Cellular Technology Institute, Otsuka Pharmaceutical Co. Ltd, 463-10 Kagasuno, Kawauchi-cho, Tokushima City, Japan
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Abstract |
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Introduction |
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Materials and methods |
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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 EcoRIHindII, PstIXbaI, AvaI HindIII, KpnBamHI and BamHIXbaI, 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 glyoxalDMSO 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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Notes |
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References |
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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, 27788.[ISI][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, 40913.[ISI][Medline]
4 . 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, 5739.[Abstract]
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, 50716.[ISI][Medline]
6 . Thomas, P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proceedings of the National Academy of Sciences, USA 77, 52015.[Abstract]
7
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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, 34552.[ISI][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, 259.[ISI][Medline]
9 . Shalit, I. (1991). Immunological aspects of new quinolones. European Journal of Clinical Microbiology and Infectious Diseases 10, 2626.[ISI][Medline]
10 . 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, 31726.[Abstract]
Received 2 July 1999; returned 11 October 1999; revised 22 December 1999; accepted 25 February 2000