromycin and clindamycin) can inhibit neutrophil functions at therapeutic plasma concentrations.5,6 On the other hand, other antibacterial agents (e.g. tetracyclines, trimethoprim, sulphamethoxazole and cephalothin) have been reported to enhance neutrophil function.7 Thus, it is important to investigate the influence of new antimicrobial agents on neutrophil function as part of a full evaluation of the drug.
Faropenem is a novel oral penem antimicrobial agent. Faropenem, which shares structural
similarities with both penicillins and cephalosporins, exhibits a broad-spectrum activity against
Gram-negative, Gram-positive and certain anaerobic bacteria and has been shown to be highly
stable against a number of ß-lactamases.8 To our
knowledge, the effect of faropenem on neutrophil function has not been evaluated previously.
Therefore, the aim of the present study was to investigate the effect of faropenem on the killing
function of human neutrophils in vitro by measuring neutrophil production of O2·-. To measure the ability of neutrophils to generate O2·-, we used Cypridina luciferin analogue (CLA;
2-methyl-6-phenyl-3,7-dihydroimidazo [1,2-]-pyrazin-3-one) as an agent to amplify
chemiluminescence. Cypridina luciferin analogue-dependent chemiluminescence
(CLA-DCL) appears to be highly dependent on O2·-
generation.9
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Materials and methods |
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Faropenem was kindly supplied by Yamanouchi Pharmaceutical Co. (Tokyo, Japan). N-formyl-Met-Leu-Phe (fMLP), 12-O-tetra-decanoylphorbolmyristate acetate (TPA) and ionomycin were obtained from Sigma Chemical Co. (St Louis, MO, USA). CLA was obtained from Tokyo Kasei Kogyo (Tokyo, Japan). Fura-2/AM was obtained from Wako Pure Chemical Industries (Hyogo, Japan).
Isolation of neutrophils
Human neutrophils were isolated from the peripheral blood of healthy volunteers. Whole blood was collected into a heparinized syringe and neutrophils were prepared using 6% (w/v) Dextran and hypotonic methods, as described previously.10 About 90 to 95% of the cells obtained were neutrophils. Cells were then suspended in Hanks' balanced salt solution (HBSS; Nissui Pharmaceutical Co., Tokyo, Japan) at a density of 1 x 106 neutrophils/mL.
Measurement of superoxide anion generation by neutrophils
The concentration of generated O2·- was measured by a Luminescence Reader (Model BLP 102; Aloka, Inc., Tokyo, Japan), as described previously.9 The standard reaction mixture for chemiluminescence assay contained 5 x 104 cells, a stimulator (100 nM fMLP, 100 nM TPA or 2.5 µM ionomycin) and 2.5 µM CLA in HBSS, in a total volume of 2 mL. All components, except the stimulator, were first preincubated for 1 min and the reaction was initiated later by the addition of the stimulator. For the pharmacokinetic studies, cells were preincubated with faropenem for 0 to 60 min followed by measurement of chemiluminescence at predetermined intervals. CLA-DCL was assessed with the maximally changed value of chemiluminescence in kilocounts (KC)/min/106 cells.
Measurement of cytosol Ca2+ concentration
Changes in intracellular Ca2+ concentrations [Ca2+]i were measured by dual excitation microfluorimetry (model F-2000 fluorescence spectrometer, Hitachi, Tokyo, Japan) after loading neutrophils with Fura-2 by exposure to 2 µM Fura-2/AM in HBSS for 15 min at room temperature. The disposable cuvettes of the fluorimeter containing neutrophils with or without faropenem were placed in a thermostatically controlled cuvette holder maintained at 37°C. [Ca2+]i was calculated from the ratio (R = F340/F380) of fluorescence.11
Statistical analysis
Cell viability was measured by trypan blue staining and confirmed to be >98% at both the beginning and end of the experiments. All data were expressed as mean ± S.E.M. Differences between two groups were examined for statistical significance using the Student's t-test or paired t-test, as indicated. Correlations were examined by simple linear regression analysis. A P value <0.05 denoted the presence of a statistically significant difference.
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Results |
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Simple linear regression analysis showed that log doses of faropenem significantly correlated with CLA-DCL (r2 = 0.65, P < 0.0001). Faropenem at 10 and 100 mg/L significantly enhanced CLA-DCL in the presence of fMLP (Figure 1, P < 0.05).
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Five minute preincubation with 10 mg/L faropenem significantly enhanced CLA-DCL by 144% (Figure 2). However, the enhanced effect gradually decreased and became similar to the initial level at 60 min. The enhanced effect of faropenem on CLA-DCL was statistically significant at 5, 15 (P < 0.01) and 30 (P < 0.05) min.
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We also examined the effect of faropenem in the presence of other stimulators. At 10 mg/L, faropenem did not enhance CLA-DCL in the presence of TPA (Figure 3a), while 10 mg/L of faropenem significantly enhanced CLA-DCL in the presence of ionomycin (P < 0.01, Figure 3b).
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Preincubation of neutrophils with 10 mg/L faropenem did not significantly modify the first and second peak responses of [Ca2+]i (Figure 4).
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Discussion |
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Agonist-induced transmembrane signalling in neutrophils consists of a complex series of reactions involving two interrelated pathways: changes in [Ca2+]i and activation of protein kinase C. After binding of fMLP to the surface receptor, phosphatidylinositol 4,5-bisphosphate is hydrolysed by phospholipase C and phosphatidylcholine by phospholipase D into two putative intracellular messengers, diacylglycerol, and in the case of phospholipase C, inositol 1,4,5-trisphosphate [(Ins(1,4,5)P3]. Ins(1,4,5)P3 mediates the release of calcium from intracellular stores, whereas diacylglycerol is considered the endogenous activator of protein kinase C.17 Although the exact mechanism has not yet been identified, activation of superoxide ion production presumably results from subsequent calcium ion- or protein kinase C-dependent phosphorylation or both.18 Examination of the possible mechanisms of action in the present study showed that the enhanced effect of faropenem was noted in the presence of fMLP or ionomycin but not TPA. Furthermore, [Ca2+]i did not change following the addition of faropenem. Combined together, our results suggest that faropenem might enhance NADPH oxidase at the site where Ca2+ regulates NADPH oxidase.
Previous studies have explored the mechanisms that improve the response to antibiotic therapy. These studies demonstrated that the ability of certain antibiotics to be concentrated within neutrophils19 and inhibition of intraphagocytic bacterial growth in low intraphagolysosomal pH20 are effective bactericidal mechanisms that improve the response to therapy. In this regard, the entry of antibiotics into neutrophils is dependent on their solubility in lipid membrane. Since faropenem has low lipid solubility, its entry into neutrophils is probably difficult as suggested earlier by Braga et al.21 and therefore faropenem is less likely to change intraphagolysosomal pH. However, active transport mechanisms might be involved in the entry of faropenem into neutrophils. Other mechanisms may also influence the effects of antibiotics. For example, Hoeben et al.22 reported that enrofloxacin might potentiate the action of phorbol 12-myristate 13-acetate (PMA) by increasing [Ca2+]i in bovine milk polymorphonuclear leucocytes. This mechanism is different from the results of our study and may represent differences in the action of antibiotics or species differences. On the other hand, Ohnishi et al.23 showed that AC-1370 (cefpimizole) enhanced macrophage functions and that heat-resistant and trypsin-sensitive soluble factors released by cefpimizole-bound macrophages enhanced neutrophil functions.
Phagocytosis represents an important function of neutrophils in host defence responses against microbes. Defective host defence responses often result in serious infections, particularly in immunocompromised patients, e.g. patients with malignancies or those treated with glucocorticoids or immunosuppressants.24 In this regard, potentiators of host defence reactions are effective in the treatment of microbial infection when used in conjunction with antimicrobial chemotherapy. Thus, antibiotics that can potentiate phagocyte functions may be more effective in the treatment of microbial infections than others. Further studies, including measurement of cytosol pH, are needed to extend our findings in vitro into clinical conditions in vivo and to assess the effect of faropenem treatment on infectious disease.
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Acknowledgments |
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Notes |
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References |
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2 . Babior, B. M. (1978). Oxygen-dependent microbial killing by phagocytes. New England Journal of Medicine 298, 65968, 7215.[ISI][Medline]
3 . Curnutte, J. T. & Babior, B. M. (1987). Chronic granulomatous disease. Advances in Human Genetics 16, 22997.[ISI][Medline]
4 . Sato, N., Kashima, K., Tanaka, Y., Shimizu, H. & Mori, M. (1997). Effect of granulocyte-colony stimulating factor on generation of oxygen-derived free radicals and myeloperoxidase activity in neutrophils from poorly controlled NIDDM patients. Diabetes 46, 1337.[Abstract]
5 . Pierce, L. A., Tarnow-Mordi, W. O. & Cree, I. A. (1995). Antibiotic effects on phagocyte chemiluminescence in vitro. International Journal of Clinical and Laboratory Research 25, 938.[Medline]
6 . Hand, W. L. & Hand, D. L. (1995). Influence of pentoxifylline and its derivatives on antibiotic uptake and superoxide generation by human phagocytic cells. Antimicrobial Agents and Chemotherapy 39, 15749.[Abstract]
7 . Venezio, F. R. & DiVincenzo, C. A. (1985). Effects of aminoglycoside antibiotics on polymorphonuclear leukocyte function in vivo. Antimicrobial Agents and Chemotherapy 27, 7124.[ISI][Medline]
8 . Woodcock, J. M., Andrews, J. M., Brenwald, N. P., Ashby, J. P. & Wise, R. (1997). The in-vitro activity of faropenem, a novel oral penem. Journal of Antimicrobial Chemotherapy 39, 3543.[Abstract]
9
.
Sugioka, K., Nakano, M., Kurashige, S., Akuzawa, Y.
& Goto, T. (1986). A chemiluminescent probe with a Cypridina
luciferin analog, 2-methyl-6-phenyl-3,7-dihydroimidazol[1,2-]-pyrazin-3-one, specific and
sensitive for O2- production in phagocytizing macrophages. FEBS Letters 197, 2730.[ISI][Medline]
10 . Sato, N. & Shimizu, H. (1993). Granulocyte-colony stimulating factor improves an impaired bactericidal function in neutrophils from STZ-induced diabetic rats. Diabetes 42, 4703.[Abstract]
11 . Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 344050.[Abstract]
12 . Nakashima, M., Uematsu, T., Yoshinaga, K., Sueyoshi, T., Kikuchi, Y., Hirabayashi T. et al. (1993). Phase I clinical study on a new oral penem antibiotic, SY5555. Chemotherapy 41, 127792.
13 . Hoeben, D., Dosogne, H., Heyneman, R. & Burvenich, C. (1997). Effect of antibiotics on the phagocytotic and respiratory burst activity of bovine granulocytes. European Journal of Pharmacology 332, 28997.[ISI][Medline]
14 . Boogaerts, M. A., Malbrain, S., Scheers, W. & Verwilghen, R. L. (1986). Effects of quinolones on granulocyte function in vitro. Infection 14, Suppl. 4, S25862.[ISI][Medline]
15 . Ueta, E., Yoneda, K., Yamamoto, T. & Osaki, T. (1992). Upregulatory effects of cefpimizole natrium on human leukocytes. International Journal of Immunopharmaceutics 14, 87785.
16 . Labro, M. T., Babin-Chevaye, C. & Hakim, J. (1986). Effects of cefotaxime and cefodizime on human granulocyte functions in vitro. Journal of Antimicrobial Chemotherapy 18, 2337.[Abstract]
17 . Nishizuka, Y. (1986). Studies and perspectives of protein kinase C. Science 233, 30512.[ISI][Medline]
18 . Pozzan, T., Lew, D. P., Wollheim, C. B. & Tsien, R. Y. (1983). Is cytosolic ionized calcium regulating neutrophil activation? Science 221, 14135.[ISI][Medline]
19 . Mandell, G. L. (1973). Interaction of intraleukocytic bacteria and antibiotics. Journal of Clinical Investigation 52, 16739.[ISI][Medline]
20 . Lam, C. & Mathison, G. E. (1983). Effect of low intraphagolysosomal pH on antimicrobial activity of antibiotics against ingested staphylococci. Journal of Medical Microbiology 16, 30916.[Abstract]
21 . Braga, P. C., Dal-Sasso, M., Maci, S., Allegra, L., Fonti, E., Ghessi, A. et al. (1995). Influence of brodimoprim on polymorphonuclear leukocyte phagocytosis and oxidant radical production. Chemotherapy 41, 3607.[ISI][Medline]
22 . Hoeben, D., Burvenich, C. & Heyneman, R. (1997). Influence of antimicrobial agents on bactericidal activity of bovine milk polymorphonuclear leukocytes. Veterinary Immunology and Immunopathology 56, 27182.[ISI][Medline]
23 . Ohnishi, H., Inaba H., Mochizuki H. & Kosuzume H. (1984). Mechanism of action of AC-1370 on phagocyte functions. Antimicrobial Agents and Chemotherapy 25,88 92.[ISI][Medline]
24 . Klainer, A. S. & Beisel, W. R. (1969). Opportunistic infection: a review. American Journal of the Medical Sciences 258, 43156.[ISI][Medline]
Received 3 December 1998; returned 11 March 1999; revised 7 April 1999; accepted 21 April 1999