Journal of Antimicrobial Chemotherapy (2000) 46, 91-94
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Effect of grepafloxacin on cytokine production in vitro
Cellular Technology Institute, Otsuka Pharmaceutical Co. Ltd, 463-10 Kagasuno, Kawauchi-cho, Tokushima City, Japan
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Abstract |
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The effect of a new quinolone antibacterial agent, grepafloxacin, on the production of cytokines was investigated using lipopolysaccharide-stimulated human peripheral blood cells. Grepafloxacin 1–30 mg/L inhibited the production of interleukin 1
(IL-1) and IL-1ß, and the expression of IL-1, IL-1ß, tumour necrosis factor (TNF), IL-6 and IL-8 mRNA. These results suggest that the inhibitory effect of grepafloxacin is exerted, in part, at the gene transcription level. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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It has been reported that quinolone antibacterial agents can either promote or inhibit the production of cytokines.1–3 In particular, inflammatory cytokines, represented by interleukin 1 (IL-1) and tumour necrosis factor
(TNF) are thought to be involved in various inflammatory processes associated with infection. In this study we evaluated the effect of a new quinolone antibacterial agent, grepafloxacin,4 on the production of inflammatory cytokines using human peripheral blood cells. |
Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Test drugs
Grepafloxacin was manufactured by Otsuka Pharmaceutical Co. Ltd (Tokushima, Japan). Ciprofloxacin and ofloxacin were extracted from Ciproxan (Bayer, Leverkusen, Germany) and Tarivid (Daiichi Pharmaceutical Co. Ltd, Tokyo, Japan), respectively.
Human blood
Human blood was collected from healthy volunteers. Written consent was obtained in all cases.
Reagents
Lipopolysaccharide (LPS) from Escherichia coli O55:B5 was purchased from Difco Laboratories (Detroit, MI, USA), concanavalin A (Con-A) from Vector Inc. (Burlingame, CA, USA), and glyoxal (40% solution) and dimethylsulphoxide (DMSO) from Wako Pure Chemical Industries (Osaka, Japan). The culture medium used for whole blood culture was RPMI-1640 (Nissui Pharmaceutical Co., Tokyo, Japan) with penicillin 100 U/mL (Meiji Seika Ltd, Tokyo, Japan) and streptomycin 0.1 mg/L (Meiji Seika Ltd). For mononuclear cell culture, 10% heat-inactivated fetal bovine serum (FBS, Flow Laboratories, North Ryde, New South Wales, Australia) was added to the whole blood culture medium.
Whole blood culture and production of cytokines
Human peripheral whole blood (10%), a test compound and LPS (1 mg/L) were added to the RPMI-1640 medium and incubated at 37°C in 5% CO2 overnight. The supernatant was collected for IL-1 and IL-1ß measurement. Similarly, the sample for IL-2 measurement was prepared by adding Con-A (10 mg/L) in place of LPS, incubating it for 3 days and collecting the culture supernatant. These samples were stored at –20°C until assayed.
Measurement of cytokines
Cytokine concentrations were determined by the sandwich ELISA method established in our laboratory.5 The lower limit of detection for human IL-1, IL-1ß and IL-2 was 10, 20 and 50 ng/L, respectively. The percentage inhibition of cytokine release (Inh%) was calculated from the following formula:
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Separation, purification and analysis of RNA
Using Ficoll-Paque (Pharmacia, Uppsala, Sweden), human mononuclear cells were separated aseptically and suspended in RPMI-1640 with 10% FBS at 1.7 x 106 cells/mL. Grepafloxacin (10 or 30 mg/L) and LPS (1 mg/L) were added to the mononuclear cells. After 4 h incubation at 37°C, the cell fraction was dissolved in Isogen (Nippon Gene Co., Tokyo, Japan) and total RNA was separated and purified according to the manufacturer's protocol. cDNAs for each cytokine were cloned and cDNA fragments for IL-1, IL-1ß, TNF, IL-6 and IL-8 were separated with the restriction enzymes EcoRI–HindII, PstI–XbaI, AvaI– HindIII, Kpn–BamHI and BamHI–XbaI, respectively. They were purified and used as DNA probes. Each DNA probe was 32P-labelled using the Multi-prime DNA labelling system (Amersham, Little Chalfont, UK). As an internal control, the cDNA fragment for human glyceraldehyde 3-phosphate dehydrogenase (G3PDH) (Clontech, Palo Alto, CA, USA) was similarly 32P-labelled.
Northern blotting, as described by Thomas,6 was used to detect mRNA. Total RNA (5 µg) was denatured with glyoxal–DMSO and then separated by 1.2% agarose gel electrophoresis. The RNA was transferred to a nylon membrane (Pall Inc., Glen Cove, NY, USA). After hybridization at 42°C for 2 days, the membrane was washed and bound probes were detected by autoradiography on X-Omat AR film (Kodak, Rochester, NY, USA) at –80°C overnight or for 3 days. The autoradiograph obtained was then scanned with an Ultrascan XL densitometer (LKB, Bromma, Sweden).
As a positive control for IL-1, IL-1ß and TNF mRNA, 2 µg of poly(A)+ RNA purified from mitogen-stimulated human histiocytic lymphoma U937 was used.7 As a positive control for IL-6 and IL-8, 10 µg of total RNA purified from human astrocytoma U373MG was used.8 The quantity of mRNA for each cytokine was calculated on the basis of the expression of mRNA for G3PDH used as the internal control. mRNA expression levels are indicated by relative values.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Human whole blood was stimulated with LPS (1 mg/L) and cultured overnight. The concentrations of IL-1
and IL-1ß in the culture supernatant were measured. Grepafloxacin, ciprofloxacin and ofloxacin inhibited the production of IL-1 and IL-1ß at final concentrations of 1–30 mg/L (see Table
). The inhibitory effect of grepafloxacin was stronger than those of ciprofloxacin and ofloxacin. Grepafloxacin also inhibited the production of TNF, IL-6 and IL-8 but this activity was not as marked (data not shown). The effect of grepafloxacin is not a result of toxicity to cells: our preliminary data showed that grepafloxacin 1–30 mg/L did not affect the viability of LPS-stimulated mononuclear cells. IL-2 in the culture supernatant was measured after 3 days of exposure to Con-A 10 mg/L in place of LPS. Grepafloxacin, ciprofloxacin and ofloxacin stimulated the production of IL-2 at final concentrations of 1–30 mg/L (Table).
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The effect of grepafloxacin on the expression of mRNA for inflammatory cytokines was investigated. The addition of grepafloxacin (30 mg/L) inhibited the expression of IL-1
, IL-1ß, TNF, IL-6 and IL-8 mRNA induced by LPS. The relative value of mRNA for each cytokine derived with the addition of grepafloxacin (30 mg/L) was 44, 54, 16, 23 and 36, respectively. The addition of grepafloxacin (10 mg/L) also inhibited the expression of mRNA for IL-1, TNF, IL-6 and IL-8 induced by LPS. The relative values were 69, 42, 60 and 38, respectively. In this case, expression of IL-1ß mRNA was not inhibited; the relative value was 104 (Figure). From the above observations, it was presumed that the inhibitory effect of grepafloxacin on the production of inflammatory cytokines was, in part, based on inhibition of the mRNA expression.
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Many of the reports regarding the effect of quinolone antibacterial agents on cytokine production involve in vitro studies with concentrations higher than those seen in clinical use, except those showing stimulation of IL-2 production.9 As grepafloxacin penetrates tissues well,10 its inhibition of IL-1 production could be important in clinical use in addition to its antibacterial effect. The combination of these two effects may make grepafloxacin useful for treating clinical infections where cytokines have been activated by endotoxin release in septicaemic states and where excessive inflammatory reaction has occurred.
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Notes |
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* Corresponding author. Tel: +81-665-2126; Fax: +81-665-6976; E-mail: y_ono{at}research.otsuka.co.jp
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Roche, Y., Fay, M. & Gougerot-Pocidalo, M. A. (1987). Effects of quinolones on interleukin 1 production in vitro by human monocytes. Immunopharmacology 13, 99–109.[Web of Science][Medline]
2
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Bailly, S., Mahe, Y., Ferrua, B., Fay, M., Tursz, T., Wakasugi, H. et al. (1990). Quinolone-induced differential modification of IL-1 and IL-1ß production by LPS-stimulated human monocytes. Cellular Immunology 128, 277–88.[Web of Science][Medline]
3 . Riesbeck, K. & Forsgren, A. (1990). Selective enhancement of synthesis of interleukin-2 in lymphocytes in the presence of ciprofloxacin. European Journal of Clinical Microbiology and Infectious Diseases 9, 409–13.[Web of Science][Medline]
4
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Imada, T., Miyazaki, S., Nishida, M., Yamaguchi, K. & Goto, S. (1992). In vitro and in vivo antibacterial activities of a new quinolone, OPC-17116. Antimicrobial Agents and Chemotherapy 36, 573–9.
5 . Kita, M., Ohmoto, Y., Hirai, Y., Yamaguchi, N. & Imanishi, J. (1992). Induction of cytokines in human peripheral blood mononuclear cells by mycoplasmas. Microbiology and Immunology 36, 507–16.[Web of Science][Medline]
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Thomas, P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proceedings of the National Academy of Sciences, USA 77, 5201–5.
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Nishida, T., Nishino, N., Takano, M., Kawai, K., Bando, K., Masui, Y. et al. (1987). cDNA cloning of IL-1 and IL-1ß from mRNA of U937 cell line. Biochemical and Biophysical Research Communications 143, 345–52.[Web of Science][Medline]
8 . Nishida, T., Nakai, S., Kawakami, T., Aihara, K., Nishino, N. & Hirai, Y. (1989). Dexamethasone regulation of the expression of cytokine mRNAs induced by interleukin-1 in the astrocytoma cell line U373MG. FEBS Letters 243, 25–9.[Web of Science][Medline]
9 . Shalit, I. (1991). Immunological aspects of new quinolones. European Journal of Clinical Microbiology and Infectious Diseases 10, 262–6.[Web of Science][Medline]
10
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Cook, P. J., Andrews, J. M., Wise, R., Honeybourne, D. & Mougdil, H. (1995). Concentrations of OPC-17116, a new fluoroquinolone antibacterial, in serum and lung compartments. Journal of Antimicrobial Chemotherapy 35, 317–26.
Received 2 July 1999; returned 11 October 1999; revised 22 December 1999; accepted 25 February 2000
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