Experimental Chemotherapy Chemotherapy 2013;59:395–401 DOI: 10.1159/000358818
Received: October 28, 2013 Accepted after revision: January 17, 2014 Published online: May 17, 2014
Effects of Erythromycin and Rifampicin on Immunomodulatory Gene Expression and Cellular Function in Human Polymorphonuclear Leukocytes Xiaoqin Mu a, b Tsuneyuki Ubagai a Takane Kikuchi-Ueda a Shigeru Tansho-Nagakawa a Ryuichi Nakano a Hirotoshi Kikuchi a Yasuo Ono a a Department of Microbiology and Immunology, Teikyo University School of Medicine, Tokyo, Japan; b Genomics Research Center (one of The State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, China
Key Words Antibiotics · Polymorphonuclear leukocytes · Real-time quantitative PCR · Immunomodulatory genes · Chemotaxis
Abstract Background: We investigated the effects of two antibiotics, erythromycin and rifampicin, on the immunomodulatory gene expression and cellular function of human polymorphonuclear leukocytes (PMNs). Methods: We used real-time quantitative PCR to examine the expression of immunomodulatory genes. The production of reactive oxygen species (ROS) was determined by fluorescence-activated cell sorting. PMN chemotaxis was analyzed using a KK chemotaxis chamber. Results: Stimulation of PMNs with lipopolysaccharide (LPS) resulted in increases in the mRNA levels of immunomodulatory genes. Rifampicin significantly inhibited the overexpression of TLR2, TLR4, CD14 and IL8Rs. However, erythromycin suppressed only the upregulation of TLR2 and TNFA. Neither antibiotic had an effect on the production of ROS. Rifampicin significantly inhibited PMN chemotaxis, but erythromycin had no effect. Conclusions: Erythromycin and rifampicin may play anti-inflammatory roles by affecting the expression levels of immunomodulatory genes or the chemotaxis of PMNs. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0009–3157/14/0596–0395$39.50/0 E-Mail [email protected] www.karger.com/che
Introduction
Polymorphonuclear leukocytes (PMNs) play a defensive role against infectious diseases [1]. In addition to fighting infections caused by pathogenic microorganisms, PMNs also cause inflammatory reactions at the infection site and trigger allergic reactions, causing immunopathological damage [2]. Pattern recognition receptors on PMNs such as Toll-like receptor (TLR)-2, TLR-4 and CD14 recognize both invading pathogens and endogenous molecules through molecular motifs [3]. They can initiate and propagate inflammation to protect the organism from infectious diseases [4]. However, excessive activation of these receptors may lead to immune disorders [5]. Reactive oxygen species (ROS) generated by PMNs play an important role in the killing of bacteria. However, this also causes damage by destroying the surrounding tissues and inducing apoptosis in other immune cells [6]. The membrane chemokine receptors of PMNs also play key roles in the inflammatory response which stimulate the PMNs in the direction for which the chemokine is bound [7]. Most studies of the anti-inflammatory effects of antibiotics have focused on immune cells such as lymphocytes [8], monocytes [9] and macrophages [10] as well as various inflammatory mediators in the serum and the Tsuneyuki Ubagai Department of Microbiology and Immunology Teikyo University School of Medicine 2-11-1 Kaga, Itabashi, Tokyo 173-8605 (Japan) E-Mail ubat @ med.teikyo-u.ac.jp
bronchoalveolar lavage fluid [11, 12]. Many studies have examined the effects of antibiotics on the function of PMNs [13–16], but few have reported on the gene expression of PMN-activating cytokines and their receptors [17]. Both erythromycin and rifampicin have good intracellular distribution properties and may exert immunomodulatory effects. Our study was designed to clarify whether these two antibiotics affect the gene expression and the cellullar function of PMNs.
Table 1. Primer sets for real-time quantitative PCR
Gene
Sequence
Product size, bp
TLR2
F: 5′-TCTGCTATGATGCATTTGTTT-3′ R: 5′-TATTGTCAATGATCCACTTGC-3′
150
TLR4
F: 5′-ATTTCAGCTCTGCCTTCACTA-3′ R: 5′-CTTCTGCAGGACAATGAAGAT-3′
212
CD14
F: 5′-CGCTCGAGGACCTAAAGATA-3′ R: 5′-CAGACAGGTCTAGGCTGGTAA-3′
243
TNFA
F: 5′-AGACCAAGGTCAACCTCCT-3′ R: 5′-AAAGTAGACCTGCCCAGAC-3′
194
IL1B
F: 5′-AATGACCTGAGCACCTTCTTT-3′ R: 5′-TCATATGGACCAGACATCACC-3′
161
IL8Rs
F: 5′-GGTCATCTTTGCTGTCGTCC-3′ R: 5′-CGTAGATGATGGGGTTGAG-3′
191
Materials and Methods Materials Erythromycin, rifampicin, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS; Escherichia coli O111:B4) and interleukin (IL)-8 were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Erythromycin and rifampicin were dissolved in 100% DMSO and adjusted to final concentrations ranging from 0.01 to 25 μg/ml by diluting with Hanks’ balanced salt solution (HBSS). PMN Preparation Human PMNs were isolated from the peripheral venous blood of healthy volunteers. Briefly, 20 ml of whole blood was mixed with 7 ml of a 6% dextran solution and 15 ml of HBSS and allowed to stand for 20 min at room temperature until stratification occurred. The upper leukocyte-rich plasma layer was removed to a new tube containing a Ficoll-Paque Plus density gradient (Amersham Bioscience, Wisc., USA) and was centrifuged at 1,600 rpm for 30 min. The cell pellet was treated with deionized water for 30 s to lyse the erythrocytes, and osmolality was restored by adding 10× HBSS. Cell purity was detected by hematology analyzer Celltac MEK 6450. Cell viability was determined by light microscopic assessment with Trypan blue exclusion staining. Cell preparations contained >95% PMNs with a viability of >97%. The cells were diluted to densities of 5 × 106 cells/ml with HBSS for the gene expression assays and 1 × 106 cells/ml for the flow cytometry assays and chemotactic analysis. All volunteers were healthy adults, 30–59 years of age, who were informed of the purpose of these experiments. Informed consent was obtained for experimentation with human subjects and all procedures were performed in compliance with relevant laws and institutional guidelines. The protocol was approved by the Ethical Review Committee at the Teikyo University School of Medicine (No. 07-104). RNA Preparation LPS from E. coli was used to mimic infections by Gram-negative bacteria and to stimulate induction of the upregulation of immunomodulatory genes. Human PMNs (5 × 106 cells/ml) were treated with LPS (100 ng/ml) for 5 min at room temperature prior to incubation with different concentrations of erythromycin or rifampicin for 1 h at 37 ° C. A control was added, with DMSO (the solvent for the antibiotics) adjusted to the concentration of 0.1%, and this was incubated for 1 h at 37 ° C. Cells were collected and washed with cold PBS. The total RNA of the PMNs was extracted using the RNeasy Plus mini kit (Qiagen, Düsseldorf, Germany). An Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn,
396
Chemotherapy 2013;59:395–401 DOI: 10.1159/000358818
MAC-1 F: 5′-AAGGTGTCCACACTCCAGAAC-3′ R: 5′-GAGGAGCAGTTTGTTTCCAAG-3′
204
ACTB
205
F: 5′-TTAAGGAGAAGCTGTGCTACG-3′ R: 5′-TTGAAGGTAGTTTCGTGGATG-3′
F = Forward primer; R = reverse primer.
Germany) was used to determine the quantity and quality of the total RNA samples. Complementary DNA Synthesis Total RNA was reverse-transcribed to cDNA following the manufacturer’s instruction using the SuperScript VILO cDNA synthesis kit (Invitrogen Life Technologies, Calif., USA). Briefly, 250 ng of total RNA was incubated with 4 μl of the 5× VILO reaction mix and 2 μl of the SuperScript enzyme mix in a 20-μl reaction volume at 25 ° C for 10 min, followed by a further incubation at 42 ° C for 2 h. The reaction was terminated by heating to 85 ° C for 5 min. The cDNA product was diluted to a final volume of 50 μl with deionized water and used for real-time quantitative PCR analysis.
Real-Time Quantitative PCR Analysis Real-time quantitative PCR was performed to determine the gene expression levels of target genes in human PMNs using a StepOne real-time PCR system (Applied Biosystems, Calif., USA) [18, 19]. The target genes included pattern recognition receptors (TLR2, TLR4 and CD14), inflammatory cytokines (TNFA and IL1B), chemokine receptors (IL8Rs and MAC-1) and a housekeeping gene ACTB. Real-time PCR reactions were carried out in 10-μl reaction volumes containing the following components: 4 μl of the cDNA solution, 5 μl of the Power SyBR Green PCR master mix, 200 nM of the primers and supplementary deionized water. The cDNA amplification conditions were: 95 ° C for 10 min, 40 cycles at 95 ° C for 12 s, 13 s at the annealing temperature (53 ° C for ACTB, 56 ° C for TLR2, 55 ° C for TLR4, 53 ° C for CD14, 58 ° C for TNFA,
Mu /Ubagai /Kikuchi-Ueda / Tansho-Nagakawa /Nakano /Kikuchi /Ono
6
6
5
5
*
*
#
2 1
5 Ctl LPS 0.01 0.1 1 Erythromycin (μg/ml)
54 ° C for IL1B, 60 ° C for IL8Rs and 48 ° C for MAC-1), 72 ° C for 15 s and 60 ° C for 1 min. The target gene expression levels were normalized against the gene expression level of ACTB. The primer sequences are shown in table 1.
*
3
0
a
#
4
Fluorescence-Activated Cell Sorting Assay The production of ROS was detected employing a fluorescence-activated cell sorter (BD Biosciences, San José, Calif., USA) according to the directions provided by the ROS/superoxide detection kit (Enzo Life Science Inc., Farmingdale, N.Y., USA) [20]. In this kit, superoxide detection (orange fluorescence) is separated from the total ROS detection (green fluorescence). Because the sensitivity of the green probe to superoxides was low, the orange probe that reacts specifically with superoxides was used instead. In brief, PMNs at a density of 1 × 106 cells/ml were treated with different concentrations of erythromycin or rifampicin (0, 0.1, 1, 6.25, 12.5 and 25 μg/ml), while the control group had DMSO added to a final concentration of 0.1%, which was then incubated at 37 ° C for 1 h. After washing with the kit-supplied wash buffer, each sample was suspended in 500 μl of the ROS/superoxide detection mix and incubated for 20 min at 37 ° C in the dark. The reaction mixture was then stimulated with 5 μl of PMA (500 ng/ml), an activator of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, to initiate the production of ROS, and incubated for 10 min continuously at 37 ° C in the dark. The mean fluorescence intensity was measured with the flow cytometer.
10
Relative level of mRNA
recognition receptors TLR2, TLR4 and CD14. a Effects of erythromycin on the mRNA levels of TLR2, TLR4 and CD14, respectively, in LPS-induced human PMNs. b Effects of rifampicin on the mRNA levels of TLR2, TLR4 and CD14, respectively, in LPS-induced human PMNs. Statistical significance was evaluated by the least significant difference test. # p < 0.05 vs. control, * p < 0.05 vs. LPS, ** p < 0.01 vs. LPS. Data represent a minimum of 3 separate experiments. Ctl = Control.
Relative level of mRNA
Fig. 1. Gene expression levels of the pattern
TLR2 TLR4 CD14
#
4 3 2 1 0
b
* #
** *
*
** ** * *
* ** **
5 Ctl LPS 0.01 0.1 1 Rifampicin (μg/ml)
10
with a charge-coupled-device camera. The migration of individual PMNs was monitored by imaging the cells in the KK chamber every 30 s. The velocity of the migrating PMNs towards the IL-8 was calculated as: velocity = 240 μm ÷ migration time. For each sample, the mean velocity of migration was calculated from the data for 20 cells. Statistical Analysis All data were expressed as the mean ± standard deviation. Statistical analysis was done by performing an ANOVA, a least significant difference test and a Dunnett t test. All statistical comparisons were performed utilizing GraphPad Prism V5 software. A two-tailed p value of
Received: October 28, 2013 Accepted after revision: January 17, 2014 Published online: May 17, 2014
Effects of Erythromycin and Rifampicin on Immunomodulatory Gene Expression and Cellular Function in Human Polymorphonuclear Leukocytes Xiaoqin Mu a, b Tsuneyuki Ubagai a Takane Kikuchi-Ueda a Shigeru Tansho-Nagakawa a Ryuichi Nakano a Hirotoshi Kikuchi a Yasuo Ono a a Department of Microbiology and Immunology, Teikyo University School of Medicine, Tokyo, Japan; b Genomics Research Center (one of The State-Province Key Laboratories of Biomedicine-Pharmaceutics of China), Harbin Medical University, Harbin, China
Key Words Antibiotics · Polymorphonuclear leukocytes · Real-time quantitative PCR · Immunomodulatory genes · Chemotaxis
Abstract Background: We investigated the effects of two antibiotics, erythromycin and rifampicin, on the immunomodulatory gene expression and cellular function of human polymorphonuclear leukocytes (PMNs). Methods: We used real-time quantitative PCR to examine the expression of immunomodulatory genes. The production of reactive oxygen species (ROS) was determined by fluorescence-activated cell sorting. PMN chemotaxis was analyzed using a KK chemotaxis chamber. Results: Stimulation of PMNs with lipopolysaccharide (LPS) resulted in increases in the mRNA levels of immunomodulatory genes. Rifampicin significantly inhibited the overexpression of TLR2, TLR4, CD14 and IL8Rs. However, erythromycin suppressed only the upregulation of TLR2 and TNFA. Neither antibiotic had an effect on the production of ROS. Rifampicin significantly inhibited PMN chemotaxis, but erythromycin had no effect. Conclusions: Erythromycin and rifampicin may play anti-inflammatory roles by affecting the expression levels of immunomodulatory genes or the chemotaxis of PMNs. © 2014 S. Karger AG, Basel
© 2014 S. Karger AG, Basel 0009–3157/14/0596–0395$39.50/0 E-Mail [email protected] www.karger.com/che
Introduction
Polymorphonuclear leukocytes (PMNs) play a defensive role against infectious diseases [1]. In addition to fighting infections caused by pathogenic microorganisms, PMNs also cause inflammatory reactions at the infection site and trigger allergic reactions, causing immunopathological damage [2]. Pattern recognition receptors on PMNs such as Toll-like receptor (TLR)-2, TLR-4 and CD14 recognize both invading pathogens and endogenous molecules through molecular motifs [3]. They can initiate and propagate inflammation to protect the organism from infectious diseases [4]. However, excessive activation of these receptors may lead to immune disorders [5]. Reactive oxygen species (ROS) generated by PMNs play an important role in the killing of bacteria. However, this also causes damage by destroying the surrounding tissues and inducing apoptosis in other immune cells [6]. The membrane chemokine receptors of PMNs also play key roles in the inflammatory response which stimulate the PMNs in the direction for which the chemokine is bound [7]. Most studies of the anti-inflammatory effects of antibiotics have focused on immune cells such as lymphocytes [8], monocytes [9] and macrophages [10] as well as various inflammatory mediators in the serum and the Tsuneyuki Ubagai Department of Microbiology and Immunology Teikyo University School of Medicine 2-11-1 Kaga, Itabashi, Tokyo 173-8605 (Japan) E-Mail ubat @ med.teikyo-u.ac.jp
bronchoalveolar lavage fluid [11, 12]. Many studies have examined the effects of antibiotics on the function of PMNs [13–16], but few have reported on the gene expression of PMN-activating cytokines and their receptors [17]. Both erythromycin and rifampicin have good intracellular distribution properties and may exert immunomodulatory effects. Our study was designed to clarify whether these two antibiotics affect the gene expression and the cellullar function of PMNs.
Table 1. Primer sets for real-time quantitative PCR
Gene
Sequence
Product size, bp
TLR2
F: 5′-TCTGCTATGATGCATTTGTTT-3′ R: 5′-TATTGTCAATGATCCACTTGC-3′
150
TLR4
F: 5′-ATTTCAGCTCTGCCTTCACTA-3′ R: 5′-CTTCTGCAGGACAATGAAGAT-3′
212
CD14
F: 5′-CGCTCGAGGACCTAAAGATA-3′ R: 5′-CAGACAGGTCTAGGCTGGTAA-3′
243
TNFA
F: 5′-AGACCAAGGTCAACCTCCT-3′ R: 5′-AAAGTAGACCTGCCCAGAC-3′
194
IL1B
F: 5′-AATGACCTGAGCACCTTCTTT-3′ R: 5′-TCATATGGACCAGACATCACC-3′
161
IL8Rs
F: 5′-GGTCATCTTTGCTGTCGTCC-3′ R: 5′-CGTAGATGATGGGGTTGAG-3′
191
Materials and Methods Materials Erythromycin, rifampicin, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS; Escherichia coli O111:B4) and interleukin (IL)-8 were purchased from Sigma-Aldrich Japan (Tokyo, Japan). Erythromycin and rifampicin were dissolved in 100% DMSO and adjusted to final concentrations ranging from 0.01 to 25 μg/ml by diluting with Hanks’ balanced salt solution (HBSS). PMN Preparation Human PMNs were isolated from the peripheral venous blood of healthy volunteers. Briefly, 20 ml of whole blood was mixed with 7 ml of a 6% dextran solution and 15 ml of HBSS and allowed to stand for 20 min at room temperature until stratification occurred. The upper leukocyte-rich plasma layer was removed to a new tube containing a Ficoll-Paque Plus density gradient (Amersham Bioscience, Wisc., USA) and was centrifuged at 1,600 rpm for 30 min. The cell pellet was treated with deionized water for 30 s to lyse the erythrocytes, and osmolality was restored by adding 10× HBSS. Cell purity was detected by hematology analyzer Celltac MEK 6450. Cell viability was determined by light microscopic assessment with Trypan blue exclusion staining. Cell preparations contained >95% PMNs with a viability of >97%. The cells were diluted to densities of 5 × 106 cells/ml with HBSS for the gene expression assays and 1 × 106 cells/ml for the flow cytometry assays and chemotactic analysis. All volunteers were healthy adults, 30–59 years of age, who were informed of the purpose of these experiments. Informed consent was obtained for experimentation with human subjects and all procedures were performed in compliance with relevant laws and institutional guidelines. The protocol was approved by the Ethical Review Committee at the Teikyo University School of Medicine (No. 07-104). RNA Preparation LPS from E. coli was used to mimic infections by Gram-negative bacteria and to stimulate induction of the upregulation of immunomodulatory genes. Human PMNs (5 × 106 cells/ml) were treated with LPS (100 ng/ml) for 5 min at room temperature prior to incubation with different concentrations of erythromycin or rifampicin for 1 h at 37 ° C. A control was added, with DMSO (the solvent for the antibiotics) adjusted to the concentration of 0.1%, and this was incubated for 1 h at 37 ° C. Cells were collected and washed with cold PBS. The total RNA of the PMNs was extracted using the RNeasy Plus mini kit (Qiagen, Düsseldorf, Germany). An Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn,
396
Chemotherapy 2013;59:395–401 DOI: 10.1159/000358818
MAC-1 F: 5′-AAGGTGTCCACACTCCAGAAC-3′ R: 5′-GAGGAGCAGTTTGTTTCCAAG-3′
204
ACTB
205
F: 5′-TTAAGGAGAAGCTGTGCTACG-3′ R: 5′-TTGAAGGTAGTTTCGTGGATG-3′
F = Forward primer; R = reverse primer.
Germany) was used to determine the quantity and quality of the total RNA samples. Complementary DNA Synthesis Total RNA was reverse-transcribed to cDNA following the manufacturer’s instruction using the SuperScript VILO cDNA synthesis kit (Invitrogen Life Technologies, Calif., USA). Briefly, 250 ng of total RNA was incubated with 4 μl of the 5× VILO reaction mix and 2 μl of the SuperScript enzyme mix in a 20-μl reaction volume at 25 ° C for 10 min, followed by a further incubation at 42 ° C for 2 h. The reaction was terminated by heating to 85 ° C for 5 min. The cDNA product was diluted to a final volume of 50 μl with deionized water and used for real-time quantitative PCR analysis.
Real-Time Quantitative PCR Analysis Real-time quantitative PCR was performed to determine the gene expression levels of target genes in human PMNs using a StepOne real-time PCR system (Applied Biosystems, Calif., USA) [18, 19]. The target genes included pattern recognition receptors (TLR2, TLR4 and CD14), inflammatory cytokines (TNFA and IL1B), chemokine receptors (IL8Rs and MAC-1) and a housekeeping gene ACTB. Real-time PCR reactions were carried out in 10-μl reaction volumes containing the following components: 4 μl of the cDNA solution, 5 μl of the Power SyBR Green PCR master mix, 200 nM of the primers and supplementary deionized water. The cDNA amplification conditions were: 95 ° C for 10 min, 40 cycles at 95 ° C for 12 s, 13 s at the annealing temperature (53 ° C for ACTB, 56 ° C for TLR2, 55 ° C for TLR4, 53 ° C for CD14, 58 ° C for TNFA,
Mu /Ubagai /Kikuchi-Ueda / Tansho-Nagakawa /Nakano /Kikuchi /Ono
6
6
5
5
*
*
#
2 1
5 Ctl LPS 0.01 0.1 1 Erythromycin (μg/ml)
54 ° C for IL1B, 60 ° C for IL8Rs and 48 ° C for MAC-1), 72 ° C for 15 s and 60 ° C for 1 min. The target gene expression levels were normalized against the gene expression level of ACTB. The primer sequences are shown in table 1.
*
3
0
a
#
4
Fluorescence-Activated Cell Sorting Assay The production of ROS was detected employing a fluorescence-activated cell sorter (BD Biosciences, San José, Calif., USA) according to the directions provided by the ROS/superoxide detection kit (Enzo Life Science Inc., Farmingdale, N.Y., USA) [20]. In this kit, superoxide detection (orange fluorescence) is separated from the total ROS detection (green fluorescence). Because the sensitivity of the green probe to superoxides was low, the orange probe that reacts specifically with superoxides was used instead. In brief, PMNs at a density of 1 × 106 cells/ml were treated with different concentrations of erythromycin or rifampicin (0, 0.1, 1, 6.25, 12.5 and 25 μg/ml), while the control group had DMSO added to a final concentration of 0.1%, which was then incubated at 37 ° C for 1 h. After washing with the kit-supplied wash buffer, each sample was suspended in 500 μl of the ROS/superoxide detection mix and incubated for 20 min at 37 ° C in the dark. The reaction mixture was then stimulated with 5 μl of PMA (500 ng/ml), an activator of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, to initiate the production of ROS, and incubated for 10 min continuously at 37 ° C in the dark. The mean fluorescence intensity was measured with the flow cytometer.
10
Relative level of mRNA
recognition receptors TLR2, TLR4 and CD14. a Effects of erythromycin on the mRNA levels of TLR2, TLR4 and CD14, respectively, in LPS-induced human PMNs. b Effects of rifampicin on the mRNA levels of TLR2, TLR4 and CD14, respectively, in LPS-induced human PMNs. Statistical significance was evaluated by the least significant difference test. # p < 0.05 vs. control, * p < 0.05 vs. LPS, ** p < 0.01 vs. LPS. Data represent a minimum of 3 separate experiments. Ctl = Control.
Relative level of mRNA
Fig. 1. Gene expression levels of the pattern
TLR2 TLR4 CD14
#
4 3 2 1 0
b
* #
** *
*
** ** * *
* ** **
5 Ctl LPS 0.01 0.1 1 Rifampicin (μg/ml)
10
with a charge-coupled-device camera. The migration of individual PMNs was monitored by imaging the cells in the KK chamber every 30 s. The velocity of the migrating PMNs towards the IL-8 was calculated as: velocity = 240 μm ÷ migration time. For each sample, the mean velocity of migration was calculated from the data for 20 cells. Statistical Analysis All data were expressed as the mean ± standard deviation. Statistical analysis was done by performing an ANOVA, a least significant difference test and a Dunnett t test. All statistical comparisons were performed utilizing GraphPad Prism V5 software. A two-tailed p value of
