Arch Dermatol Res (1991) 283:524-528
AmNves of
9 Springer-Verlag 1991
Effects of subminimal inhibitory concentrations of minocycline on neutrophil chemotactic factor production in comedonal bacteria, neutrophil phagocytosis and oxygen metabolism H. Akamatsu 1, y. Niwa 2, I. Kurokawa 1, R. Masnda 1, S. Nishijima 1, and Yasuo Asada 1 1 Department of Dermatology, Kansai Medical University, 1 Fumizono-cho, Moriguchi, Osaka, 570 Japan 2 Niwa Institute for Immunology, Kochi, 787-03 Japan Received March 9, 1991
Summary. Comedonai bacteria, Propionibacterium acnes, P. granulosum and coagnlase-negative staphylococci (CNS) seem to play an important initiating role in the inflammatory process by producing neutrophil chemotactic factors. The attracted neutrophils, after phagocytosis, release inflammatory factors such as reactive oxygen species (ROS). We investigated the effects of minocycline at subminimal inhibitory concentrations (sub-MIC), i.e. one-tenth MIC, on the production of human neutrophil chemotactic factors in comedonal bacteria, and on several inflammatory parameters of neutrophils, including neutrophil phagocytosis and generation of ROS (O~, H2Oz, OH'). ROS generation in a cell-free, xanthinexanthine oxidase system was also assessed. Production of neutrophil chemotactic factors in all strains of P. acnes, P. granulosum and CNS were significantly suppressed by subMIC minocycline. Sub-MIC minocycline effectively reduced three kinds of neutrophil-generated ROS (O~, H202, OH'). However, neutrophil phagocytosis and the ROS generated in a cell-free system were not markedly changed in the presence of sub-MIC minocycline. The results suggest that sub-MIC minocycline has an anti-inflammatory effect by inhibiting the production of neutrophil chemotactic factors in comedonal bacteria as well as ROS generated by neutrophils in the inflammatory process of acne. Key words: Minocycline - Subminimal inhibitory concentrations - A c n e - Neutrophil chemotactic factors Neutrophil oxygen metabolism
produced by these bacteria play an important initiating role in the inflammatory process of acne. In this process, the neutrophils attracted to the site of ache comedones by these bacterial chemoattractants are thought to release lysosomal enzymes and, additionally, reactive oxygen species (ROS) [18, 24, 26], which are among the most potent inflammatory mediators, resulting in damage to the follicular epithelium and extrusion of the intrafollicular contents into the dermis. Oral administration and topical application of certain antibiotics have been considered to be clinically effective in treating inflammatory acne. These drugs suppress neutrophil chemotaxis in vitro [5, 7, 16] and in vivo [6] and it has therefore been suggested that this effect might partially account for the efficacy of these drugs in the treatment of inflammatory acne [5, 6]. Esterly et al. [5] have reported that tetracycline, erythromycin and clindamycin, at concentrations below therapeutic blood levels, suppress neutrophil chemotaxis. It has been demonstrated that subminimal inhibitory concentrations (sub-MIC) oferythromycin, tetracycline and minocycline inhibit the production of neutrophil chemotactic factors in P. acnes, indicating that sub-MIC of these drugs are capable of reducing the inflammatory capacity of P. acnes leading to clinical improvement [27]. In the present study, we investigated the effect of subMIC minocycline on the production of human neutrophil chemotactic factors in comedonal bacteria (P. acnes, P. granulosum and CNS strains), and on neutrophil function, especially on ROS generated both by neutrophils and in a cell-free, xanthine-xanthine oxidase system.
Materials and methods The bacterial flora in acne lesions is mainly composed of Propionibacterium acnes (P. aenes), P. granulosum and coagulase-negative staphylococci (CNS) [15]. It is well known that low-molecular-weight chemotactic factors Offprint requests to: H. Akamatsu
Organism
Four strains each of P. acnes, P. granulosum and CNS were used in this study. One strain of P. aches was ATCC 11828. Three strains of P. aches and all strains of P. granulosum and CNS were isolated in our laboratory from comedones of patients with acne. All were identified by biochemical tests [2, 11].
H. Akamatsu et al. : Sub-MIC of minocycline and ache
525
Minimum inhibitory concentration
Table 1. MIC of minocycline for P. aches, P. granulosum and CNS strains
MIC of minocycline (Japan Lederle Ltd., Tokyo, Japan) was determined by the medium dilution method as described by the Japanese Society of Chemotherapy [8, 14].
P. acnes
Determination of the concentration of minocycline that just affected the growth curve of the test bacteria Test strains were inoculated into brain heart infusion (BHI) broth (Nissui, Tokyo, Japan) containing various sub-MIC concentrations of minocycline. P. acnes and P. granulosum were incubated anaerobically and CNS was incubated aerobically in an atmosphere of 10% COz and 90% N2 at 37~ The turbidity of the cultures was measured at 660 nm using a spectrophotometer (Hitachi, Tokyo, Japan).
Chemoattractants Test strains were inoculated into tubes of BHI broth with and without (control) the concentration of minocycline that just affected the growth curve. P. aenes and P. granulosumwere incubated anaerobically for 5 days and CNS aerobically for 2 days. The cultures were centrifuged at 3000 g for 20 rain and the upper layer was filtered through a membrane filter of 0.45 gin pore size 8 (Gelman Sciences, Mich., USA). The clear filtrates were used as chemoattractants.
Neutrophil preparation Neutrophils were isolated from heparinized venous blood from seven healthy volunteers as described previously [19]. With this procedure, the viability of the harvested neutrophils was always greater than 99% by the trypan blue exclusion test, and [14C]inulin uptake, which measures phagocytic activity, was greater than 900 dpm [22]. The neutrophils were resuspended in media appropriate for their subsequent use: RPMI for assay ofchemotactic activity; Krebs Ringer phosphate (KRP) buffer [9] for phagocytosis; KRP containing glucose (5 raM) for OH" generation; and KRP buffer containing glucose (5 raM) and gelatin (1 mg/ml) for the assay of O2 and HzO2 generation.
Viability and phagocytic function of neutrophils Neutrophil viability after incubation with the agents was determined by trypan-blue exclusion. Phagocytic function was measured by zymosan-induced stimulation of [14C]inulin uptake [22]. When over 2 % of the neutrophils were stained by trypan-bhie, or when neutrophils showed less than 600 dpm [14C]inulin uptake/mg protein, their function was considered to have been impaired and the results were discarded.
Neutrophil chemotaxis assay Neutrophil chemotaxis was assayed by the Boyden chamber method [4] with slight modifications. Briefly, the neutrophils were resuspended in RPMI 1640 containing 0.5% bovine serum albumin at a final concentration of 1.5 x 106 cells/ml. The cell suspensions were placed in the upper compartment of a modified Boyden chemotactic chamber separated from chemoattractant by a polycarbonate filter with pore size 3 gm (Millipore Corporation, Yonezawa, Japan). The chamber was incubated at 37~ for 60 rain in an atmosphere of 5%
P. granulosum
CNS
Strain
MIC (gg/ml)
Strain
MIC (Ixg/ml)
Strain
MIC (gg/ml)
ATCCl1828 I II III
0.20 0.20 0.20 0.10
I II III IV
0.10 0.20 0.10 0.20
I II III IV
0.20 0.39 0.20 0.20
COz, the filters were then removed, fixed with methanol for 1 rain and stained with Giemsa. The number of neutrophils migrating through the filter (termed the chemotactic index) was expressed as an average number of cells per filter determined from the count in ten randomly selected high power microscope fields for each filter. Chemotactic activity [27] was taken as the chemotactic index divided by the optical density (at 660 nm) of the culture which produced the chemotactic supernatant.
Neutrophil phagocytosis assay Emulsions of paraffin oil containing oil-red-O were prepared as previously described [23], except that the lipopolysaccharide solution (endotoxin) was replaced with normal human serum. The emulsion was incubated with an equal volume of normal human serum at 37~ for 30 min for opsonization. Neutrophils (2 x 107 cells/ 0.9 ml KRP) which had been preincubated for 5 min with each concentration of minocycline were added with 0.1 ml of the opsonized emulsion. The mixture was incubated for 5 min at 37~C, and then 9 ml ice-cold KRP was added to the solution to stop the reaction. The cells were washed three times with ice-cold KRP to remove the paraffin oil droplets that had not been ingested. Paraffin oil containing oil-red-O was extracted from the cells by the method of Bligh and Dyer [3] using chloroform and methanol (v/v, 1 : 2), and the optical density of the chloroform layer was determined at a wavelength of 525 nm. The mean optical density of oil-red-O extracted from 2 x 10v neutrophils incubated with opsonized paraffin oil droplets was 0.0143 + 0.0016 (average • SD of five experiments). Microscopic examination revealed that a majority of neutrophils was heavily loaded with oil droplets. In contrast, when non-opsonized paraffin oil droplets were incubated with neutrophils, the optical density was less than 0.005, and only a few neutrophils were loaded. These findings confirm that most of the extracted oil-red-O represented droplets ingested by the neutrophils.
Neutrophil R O S generation assay In studies of O2 formation, I x 1 0 6 neutrophils were preincubated at 37~ for 10 min with 1 mg/ml opsonized zymosan (Sigma, St. Louis, Mo., USA) at each concentration of minocycline, then 0.1 mM ferricytochrome c (type III, Sigma) was added. The neutrophils were incubated for another 30 rain. Immediately after sedimentation of the neutrophils and opsonized zymosan by centrifugation, 0.1 ml of the supernatant was assayed for reduced cytochrome c by measuring absorbance at 550 nm [10, 17] in 2 ml 100 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA (pH 7.8). The results were converted to nmol of reduced cytochrome c, using AE550 n m = 2.1 x 104/M/cm [21]. HzO2 generation was measured by quantifying the decrease in fluorescence intensity of scopoletin (Sigma) due to its peroxidasemediated oxidation by HaOz [21]. After incubation of 2.5 x 106
H. Akamatsu et al.: Sub-MIC of minocycline and acne
526
3,0-
100-
2.080-
1,0-
t
s E
coel3 ~o
X
1
s O
0.1-
oo 60-
%
*
t*
I
~*
E
0_
40-
20-
control ATCC 11828 i ]I P. aches strains
2'4
4'8 7'2 9'6 1")0 1~,4 168 Incubation time (hours)
Fig. 1. Growth curve of P. acnes ATCC 11828 in the presence of sub-MIC minocycline. 0 , no antibiotic; • one-tenth MIC minocycline
Fig. 2. Effect of sub-MIC minocycline on neutrophil chemotactic factor production in P. a c n e s strains. Results are expressed as percent of chemotactic activity without minocycline. * p < 0.05 vs control, ** p < 0.01
100-
neutrophils for 10 min at room temperature in KRP containing 5 mM glucose and 0.1 rag/mr gelatin in the presence of each concentration of minocycline and 1 mg/ml opsonized zymosan, 0.I ml 50 mM scopoletin in KRP and 0.05 ml 1 mg/ml horseradish peroxidase (type II; Sigma) in phosphate-buffered saline (PBS) were added. The HzOz-plus-peroxidase-inducedrate of decrease in fluorescence intensity of the scopoletin within 30 rain was quantified using a fluorescence spectrophotometer (Hitachi). To calculate the HzO2 concentration, we assumed that 1 mol H20 2 oxidized 1 mol scopoletin [21]. OH" was quantified by the amount of ethylene formed from ~-keto-methiolbutyric acid (KMB) (Sigma) plus OH" generated by neutrophils [12]. Neutrophils (2 x 106) in 2 ml KRP containing glucose were preincubated with 1 mM KMB and each concentration of minocycline in a stoppered tube and gently mixed in a 37~ shaker bath for 5 min. Opsonized zymosan was then added, and the cells were incubated for 10 min. Samples of gas in the tube were then obtained using a gas-tight syringe, and the ethylene content was determined by gas chromatography (Hitachi). The total amount of ethylene formed during 10, 20 and 30 min served as the OH" value.
R O S generation assay in the xanthine-xanthine oxidase system All ROS were also measured in the xanthine-xanthine oxidase system. Instead of adding neutrophils and opsonized zymosan, 0.1 mM hypoxanthine, 1.25 mM EDTA and 16.5 p~Mferricytochrome c were mixed in a total volume of 2 ml (I25 mM phosphate buffer). After the addition of each concentration of minocyctine, approximately 0.006 U/ml dialysed xanthine oxidase was added to generate ROS [20]. All assays were performed in triplicate, The results are expressed as the mean _+SD of replicate assays. Statistical significance was determined using Student's t-test.
800~
s ~60
~6 E 40-
20-
control
1~ IlI P. granulosum strains
Fig. 3. Effect of sub-MiC minocyclinc on neutrophil chemotactic factor production in P. granulosum. Results are expressed as percent of chemotactic activity without minocycline. * p < 0.05 vs control, 9* p < 0.01
Results M I C o f m i n o c y c l i n e for four strains each o f P. acnes, P.
granulosum a n d C N S are s h o w n in Table 1. T h e c o n c e n t r a t i o n s o f m i n o c y c l i n e t h a t j u s t affected the g r o w t h curve o f P. acnes, P. granulosum a n d C N S were o n e - t e n t h o f the M I C s . A sample g r o w t h curve is presented i n Fig. 1.
H. Akamatsu et al. : Sub-MIC of minocycline and acne
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AmNves of
9 Springer-Verlag 1991
Effects of subminimal inhibitory concentrations of minocycline on neutrophil chemotactic factor production in comedonal bacteria, neutrophil phagocytosis and oxygen metabolism H. Akamatsu 1, y. Niwa 2, I. Kurokawa 1, R. Masnda 1, S. Nishijima 1, and Yasuo Asada 1 1 Department of Dermatology, Kansai Medical University, 1 Fumizono-cho, Moriguchi, Osaka, 570 Japan 2 Niwa Institute for Immunology, Kochi, 787-03 Japan Received March 9, 1991
Summary. Comedonai bacteria, Propionibacterium acnes, P. granulosum and coagnlase-negative staphylococci (CNS) seem to play an important initiating role in the inflammatory process by producing neutrophil chemotactic factors. The attracted neutrophils, after phagocytosis, release inflammatory factors such as reactive oxygen species (ROS). We investigated the effects of minocycline at subminimal inhibitory concentrations (sub-MIC), i.e. one-tenth MIC, on the production of human neutrophil chemotactic factors in comedonal bacteria, and on several inflammatory parameters of neutrophils, including neutrophil phagocytosis and generation of ROS (O~, H2Oz, OH'). ROS generation in a cell-free, xanthinexanthine oxidase system was also assessed. Production of neutrophil chemotactic factors in all strains of P. acnes, P. granulosum and CNS were significantly suppressed by subMIC minocycline. Sub-MIC minocycline effectively reduced three kinds of neutrophil-generated ROS (O~, H202, OH'). However, neutrophil phagocytosis and the ROS generated in a cell-free system were not markedly changed in the presence of sub-MIC minocycline. The results suggest that sub-MIC minocycline has an anti-inflammatory effect by inhibiting the production of neutrophil chemotactic factors in comedonal bacteria as well as ROS generated by neutrophils in the inflammatory process of acne. Key words: Minocycline - Subminimal inhibitory concentrations - A c n e - Neutrophil chemotactic factors Neutrophil oxygen metabolism
produced by these bacteria play an important initiating role in the inflammatory process of acne. In this process, the neutrophils attracted to the site of ache comedones by these bacterial chemoattractants are thought to release lysosomal enzymes and, additionally, reactive oxygen species (ROS) [18, 24, 26], which are among the most potent inflammatory mediators, resulting in damage to the follicular epithelium and extrusion of the intrafollicular contents into the dermis. Oral administration and topical application of certain antibiotics have been considered to be clinically effective in treating inflammatory acne. These drugs suppress neutrophil chemotaxis in vitro [5, 7, 16] and in vivo [6] and it has therefore been suggested that this effect might partially account for the efficacy of these drugs in the treatment of inflammatory acne [5, 6]. Esterly et al. [5] have reported that tetracycline, erythromycin and clindamycin, at concentrations below therapeutic blood levels, suppress neutrophil chemotaxis. It has been demonstrated that subminimal inhibitory concentrations (sub-MIC) oferythromycin, tetracycline and minocycline inhibit the production of neutrophil chemotactic factors in P. acnes, indicating that sub-MIC of these drugs are capable of reducing the inflammatory capacity of P. acnes leading to clinical improvement [27]. In the present study, we investigated the effect of subMIC minocycline on the production of human neutrophil chemotactic factors in comedonal bacteria (P. acnes, P. granulosum and CNS strains), and on neutrophil function, especially on ROS generated both by neutrophils and in a cell-free, xanthine-xanthine oxidase system.
Materials and methods The bacterial flora in acne lesions is mainly composed of Propionibacterium acnes (P. aenes), P. granulosum and coagulase-negative staphylococci (CNS) [15]. It is well known that low-molecular-weight chemotactic factors Offprint requests to: H. Akamatsu
Organism
Four strains each of P. acnes, P. granulosum and CNS were used in this study. One strain of P. aches was ATCC 11828. Three strains of P. aches and all strains of P. granulosum and CNS were isolated in our laboratory from comedones of patients with acne. All were identified by biochemical tests [2, 11].
H. Akamatsu et al. : Sub-MIC of minocycline and ache
525
Minimum inhibitory concentration
Table 1. MIC of minocycline for P. aches, P. granulosum and CNS strains
MIC of minocycline (Japan Lederle Ltd., Tokyo, Japan) was determined by the medium dilution method as described by the Japanese Society of Chemotherapy [8, 14].
P. acnes
Determination of the concentration of minocycline that just affected the growth curve of the test bacteria Test strains were inoculated into brain heart infusion (BHI) broth (Nissui, Tokyo, Japan) containing various sub-MIC concentrations of minocycline. P. acnes and P. granulosum were incubated anaerobically and CNS was incubated aerobically in an atmosphere of 10% COz and 90% N2 at 37~ The turbidity of the cultures was measured at 660 nm using a spectrophotometer (Hitachi, Tokyo, Japan).
Chemoattractants Test strains were inoculated into tubes of BHI broth with and without (control) the concentration of minocycline that just affected the growth curve. P. aenes and P. granulosumwere incubated anaerobically for 5 days and CNS aerobically for 2 days. The cultures were centrifuged at 3000 g for 20 rain and the upper layer was filtered through a membrane filter of 0.45 gin pore size 8 (Gelman Sciences, Mich., USA). The clear filtrates were used as chemoattractants.
Neutrophil preparation Neutrophils were isolated from heparinized venous blood from seven healthy volunteers as described previously [19]. With this procedure, the viability of the harvested neutrophils was always greater than 99% by the trypan blue exclusion test, and [14C]inulin uptake, which measures phagocytic activity, was greater than 900 dpm [22]. The neutrophils were resuspended in media appropriate for their subsequent use: RPMI for assay ofchemotactic activity; Krebs Ringer phosphate (KRP) buffer [9] for phagocytosis; KRP containing glucose (5 raM) for OH" generation; and KRP buffer containing glucose (5 raM) and gelatin (1 mg/ml) for the assay of O2 and HzO2 generation.
Viability and phagocytic function of neutrophils Neutrophil viability after incubation with the agents was determined by trypan-blue exclusion. Phagocytic function was measured by zymosan-induced stimulation of [14C]inulin uptake [22]. When over 2 % of the neutrophils were stained by trypan-bhie, or when neutrophils showed less than 600 dpm [14C]inulin uptake/mg protein, their function was considered to have been impaired and the results were discarded.
Neutrophil chemotaxis assay Neutrophil chemotaxis was assayed by the Boyden chamber method [4] with slight modifications. Briefly, the neutrophils were resuspended in RPMI 1640 containing 0.5% bovine serum albumin at a final concentration of 1.5 x 106 cells/ml. The cell suspensions were placed in the upper compartment of a modified Boyden chemotactic chamber separated from chemoattractant by a polycarbonate filter with pore size 3 gm (Millipore Corporation, Yonezawa, Japan). The chamber was incubated at 37~ for 60 rain in an atmosphere of 5%
P. granulosum
CNS
Strain
MIC (gg/ml)
Strain
MIC (Ixg/ml)
Strain
MIC (gg/ml)
ATCCl1828 I II III
0.20 0.20 0.20 0.10
I II III IV
0.10 0.20 0.10 0.20
I II III IV
0.20 0.39 0.20 0.20
COz, the filters were then removed, fixed with methanol for 1 rain and stained with Giemsa. The number of neutrophils migrating through the filter (termed the chemotactic index) was expressed as an average number of cells per filter determined from the count in ten randomly selected high power microscope fields for each filter. Chemotactic activity [27] was taken as the chemotactic index divided by the optical density (at 660 nm) of the culture which produced the chemotactic supernatant.
Neutrophil phagocytosis assay Emulsions of paraffin oil containing oil-red-O were prepared as previously described [23], except that the lipopolysaccharide solution (endotoxin) was replaced with normal human serum. The emulsion was incubated with an equal volume of normal human serum at 37~ for 30 min for opsonization. Neutrophils (2 x 107 cells/ 0.9 ml KRP) which had been preincubated for 5 min with each concentration of minocycline were added with 0.1 ml of the opsonized emulsion. The mixture was incubated for 5 min at 37~C, and then 9 ml ice-cold KRP was added to the solution to stop the reaction. The cells were washed three times with ice-cold KRP to remove the paraffin oil droplets that had not been ingested. Paraffin oil containing oil-red-O was extracted from the cells by the method of Bligh and Dyer [3] using chloroform and methanol (v/v, 1 : 2), and the optical density of the chloroform layer was determined at a wavelength of 525 nm. The mean optical density of oil-red-O extracted from 2 x 10v neutrophils incubated with opsonized paraffin oil droplets was 0.0143 + 0.0016 (average • SD of five experiments). Microscopic examination revealed that a majority of neutrophils was heavily loaded with oil droplets. In contrast, when non-opsonized paraffin oil droplets were incubated with neutrophils, the optical density was less than 0.005, and only a few neutrophils were loaded. These findings confirm that most of the extracted oil-red-O represented droplets ingested by the neutrophils.
Neutrophil R O S generation assay In studies of O2 formation, I x 1 0 6 neutrophils were preincubated at 37~ for 10 min with 1 mg/ml opsonized zymosan (Sigma, St. Louis, Mo., USA) at each concentration of minocycline, then 0.1 mM ferricytochrome c (type III, Sigma) was added. The neutrophils were incubated for another 30 rain. Immediately after sedimentation of the neutrophils and opsonized zymosan by centrifugation, 0.1 ml of the supernatant was assayed for reduced cytochrome c by measuring absorbance at 550 nm [10, 17] in 2 ml 100 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA (pH 7.8). The results were converted to nmol of reduced cytochrome c, using AE550 n m = 2.1 x 104/M/cm [21]. HzO2 generation was measured by quantifying the decrease in fluorescence intensity of scopoletin (Sigma) due to its peroxidasemediated oxidation by HaOz [21]. After incubation of 2.5 x 106
H. Akamatsu et al.: Sub-MIC of minocycline and acne
526
3,0-
100-
2.080-
1,0-
t
s E
coel3 ~o
X
1
s O
0.1-
oo 60-
%
*
t*
I
~*
E
0_
40-
20-
control ATCC 11828 i ]I P. aches strains
2'4
4'8 7'2 9'6 1")0 1~,4 168 Incubation time (hours)
Fig. 1. Growth curve of P. acnes ATCC 11828 in the presence of sub-MIC minocycline. 0 , no antibiotic; • one-tenth MIC minocycline
Fig. 2. Effect of sub-MIC minocycline on neutrophil chemotactic factor production in P. a c n e s strains. Results are expressed as percent of chemotactic activity without minocycline. * p < 0.05 vs control, ** p < 0.01
100-
neutrophils for 10 min at room temperature in KRP containing 5 mM glucose and 0.1 rag/mr gelatin in the presence of each concentration of minocycline and 1 mg/ml opsonized zymosan, 0.I ml 50 mM scopoletin in KRP and 0.05 ml 1 mg/ml horseradish peroxidase (type II; Sigma) in phosphate-buffered saline (PBS) were added. The HzOz-plus-peroxidase-inducedrate of decrease in fluorescence intensity of the scopoletin within 30 rain was quantified using a fluorescence spectrophotometer (Hitachi). To calculate the HzO2 concentration, we assumed that 1 mol H20 2 oxidized 1 mol scopoletin [21]. OH" was quantified by the amount of ethylene formed from ~-keto-methiolbutyric acid (KMB) (Sigma) plus OH" generated by neutrophils [12]. Neutrophils (2 x 106) in 2 ml KRP containing glucose were preincubated with 1 mM KMB and each concentration of minocycline in a stoppered tube and gently mixed in a 37~ shaker bath for 5 min. Opsonized zymosan was then added, and the cells were incubated for 10 min. Samples of gas in the tube were then obtained using a gas-tight syringe, and the ethylene content was determined by gas chromatography (Hitachi). The total amount of ethylene formed during 10, 20 and 30 min served as the OH" value.
R O S generation assay in the xanthine-xanthine oxidase system All ROS were also measured in the xanthine-xanthine oxidase system. Instead of adding neutrophils and opsonized zymosan, 0.1 mM hypoxanthine, 1.25 mM EDTA and 16.5 p~Mferricytochrome c were mixed in a total volume of 2 ml (I25 mM phosphate buffer). After the addition of each concentration of minocyctine, approximately 0.006 U/ml dialysed xanthine oxidase was added to generate ROS [20]. All assays were performed in triplicate, The results are expressed as the mean _+SD of replicate assays. Statistical significance was determined using Student's t-test.
800~
s ~60
~6 E 40-
20-
control
1~ IlI P. granulosum strains
Fig. 3. Effect of sub-MiC minocyclinc on neutrophil chemotactic factor production in P. granulosum. Results are expressed as percent of chemotactic activity without minocycline. * p < 0.05 vs control, 9* p < 0.01
Results M I C o f m i n o c y c l i n e for four strains each o f P. acnes, P.
granulosum a n d C N S are s h o w n in Table 1. T h e c o n c e n t r a t i o n s o f m i n o c y c l i n e t h a t j u s t affected the g r o w t h curve o f P. acnes, P. granulosum a n d C N S were o n e - t e n t h o f the M I C s . A sample g r o w t h curve is presented i n Fig. 1.
H. Akamatsu et al. : Sub-MIC of minocycline and acne
527 10
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-400 O_
80-
o
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~
-350 ~"
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