Journal of Chemotherapy
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Effects of Miocamycin and Erythromycin on Polymorphonuclear Cell Function P. Bacci, A. Grossi, A.M. Vannucchi, D. Rafanelli, A. Casini & M. De Luca To cite this article: P. Bacci, A. Grossi, A.M. Vannucchi, D. Rafanelli, A. Casini & M. De Luca (1992) Effects of Miocamycin and Erythromycin on Polymorphonuclear Cell Function, Journal of Chemotherapy, 4:5, 268-270, DOI: 10.1080/1120009X.1992.11739175 To link to this article: http://dx.doi.org/10.1080/1120009X.1992.11739175
Published online: 15 Jul 2016.
Submit your article to this journal
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Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [Australian Catholic University]
Date: 08 August 2017, At: 21:59
Downloaded by [Australian Catholic University] at 21:59 08 August 2017
Journal of Chemotherapy
Effects of Miocamycin and Erythromycin on Polymorphonuclear Cell Function P. BACCI - A. GROSSI - A.M. VANNUCCHI D. RAFANELLI- A. CASINI * - M. DELUCA *
Vol. 4 - n. 5 (268-270) - 1992
INTRODUCTION
It is well known that antibiotics, beyond their bactericidal or bacteriostatic activity, exert different actions on specific and aspecific defenses of the host. Among the different classes of antibiotics, macrolides, also thanks to their ability to enter eukaryotic cells, seem to stimulate macrophage and polymorphonuclear (PMN) fucntions. Most studies have focused on some of their functions, while we studied the global activity of PMN (leukotriene B4 (L TB4) production, chemotaxis, killing of Candida albicans and respiratory burst) under the influence of two antibiotics of the macrolide family: a 14-membered macrolide (erythromycin) and a 16-membered macrolide (miocamycin) .
Summary ---------------- ---------------- Previous studies have shown that erythromycin can enter phagocytic cells, stimulating their functional activity. In this work we compared the effects of erythromycin and another newer macrolide antibiotic, miocamycin, on a series of in vitro tests aimed at evaluating their influence on polymorphonuclear cell (PMN) functions. Results indicate that erythromycin induces an increase in leukotriene B4 production in PMNs, while chemotaxis, killing of Csndids slbicsns and respiratory burst are not influenced, at least at the doses used in this study. On the contrary, all these activities are significandy enhanced following incubation with miocamycin, and the response varies according to the antibiotic concentration. Key words: miocamycin, erythromycin, macrolide, polymorphonucleates.
Division of Hematology, University of Florence and USL 10/D. * Menarini s.r. l. , Medical Department, Florence, Italy. Correspondence to: Dr. A. Grossi, Unita Operativa di Ematologia, Viale Morgagni n. 85, Firenze, Italy. © Edizioni Riviste Scientifiche - Firenze
MATERIALS AND METHODS
Granulocyte enrichment
Buffy-coats obtained from blood bank were overlaid on Lymphoprep (Nycomed, Oslo, Norway) (density 1077 g/ml) and centrifuged at 250xg for 20 min. PMNs sedimented to the bottom of the tube, and red cell contamination, when present, was eliminated by lysing the cells with 1% ammonium chloride, obtaining a 9599% pure granulocyte population. In each experiment described below, cells were incubated with erythromycin or miocamycin at three different final concentrations -2.5, 5 and 10 pg/ml -- for 30 minutes. Results were compared to those obtained using a population of cells not incubated with antibiotics. ISSN 1120-009X
269
EFFECTS OF MIOCAMYCIN AND ERYTHROMYCIN ON POLYMORPHONUCLEAR CELL FUNCTIO N
Chemotaxis
Statistical studies
Chemotaxis was evaluated with Boyden chamber (Arnika Milano, Italy) method, as described by Territo 4 • Cells incubated with erythromycin or miocamycin and control samples were put in the presence of N-peptide. Chemotactic index was calculated as follows:
Five samples of each group were prepared for each test. Student's t test was used .
Downloaded by [Australian Catholic University] at 21:59 08 August 2017
CI
= -
c
X
100
R where C = mean value of CI in the presence of chemotactic peptide. R = mobility in the absence of stimulus. Both C and R are expressed in I! (micron).
LTB4 production Cells were used at the concentration of 10 6/ml. Production was measured by a radioimmunoassay (MEN DuPont de Nemours, Bad Homburg, West Germany) after stimulation of cells with ionophore A23187, in the presence or absence of erythromycin and miocamycin at the same concentrations as for chemotactic experiments.
RESULTS
Chemotaxis As shown in Table 1, 2.5, 5 and 10 pg/ml of erythromycin were unable to induce a modification in the chemotactic index if compared to controls, while 5 and 10 pg/ml of miocamycin determined a significant increase.
LTB4 production If compared to unstimulated cells, PMNs incubated with 5 and 10 pg/ml of erythromycin or miocamycin significantly increased their LTB4 production, while both antibiotics were ineffective at the lowest concentration used (2.5 pg) in our experiments (Table 2). TABLE 1 - Chemotactic Index. M .*
S.D .
160
10
2.5 pg 5.0 pg 10.0 pg
180 260 442
20 19 22
2.5 pg 5.0 pg 10.0 pg
167 171 178
20 22 22
Controls
Killing of Candida albicans 0.5 ml of neutrophil suspension (5 X 10 6/ml) were incubated with 0.5 ml of Candida (5 X 10 6 ) in the presence or not of antibiotics. 0.1 ml of the mixture and 0.3 ml of phosphate buffered saline (PBS) containing 15% fetal calf serum (FCS) were mixed, and cells finally cytocentrifuged. The smears were fixed in air, then with absolute methanol and stained with Giemsa. Dead Candida were recognized by the failure to take up stain and/or the presence of other morphological abnormalities.
Respiratory burst Cells (1 X 10 6/ml) were incubated with nitroblutetrazolium (Sigma Chemical Co, St. Louis, USA) for 30 min at 37°C. Cells were cytocentrifuged, air dried and fixed in absolute methanol for 1 min, and thereafter stained with safranin (lstituto Behring, Scoppito, Italy). The test was considered positive if more than 10% of cells contained formaz an precipitate .
Miocamycin >>
»
Erythromycin » »
* (mea n values from 3 experiments) .
TABLE 2 - Leukotriene B4 production (pgjm/) .
KI** KA-** Miocamycin » »
Erythromycin » »
M .*
S.D .
2.5 pg 5.0 pg 10.0 pg
0 51 57 225 398
2.3 2.7 4.2 3.7
2 .5 pg 5.0 pg 10.0 pg
54 151 243
2.8 3.2 4.5
* (mean values from 3 experiments) . ** KI Unstimulated Cells. KA- Stimulated cells not incubated in the presence of miocamycin or erythromycin .
270
P. BACCI - A. GROSSI - A.M. VANNUCCHI - D. RAFANELU - A. CASINI - M. DE LUCA
TABLE 3 - Killing of Candida albicans. Live
Controls Miocamycin
mean*
S.D.
mean
S.D
61
4
40
3
5 5
2.5 pg
55
3
45
))
5.0 pg
5
))
10.0 pg
38 26
8
62 74
60 61 58
3 4 6
40 38 42
Erythromycin ))
))
Downloaded by [Australian Catholic University] at 21:59 08 August 2017
Dead
2.5 pg 5.0 pg 10.0 pg
10
5 4
5
* (mean values from 3 experiments).
Killing of Candida albicans Miocamycin, but not erythromycin, was able to increase the killing of Candida. Again a concentration of at least 5 pg/ml of antibiotic was necessary to elicit the response (Table 3).
Respiratory burst
tration of 500 mg of erythromycin or 600 mg of miocamycin 6 • In our experiments concentrations as low as 5 and 10 pg/ml of miocamycin induced a significant increase in the chemotaxis index, killing of Candida; and LTB4 production. Respiratory burst required a concentration of 10 pg/ml to be elicited. Although erythromycin has been reported to stimulate the killing activity in PMNs, we could not confirm this effect, while LTB4 production was significantly increased. Because this is a stimulator of PMN migration, it may seem contradictory that chemotaxis was unmodified after exposure to the antibiotic. Perhaps higher erythromycin concentrations are necessary to exert an effect on PMN activity and, although increased, LTB4 levels reached during the incubation with the antibiotic may have not been sufficient to influence chemotaxis. In conclusion, our data demonstrate that miocamycin, but not erythromycin, is active on PMN function even at very low concentrations.
REFERENCES
Using NBT (nitroblue tetrazolium) reduction test, formazan precipitate was observed in more than 10% of cells only when a concentration of 10 pg/ml of miocamycin was used, while the same amount of erythromycin induced only a modest, not significant response.
DISCUSSION
The main difference between macrolides and other classes of antibiotics is their ability to enter phagocytic cells, stimulating their functional activities 1 • 2 • 3 • Experiments published so far have mainly addressed the uptake and killing of bacteria s •6 • '. In this paper we have tested the capability of two macrolides, erythromycin and miocamycin, to increase chemotaxis, respiratory burst, and production of LTB4, a potent mediator of inflammatory reactions, which promotes cell migration in vivo and in vitro 8 • 9 • The final antibiotic concentrations used in our experiments were lower than those usually reached 1 hour and 8 hours after the adminis-
' Prokesch RC, Hand L. Antibiotic entry into human polymorphonuclear leucocytes. Antimicrob Agents Chemother 1982; 21: 373-380. 'Johnson JD, Hand WL, Francis JB, King-Thompson N, Crowin RW . Antibiotic uptake by alveolar macrophages. J Lab Clin Med 1980; 95: 4429-4439. 'Van Rensburg CEJ, Anderson R, Joone G, Van Der Merwe AJ, Van Rensburg AJ. Effects of Erythromycyn on cellular functions in vitro and in vivo . J Antimicrob Chemother 1981; 8: 467-474 . • Territo MC . Chemotaxis. In : Cline MJ ed. Leukocyte Function. Churchill Livingstone, Edinburgh: 1981; 39-52. 'Lee Hand W, King-Thompson NL, Johnson JD. Influence of bacteri al-antibiotic interactions on subsequent antimicrobial activity of alveolar macrophages. J Infect Dis 1984; 149: 271-276. • Capelli A, Capelli 0 , Azzolini L, Richeldi L, Prandi E, Velluti G . Activities of human alveolar macrophages (HAMs). Note 1: observations on phagocytosis and bacterial killing in the presence of Miocamycin. Chemioterapia 1988; 7: 89-95 . ' Fraschini F, Scaglione F, Ferrara F, Dugnani S, Falchi M, Cattaneo G . Miocamycin and leukocyte activity in man. Chemotherapy 1989; 35: 289-295. 'Ford-Hutchinson AW, Bray MA, Doing MV, Shipley ME, Smith JH . Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 1980; 286: 264-265 . • Palmblad J, Malmsten CL, Uden AM, Radmark 0, Engstedt L, Samuelsson B. Leukotriene B4 is potent and stereospecific stimulator of neutrophil chemotaxis and adherence. Blood 198 1; 58: 658-661.
ISSN: 1120-009X (Print) 1973-9478 (Online) Journal homepage: http://www.tandfonline.com/loi/yjoc20
Effects of Miocamycin and Erythromycin on Polymorphonuclear Cell Function P. Bacci, A. Grossi, A.M. Vannucchi, D. Rafanelli, A. Casini & M. De Luca To cite this article: P. Bacci, A. Grossi, A.M. Vannucchi, D. Rafanelli, A. Casini & M. De Luca (1992) Effects of Miocamycin and Erythromycin on Polymorphonuclear Cell Function, Journal of Chemotherapy, 4:5, 268-270, DOI: 10.1080/1120009X.1992.11739175 To link to this article: http://dx.doi.org/10.1080/1120009X.1992.11739175
Published online: 15 Jul 2016.
Submit your article to this journal
View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yjoc20 Download by: [Australian Catholic University]
Date: 08 August 2017, At: 21:59
Downloaded by [Australian Catholic University] at 21:59 08 August 2017
Journal of Chemotherapy
Effects of Miocamycin and Erythromycin on Polymorphonuclear Cell Function P. BACCI - A. GROSSI - A.M. VANNUCCHI D. RAFANELLI- A. CASINI * - M. DELUCA *
Vol. 4 - n. 5 (268-270) - 1992
INTRODUCTION
It is well known that antibiotics, beyond their bactericidal or bacteriostatic activity, exert different actions on specific and aspecific defenses of the host. Among the different classes of antibiotics, macrolides, also thanks to their ability to enter eukaryotic cells, seem to stimulate macrophage and polymorphonuclear (PMN) fucntions. Most studies have focused on some of their functions, while we studied the global activity of PMN (leukotriene B4 (L TB4) production, chemotaxis, killing of Candida albicans and respiratory burst) under the influence of two antibiotics of the macrolide family: a 14-membered macrolide (erythromycin) and a 16-membered macrolide (miocamycin) .
Summary ---------------- ---------------- Previous studies have shown that erythromycin can enter phagocytic cells, stimulating their functional activity. In this work we compared the effects of erythromycin and another newer macrolide antibiotic, miocamycin, on a series of in vitro tests aimed at evaluating their influence on polymorphonuclear cell (PMN) functions. Results indicate that erythromycin induces an increase in leukotriene B4 production in PMNs, while chemotaxis, killing of Csndids slbicsns and respiratory burst are not influenced, at least at the doses used in this study. On the contrary, all these activities are significandy enhanced following incubation with miocamycin, and the response varies according to the antibiotic concentration. Key words: miocamycin, erythromycin, macrolide, polymorphonucleates.
Division of Hematology, University of Florence and USL 10/D. * Menarini s.r. l. , Medical Department, Florence, Italy. Correspondence to: Dr. A. Grossi, Unita Operativa di Ematologia, Viale Morgagni n. 85, Firenze, Italy. © Edizioni Riviste Scientifiche - Firenze
MATERIALS AND METHODS
Granulocyte enrichment
Buffy-coats obtained from blood bank were overlaid on Lymphoprep (Nycomed, Oslo, Norway) (density 1077 g/ml) and centrifuged at 250xg for 20 min. PMNs sedimented to the bottom of the tube, and red cell contamination, when present, was eliminated by lysing the cells with 1% ammonium chloride, obtaining a 9599% pure granulocyte population. In each experiment described below, cells were incubated with erythromycin or miocamycin at three different final concentrations -2.5, 5 and 10 pg/ml -- for 30 minutes. Results were compared to those obtained using a population of cells not incubated with antibiotics. ISSN 1120-009X
269
EFFECTS OF MIOCAMYCIN AND ERYTHROMYCIN ON POLYMORPHONUCLEAR CELL FUNCTIO N
Chemotaxis
Statistical studies
Chemotaxis was evaluated with Boyden chamber (Arnika Milano, Italy) method, as described by Territo 4 • Cells incubated with erythromycin or miocamycin and control samples were put in the presence of N-peptide. Chemotactic index was calculated as follows:
Five samples of each group were prepared for each test. Student's t test was used .
Downloaded by [Australian Catholic University] at 21:59 08 August 2017
CI
= -
c
X
100
R where C = mean value of CI in the presence of chemotactic peptide. R = mobility in the absence of stimulus. Both C and R are expressed in I! (micron).
LTB4 production Cells were used at the concentration of 10 6/ml. Production was measured by a radioimmunoassay (MEN DuPont de Nemours, Bad Homburg, West Germany) after stimulation of cells with ionophore A23187, in the presence or absence of erythromycin and miocamycin at the same concentrations as for chemotactic experiments.
RESULTS
Chemotaxis As shown in Table 1, 2.5, 5 and 10 pg/ml of erythromycin were unable to induce a modification in the chemotactic index if compared to controls, while 5 and 10 pg/ml of miocamycin determined a significant increase.
LTB4 production If compared to unstimulated cells, PMNs incubated with 5 and 10 pg/ml of erythromycin or miocamycin significantly increased their LTB4 production, while both antibiotics were ineffective at the lowest concentration used (2.5 pg) in our experiments (Table 2). TABLE 1 - Chemotactic Index. M .*
S.D .
160
10
2.5 pg 5.0 pg 10.0 pg
180 260 442
20 19 22
2.5 pg 5.0 pg 10.0 pg
167 171 178
20 22 22
Controls
Killing of Candida albicans 0.5 ml of neutrophil suspension (5 X 10 6/ml) were incubated with 0.5 ml of Candida (5 X 10 6 ) in the presence or not of antibiotics. 0.1 ml of the mixture and 0.3 ml of phosphate buffered saline (PBS) containing 15% fetal calf serum (FCS) were mixed, and cells finally cytocentrifuged. The smears were fixed in air, then with absolute methanol and stained with Giemsa. Dead Candida were recognized by the failure to take up stain and/or the presence of other morphological abnormalities.
Respiratory burst Cells (1 X 10 6/ml) were incubated with nitroblutetrazolium (Sigma Chemical Co, St. Louis, USA) for 30 min at 37°C. Cells were cytocentrifuged, air dried and fixed in absolute methanol for 1 min, and thereafter stained with safranin (lstituto Behring, Scoppito, Italy). The test was considered positive if more than 10% of cells contained formaz an precipitate .
Miocamycin >>
»
Erythromycin » »
* (mea n values from 3 experiments) .
TABLE 2 - Leukotriene B4 production (pgjm/) .
KI** KA-** Miocamycin » »
Erythromycin » »
M .*
S.D .
2.5 pg 5.0 pg 10.0 pg
0 51 57 225 398
2.3 2.7 4.2 3.7
2 .5 pg 5.0 pg 10.0 pg
54 151 243
2.8 3.2 4.5
* (mean values from 3 experiments) . ** KI Unstimulated Cells. KA- Stimulated cells not incubated in the presence of miocamycin or erythromycin .
270
P. BACCI - A. GROSSI - A.M. VANNUCCHI - D. RAFANELU - A. CASINI - M. DE LUCA
TABLE 3 - Killing of Candida albicans. Live
Controls Miocamycin
mean*
S.D.
mean
S.D
61
4
40
3
5 5
2.5 pg
55
3
45
))
5.0 pg
5
))
10.0 pg
38 26
8
62 74
60 61 58
3 4 6
40 38 42
Erythromycin ))
))
Downloaded by [Australian Catholic University] at 21:59 08 August 2017
Dead
2.5 pg 5.0 pg 10.0 pg
10
5 4
5
* (mean values from 3 experiments).
Killing of Candida albicans Miocamycin, but not erythromycin, was able to increase the killing of Candida. Again a concentration of at least 5 pg/ml of antibiotic was necessary to elicit the response (Table 3).
Respiratory burst
tration of 500 mg of erythromycin or 600 mg of miocamycin 6 • In our experiments concentrations as low as 5 and 10 pg/ml of miocamycin induced a significant increase in the chemotaxis index, killing of Candida; and LTB4 production. Respiratory burst required a concentration of 10 pg/ml to be elicited. Although erythromycin has been reported to stimulate the killing activity in PMNs, we could not confirm this effect, while LTB4 production was significantly increased. Because this is a stimulator of PMN migration, it may seem contradictory that chemotaxis was unmodified after exposure to the antibiotic. Perhaps higher erythromycin concentrations are necessary to exert an effect on PMN activity and, although increased, LTB4 levels reached during the incubation with the antibiotic may have not been sufficient to influence chemotaxis. In conclusion, our data demonstrate that miocamycin, but not erythromycin, is active on PMN function even at very low concentrations.
REFERENCES
Using NBT (nitroblue tetrazolium) reduction test, formazan precipitate was observed in more than 10% of cells only when a concentration of 10 pg/ml of miocamycin was used, while the same amount of erythromycin induced only a modest, not significant response.
DISCUSSION
The main difference between macrolides and other classes of antibiotics is their ability to enter phagocytic cells, stimulating their functional activities 1 • 2 • 3 • Experiments published so far have mainly addressed the uptake and killing of bacteria s •6 • '. In this paper we have tested the capability of two macrolides, erythromycin and miocamycin, to increase chemotaxis, respiratory burst, and production of LTB4, a potent mediator of inflammatory reactions, which promotes cell migration in vivo and in vitro 8 • 9 • The final antibiotic concentrations used in our experiments were lower than those usually reached 1 hour and 8 hours after the adminis-
' Prokesch RC, Hand L. Antibiotic entry into human polymorphonuclear leucocytes. Antimicrob Agents Chemother 1982; 21: 373-380. 'Johnson JD, Hand WL, Francis JB, King-Thompson N, Crowin RW . Antibiotic uptake by alveolar macrophages. J Lab Clin Med 1980; 95: 4429-4439. 'Van Rensburg CEJ, Anderson R, Joone G, Van Der Merwe AJ, Van Rensburg AJ. Effects of Erythromycyn on cellular functions in vitro and in vivo . J Antimicrob Chemother 1981; 8: 467-474 . • Territo MC . Chemotaxis. In : Cline MJ ed. Leukocyte Function. Churchill Livingstone, Edinburgh: 1981; 39-52. 'Lee Hand W, King-Thompson NL, Johnson JD. Influence of bacteri al-antibiotic interactions on subsequent antimicrobial activity of alveolar macrophages. J Infect Dis 1984; 149: 271-276. • Capelli A, Capelli 0 , Azzolini L, Richeldi L, Prandi E, Velluti G . Activities of human alveolar macrophages (HAMs). Note 1: observations on phagocytosis and bacterial killing in the presence of Miocamycin. Chemioterapia 1988; 7: 89-95 . ' Fraschini F, Scaglione F, Ferrara F, Dugnani S, Falchi M, Cattaneo G . Miocamycin and leukocyte activity in man. Chemotherapy 1989; 35: 289-295. 'Ford-Hutchinson AW, Bray MA, Doing MV, Shipley ME, Smith JH . Leukotriene B, a potent chemokinetic and aggregating substance released from polymorphonuclear leukocytes. Nature 1980; 286: 264-265 . • Palmblad J, Malmsten CL, Uden AM, Radmark 0, Engstedt L, Samuelsson B. Leukotriene B4 is potent and stereospecific stimulator of neutrophil chemotaxis and adherence. Blood 198 1; 58: 658-661.
