Journal of Antimicrobial Chemotherapy (1990) 26, 683-6*8
Influence of teicoplanin and vancomycin on degranulation by polymorphonuclear leucocytes stimulated by various agonists: an in-vitro study P. Van der Auwera, M. Bonnet and M. Husson
The interaction between vancomycin and teicoplanin (50 and 100 mg/1) on human neutrophils was studied using fMLP- and A23187-induced degranulation of elastase, /J-glucuronidase and vitamin B12-binding-protcin. Each of these proteins is a marker of a different population of granules. fMLP-induced degranulation of /J-glucuronidase was significantly impaired by pre-incubating the neutrophils with teicoplanin (without affecting cell viability) although the inhibition was at most 23% at 100 mg/1, a concentration not achieved in the serum of patients after normal doses. Calcium ionophore-induced degranulation of /7-glucuronidase and elastase were slightly impaired (Student two-tailed; P < 0-1) by both glycopeptides (/?-glucuronidase) and teicoplanin only (elastase). Again, inhibition remained below 25%. Vancomycin and teicoplanin at high concentrations, seldom achieved in patients, can moderately impair neutrophil degranulation. Introduction Vancomycin (Van der Auwera, Matsumoto & Husson, 1988) and teicoplanin (Pascual et al., 1987; Van der Auwera et al., 1988) are rapidly accumulated by polymorphonuclear leucocytes and macrophages with cell-associated/extracellular concentration ratio >10. Accumulation of teicoplanin by macrophages is a rapid ( ^ 3 0 min) and passive phenomenon (Carlone et al, 1989). As far as intracellular bioactivity of glycopeptide is concerned, conflicting results have been reported. Carper, Sullivan & Mandell (1987) showed no enhancement of the intracellular killing of Staphylococcus aureus after exposure of polymorphonuclear leucocytes to vancomycin and teicoplanin even at very high concentration (100 mg/1). Fietta et al. (1986) showed that teicoplanin but not vancomycin increased intracellular killing of 5. aureus by polymorphonuclear leucocytes. Pascual et al. (1987) showed a significant increase in intracellular killing of S. aureus but not S. epiaermidisr, however, the increase was modest by comparison with the very high cell-associated antimicrobial concentration. Carlone et al. (1989) showed that intracellular killing of S. aureus by macrophages was slightly but significantly increased in the presence of teicoplanin at half the MIC. Other antimicrobials which are strongly accumulated by polymorphonuclear leucocytes (i.e. macrolides, coumermycin, amphotericin B) have been shown to impair several of their functions (Van der Auwera et al., 1986; Labro, el Benna & Babin-Chevaye, 1989; Van der Auwera & 683 03O5-7453/9O/I10683 + O6 $02.00
© 1990 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
Service de Medecine et Laboratoire a"Investigation Clinique H. J. Tagnon, (Clinique des Maladies Infectieuses et Laboratoire de Microbiologie) Institut Jules Bordet, Centre des Tumeurs de I'Universite Libre de Bruxelles, rue Heger-Bordet, J B-1000 Bruxelles, Belgium
684
P. Van der Anwera et aL
Materials and methods Volunteers Healthy volunteers (source of the neutrophils) were tested after having given their written informed consent. Medication was not allowed for a period of one month before the experiments. Each test was performed with the neutrophils of at least four different volunteers. Isolation of the polymorphonuclear leucocytes The polymorphonuclear leucocytes were isolated as previously described (Van der Auwera et al., 1.988), by dextran sedimentation and Ficoll-Hypaque density centrifugation. Viability was checked before and after each test by trypan blue exclusion and LDH release. Purity (May-Grunwald) of the neutrophil preparation was ^ 9 7 % and viability was >97%. Degranulation This was done according to Metcalf et al. (1986) using a suspension of 3-5 107 neutrophils/ml) preincubated for 5 min with cytochalasin B (5 mg/1). Two stimuli were tested: f-MLP (10~5 M, final concentration) and the calcium ionophore A-23187 (6 x 10~6 M, final concentration). The following proteins were measured in the supernatant (10 sec at 12000 g) to measure degranulation of the different granules: /?-glucuronidase (a marker of tertiary granules—peroxidase-negative), elastase (a marker of primary azurophilic granules) and vitamin B12 binding protein (a marker of secondary specific granules). /J-Glucuronidase was measured spectrophotometrically (Metcalf et al., 1986) by a microtitre plate method. Briefly, 60 /xl of supernatant from degranulated or control cells was transferred into flat-bottom microtitre plates (96 wells, Nunc) to which 75 /J of acetate buffer (0-2 M, pH 50) was added. The substrate was then added (15 /d of 001 M />-nitrophenyl-/?-D glucuronide). After 2 h incubation at 37°C, the reaction was stopped with 150 /d NaOH 0-2 M OD was read in a microtitre plate
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
Meunier, 1989; Hand, Hand & King-Thompson, 1990). These results suggest that glycopeptides may have a toxic effect on phagocytic functions. We have reported that vancomycin and teicoplanin does not modify superoxide generation (Van der Auwera et al., 1986), random migration and C5a- and fMLPinduced chemotaxis in agar, adherence to polystyrene surface, MTT reduction (Van der Auwera & Husson, 1989), and phagocytosis of opsonized radiolabelled S. aureus by polymorphonuclear leucocytes preincubated with clinically relevant concentrations (Van der Auwera, Husson & Fruhling, 1987). At very high concentration (250 mg/1), teicoplanin can impair degranulation and chemiluminescence ( ^ 50 mg/1) (Schumacher-Perdreau et al., 1989), and chloramine production (Verrilli et al., 1987) by human neutrophils, although the mechanism of inhibition has not been studied. Degranulation of proteins specific for different intracellular granules may be induced by several agonists which are associated with specific membrane receptors (as fMLP) or intracellular calcium mobilization (as ionophore A23187) (Metcalf et al., 1986; Steadman et al., 1988). This may be used as a model to investigate eventual toxicity of glycopeptides on neutrophils. The purpose of the present study is to investigate the eventual inhibition of degranulation by the glycopeptides.
Degramdfltion of PMNs
685
Influence of vancomycin and teicoplanin on degranulation
Polymorphonuclear leucocytes were preincubated with the test antibiotic (50 and 100 mg/1) for 30 min at 37°C before cytochalasin B was added and the stimulus was applied. Unstimulated PMNs were included to control for toxicity.
Results Viability Vancomycin and teicoplanin (up to 100 mg/1) are not toxic for the PMNs as measured by trypan blue exclusion, release of LDH and release of granule markers (vitamin B12-binding protein, elastase and /?-glucuronidase). Influence of vancomycin and teicoplanin
Teicoplanin was slightly, although significantly, inhibitory to the degranulation of /?-glucuronidase induced by the two stimuli (Table I). Inhibition remained below 20% of the control. Degranulation of elastase was variably affected by the two antibiotics. Degranulation of vitamin B12 binding protein was unaffected by the two antibiotics whether induced by fMLP or A23187. Possible mechanisms
Several possible mechanisms were explored. Inactivation of fMLP by the glycopeptides was ruled out by preparing fMLP in vancomycin or teicoplanin 100 mg/1 and showing that degranulation of control neutrophils remained unchanged. Binding of test proteins (/?-glucuronidase and elastase) by the glycopeptide was ruled out, since the recovery of these two proteins was 100% when assayed in the presence of 100 mg/1 of the two antimicrobial agents.
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
spectrophotometer reader at 405 nm. Elastase was measured by a modified ELISA procedure (PMN-elastase; Merck, Darmstadt) using alpha 1-anti-trypsin (10 mg/1; Sigma A9024, St Louis, USA) to complex the secreted enzyme. Vitamin B12 binding protein was measured by a radioisotopic method using ("CoVcyanocobalamine (Amersham International pic, Cardiff, United Kingdom) (Steadman et al., 1988). Briefly, 250 /xl "co-vitamin B12 (Amersham CT 12) 4-44 ng/ml (67 ncurie/ml) in borate buffer pH 9-3 was added to 100 /J of degranulate and left for 30 min at room temperature. Charcoal solution (1 ml at 2-5% [wt/v], dextran T70 0-25%, BSA 0-1%) was added to eliminate the unbound ligand (10 min). Supernatant (2500 g, 10 min, 6°Q was counted in a gamma counter (Hewlett-Packard). The test was linear in the range of 106—3 x 107 polymorphonuclear leucocytes. Unstimulated cells were used as control. Unstimulated neutrophils were sonicated in the presence of 2-5 mg/107 cells to obtain the total amount of each of the three proteins tested. Degranulation (%) was expressed as ((test protein in supernatant from stimulated cells)—(test protein in supernatant from unstimulated cells)) x 100/(test protein in supernatant from unstimulated sonicated cells).
686
P. Van der Anwera et aL
Table I. Degranulation of polymorphonudcar leucocytes induced by fMLP and calcium ionophore A23187: influence of vancomycin and teicoplanin. Degranulation (% of total content) expressed as mean ± SEM Control («)
Teicoplanin
Vancomycin 100
fMLP /J-glucuronidase (16) 27-6 ±2-9 28-2 ±2-3 elastase (8) 49-8 ±7-5 391 ±6-9 vitamin B12-binding protein (9) 29-7 ±4-3 30-3 ±3-9
100
27-6 ± 3 4
21-3±2-2"
23-1 ±1-9*
45-7 ±6-6
38-9 ±71
44-9 ±5-7
291 ±5-6
29-8 ±3-3
31-O±3-4
18-7 ±4-7*
15-7 ±2-9*
14O±31*
460 ±7-9
26-7±51*
24-2 ±3-9*
5O0±31
42-7 ±4-9
43-5 ±6-8
'P < (M)5 (Student paired test, two tailed, w control). */ > 0-5 (JM) is a better inducer of primary and tertiary granule release. Therefore, any interaction with one of the steps leading to degranulation would be detected. In addition, elastase is considered to be the major mediator of endothelial injury caused by stimulated neutrophils and probably this applies to most tissues (Henson & Johnson 1987). We have shown that high concentrations (seldom achieved in clinical use) of teicoplanin and vancomycin may impair the degranulation of polymorphonuclear leucocytes. Both receptor-dependent and receptor-independent mechanisms were affected. Inhibition seemed to be specific to one of the granules investigated (/?-glucuronidase containing granules) since degranulation of azurophilic (as explored by elastase) and
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
A23187 /?-g]ucuronidase (4) 17-7 ±4-7* 20-35 ±3-5 elastase (4) 45-5 ±10-0 34-2 ±3-8 vitamin B12-binding protein (4) 53-5 ±3-3 46-2 ±5-5
50mg/l
50
Degradation of PMNs
687
Acknowledgements We acknowledge Mrs V. Duchateau and A. van Praet for their technical assistance. This study was supported by a grant from Merrell-Dow Lepetit. References Carlone, N. A. Cuffini, A. M., Ferrero, M., Tullio, V. & Avetta, G. (1989). Cellular uptake, and intracellular bactericidal activity of teicoplanin in human macrophages. Journal of Antimicrobial Chemotherapy 23, 849-59. Carper, H. T., Sullivan, G. W. & Mandell, G. L. (1987). Teicoplanin, vancomycin,rifampicin:invivo and in-vitro studies with Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 19, 659-62. Fietta, A., Bersani, C, De Rose, V., Grassi, F. M. & Grassi, G. G. (1986). The effect of teicoplanin on leukocylic activity and intraleukocytk micro-organisms. Journal of Hospital Infection 7, Suppl. A, 57-63. Hand, W. L., Hand, D. L. & King-Thompson, N. L. (1990). Antibiotic inhibition of the respiratory burst response in human polymorphonuclear leukocytes. Antimicrobial Agents and Chemotherapy 34, 863-70. Henson, P. M. & Johnston, R. B. (1987). Tissue injury and inflammation. Oxidants, proteinases, and cationic proteins. Journal of Clinical Investigation. 79, 669-74.
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
specific granules (as explored by vitamin B12 binding protein) were not affected, with the exception of the slight inhibition by teicoplanin of elastase degranulation induced by A23187. Inactivation of the two agonists by the glycopeptides was ruled out as well as the interaction with the assay methods of ^-glucuronidase and elastase. Interaction with microtubules can be excluded, since teicoplanin did not affect the secretion of vitamin B12 binding protein. Interaction with microfiJaments can be excluded, since teicoplanin did not affect random migration and chemotaxis induced by fMLP and zymosan-activated serum (Van der Auwera & Husson, 1989). In the study by Schumacher-Perdreau et al. (1989), teicoplanin (250 mg/1), but not vancomycin, inhibited 44-0-45-3 % of lysozyme and /?-glucuronidase release by neutrophils stimulated by fMLP and 21-9% of /J-glucuronidase release after stimulation with opsonized zymosan. This is in contrast to our results, in which the release of vitamin B12-binding protein (a marker of specific granules, like lysozyme) was not impaired by teicoplanin. This discrepancy might be due to the very high and clinically irrelevant concentration tested by Schumacher-Perdreau et al. (1989). The inhibition of 0-glucuronidase-containing granules might be a consequence of the binding of the glycopeptides to a cellular site, presumably the plasma or the granule membrane of the neutrophil. This binding site has not been identified yet, since radiolabelled vancomycin and teicoplanin (with high specific activity) are not available. Phagolysosomes are probably not a significant binding site since intracellular killing of S. aureus was not, or only modestly, increased (Fietta et al., 1986; Carper et al., 1987; Pascual et al. 1987; Carlone et al., 1989). Subcellular fractionation (Tulkens, 1985) would probably identify the binding site, although the available assay methods of these antimicrobials are not sensitive enough. Intracellular metabolism with generation of a toxic metabolite is also a possibility as yet unexplored. The clinical consequences of the inhibition of degranulation by the glycopeptides (at most 25% inhibition) are probably limited since the concentration at which inhibition was observed was fairly high, and above clinically achievable concentrations with the recommended doses.
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{Received 9 June 1990; accepted 10 July 1990)
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Labro, M. T., cl Bcnna, J. & Babin-Chcvaye, C. (1989). Comparison of the in-vitro effect of several macrolides on the oxidative burst of human neutrophils. Journal of Antimicrobial Chemotherapy 24, 561-72. Metcalf, J. A., Gallin, J. I., Nauseef, W. M. & Root, R. K. (1986). Laboratory Manual of Neutrophii Function. Raven Press, New York. Pascual, A., Tsukayama, D., Kovarik, J., Gekker, G. & Peterson, P. (1987). Uptake and activity of rifapentine in human peritoneal macrophages and polymorphonuclear leukocytes. European Journal of Clinical Microbiology 6, 152-7. Schumacher-Perdreau, F., Schell-Frederick, E., Peters, G. & Pulverer, G. (1987). Effects of glycopeptide and lipopcptide antibiotics on granulocyte function in vitro. In The Influence of Antibiotics on the Host-Parasite Relationship III (Gillessen, G., Opferkuch, W., Peters, G. & Pulverer, G., Eds), pp. 144-51. Springer-Verlag, Berlin. Snyderman, R. & Verghese, M. W. (1987). Leukocyte activation by chemoattractant receptors: roles of a guanine nuclcotide regulatory protein and polyphosphoinositide metabolism. Reviews of Infectious Diseases 9, Suppl. 5, S562-9. Spitznagel, J. K. & Shafer, W. M. (1985). Neutrophii killing of bacteria by oxygen-independent mechanisms: a historical summary. Reviews of Infectious Diseases 7, 398-403. Steadman, R., Topley, N., Jenner, D. E., Davies, M. & Williams, J. D. (1988). Type 1 fimbriate Escherichia coli stimulates a unique pattern of degranulation by human polymorphonuclear leukocytes. Infection and Immunity 56, 815-22. Tulkens, P. M. (1985). The design of antibiotics capable of an intracellular action: aims, potentialities and problems. In Drug Targeting (Bun, P. & Gumma, A., Eds), pp. 179-94. Elsevier Science Publishers, Amsterdam. Van der Auwera, P. & Husson, M. (1989). Influence of antibiotics on motility and adherence of human neutrophils studied in vitro. Drugs Under Experimental and Clinical Research 15, 211-8. Van der Auwera, P., Husson, M. & Fruhling, J. (1987). Influence of various antibiotics on phagocytosis of Staphylococcus aweus by human polymorphonuclear leucocytes. Journal of Antimicrobial Chemotherapy 20, 399-404. Van der Auwera, P., Matsumoto, T. & Husson, M. (1988). Intraphagocytic penetration of antibiotics. Journal of Antimicrobial Chemotherapy 22, 185-92. Van der Auwera, P. & Meunier, F. (1989). In-vitro effects of cilofungin (LY 121019), amphotericin B and amphotericin B-deoxycholate on human polymorphonuclear leucocytes. Journal of Antimicrobial Chemotherapy 24, 747-63. Van der Auwera, P., Petrikkos, G., Husson, M. & Klastersky, J. (1986). Influence of various antibiotics on superoxide generation by normal human neutrophils. Archives Internationales de Physiologie et de Biochimie 94, S23-8. Van der Auwera, P., Petrikkos, G., Matsumoto, T. & Husson, M. (1988). Influence of LY146032 on human polymorphonuclear leucocytes in vitro. Journal of Antimicrobial Chemotherapy 21, 57-63. Verrilli, M., Breaux, S., Dickey, J. & Maderazo, E. G. (1987). Characterization of the inhibitory effects of teicoplanin on the bactericidal activity of human granulocytes. In Program and Abstracts of the Twenty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, NY. 1987. Abstract 880, p. 250. American Society for Microbiology, Washington, DC.
Influence of teicoplanin and vancomycin on degranulation by polymorphonuclear leucocytes stimulated by various agonists: an in-vitro study P. Van der Auwera, M. Bonnet and M. Husson
The interaction between vancomycin and teicoplanin (50 and 100 mg/1) on human neutrophils was studied using fMLP- and A23187-induced degranulation of elastase, /J-glucuronidase and vitamin B12-binding-protcin. Each of these proteins is a marker of a different population of granules. fMLP-induced degranulation of /J-glucuronidase was significantly impaired by pre-incubating the neutrophils with teicoplanin (without affecting cell viability) although the inhibition was at most 23% at 100 mg/1, a concentration not achieved in the serum of patients after normal doses. Calcium ionophore-induced degranulation of /7-glucuronidase and elastase were slightly impaired (Student two-tailed; P < 0-1) by both glycopeptides (/?-glucuronidase) and teicoplanin only (elastase). Again, inhibition remained below 25%. Vancomycin and teicoplanin at high concentrations, seldom achieved in patients, can moderately impair neutrophil degranulation. Introduction Vancomycin (Van der Auwera, Matsumoto & Husson, 1988) and teicoplanin (Pascual et al., 1987; Van der Auwera et al., 1988) are rapidly accumulated by polymorphonuclear leucocytes and macrophages with cell-associated/extracellular concentration ratio >10. Accumulation of teicoplanin by macrophages is a rapid ( ^ 3 0 min) and passive phenomenon (Carlone et al, 1989). As far as intracellular bioactivity of glycopeptide is concerned, conflicting results have been reported. Carper, Sullivan & Mandell (1987) showed no enhancement of the intracellular killing of Staphylococcus aureus after exposure of polymorphonuclear leucocytes to vancomycin and teicoplanin even at very high concentration (100 mg/1). Fietta et al. (1986) showed that teicoplanin but not vancomycin increased intracellular killing of 5. aureus by polymorphonuclear leucocytes. Pascual et al. (1987) showed a significant increase in intracellular killing of S. aureus but not S. epiaermidisr, however, the increase was modest by comparison with the very high cell-associated antimicrobial concentration. Carlone et al. (1989) showed that intracellular killing of S. aureus by macrophages was slightly but significantly increased in the presence of teicoplanin at half the MIC. Other antimicrobials which are strongly accumulated by polymorphonuclear leucocytes (i.e. macrolides, coumermycin, amphotericin B) have been shown to impair several of their functions (Van der Auwera et al., 1986; Labro, el Benna & Babin-Chevaye, 1989; Van der Auwera & 683 03O5-7453/9O/I10683 + O6 $02.00
© 1990 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
Service de Medecine et Laboratoire a"Investigation Clinique H. J. Tagnon, (Clinique des Maladies Infectieuses et Laboratoire de Microbiologie) Institut Jules Bordet, Centre des Tumeurs de I'Universite Libre de Bruxelles, rue Heger-Bordet, J B-1000 Bruxelles, Belgium
684
P. Van der Anwera et aL
Materials and methods Volunteers Healthy volunteers (source of the neutrophils) were tested after having given their written informed consent. Medication was not allowed for a period of one month before the experiments. Each test was performed with the neutrophils of at least four different volunteers. Isolation of the polymorphonuclear leucocytes The polymorphonuclear leucocytes were isolated as previously described (Van der Auwera et al., 1.988), by dextran sedimentation and Ficoll-Hypaque density centrifugation. Viability was checked before and after each test by trypan blue exclusion and LDH release. Purity (May-Grunwald) of the neutrophil preparation was ^ 9 7 % and viability was >97%. Degranulation This was done according to Metcalf et al. (1986) using a suspension of 3-5 107 neutrophils/ml) preincubated for 5 min with cytochalasin B (5 mg/1). Two stimuli were tested: f-MLP (10~5 M, final concentration) and the calcium ionophore A-23187 (6 x 10~6 M, final concentration). The following proteins were measured in the supernatant (10 sec at 12000 g) to measure degranulation of the different granules: /?-glucuronidase (a marker of tertiary granules—peroxidase-negative), elastase (a marker of primary azurophilic granules) and vitamin B12 binding protein (a marker of secondary specific granules). /J-Glucuronidase was measured spectrophotometrically (Metcalf et al., 1986) by a microtitre plate method. Briefly, 60 /xl of supernatant from degranulated or control cells was transferred into flat-bottom microtitre plates (96 wells, Nunc) to which 75 /J of acetate buffer (0-2 M, pH 50) was added. The substrate was then added (15 /d of 001 M />-nitrophenyl-/?-D glucuronide). After 2 h incubation at 37°C, the reaction was stopped with 150 /d NaOH 0-2 M OD was read in a microtitre plate
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
Meunier, 1989; Hand, Hand & King-Thompson, 1990). These results suggest that glycopeptides may have a toxic effect on phagocytic functions. We have reported that vancomycin and teicoplanin does not modify superoxide generation (Van der Auwera et al., 1986), random migration and C5a- and fMLPinduced chemotaxis in agar, adherence to polystyrene surface, MTT reduction (Van der Auwera & Husson, 1989), and phagocytosis of opsonized radiolabelled S. aureus by polymorphonuclear leucocytes preincubated with clinically relevant concentrations (Van der Auwera, Husson & Fruhling, 1987). At very high concentration (250 mg/1), teicoplanin can impair degranulation and chemiluminescence ( ^ 50 mg/1) (Schumacher-Perdreau et al., 1989), and chloramine production (Verrilli et al., 1987) by human neutrophils, although the mechanism of inhibition has not been studied. Degranulation of proteins specific for different intracellular granules may be induced by several agonists which are associated with specific membrane receptors (as fMLP) or intracellular calcium mobilization (as ionophore A23187) (Metcalf et al., 1986; Steadman et al., 1988). This may be used as a model to investigate eventual toxicity of glycopeptides on neutrophils. The purpose of the present study is to investigate the eventual inhibition of degranulation by the glycopeptides.
Degramdfltion of PMNs
685
Influence of vancomycin and teicoplanin on degranulation
Polymorphonuclear leucocytes were preincubated with the test antibiotic (50 and 100 mg/1) for 30 min at 37°C before cytochalasin B was added and the stimulus was applied. Unstimulated PMNs were included to control for toxicity.
Results Viability Vancomycin and teicoplanin (up to 100 mg/1) are not toxic for the PMNs as measured by trypan blue exclusion, release of LDH and release of granule markers (vitamin B12-binding protein, elastase and /?-glucuronidase). Influence of vancomycin and teicoplanin
Teicoplanin was slightly, although significantly, inhibitory to the degranulation of /?-glucuronidase induced by the two stimuli (Table I). Inhibition remained below 20% of the control. Degranulation of elastase was variably affected by the two antibiotics. Degranulation of vitamin B12 binding protein was unaffected by the two antibiotics whether induced by fMLP or A23187. Possible mechanisms
Several possible mechanisms were explored. Inactivation of fMLP by the glycopeptides was ruled out by preparing fMLP in vancomycin or teicoplanin 100 mg/1 and showing that degranulation of control neutrophils remained unchanged. Binding of test proteins (/?-glucuronidase and elastase) by the glycopeptide was ruled out, since the recovery of these two proteins was 100% when assayed in the presence of 100 mg/1 of the two antimicrobial agents.
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
spectrophotometer reader at 405 nm. Elastase was measured by a modified ELISA procedure (PMN-elastase; Merck, Darmstadt) using alpha 1-anti-trypsin (10 mg/1; Sigma A9024, St Louis, USA) to complex the secreted enzyme. Vitamin B12 binding protein was measured by a radioisotopic method using ("CoVcyanocobalamine (Amersham International pic, Cardiff, United Kingdom) (Steadman et al., 1988). Briefly, 250 /xl "co-vitamin B12 (Amersham CT 12) 4-44 ng/ml (67 ncurie/ml) in borate buffer pH 9-3 was added to 100 /J of degranulate and left for 30 min at room temperature. Charcoal solution (1 ml at 2-5% [wt/v], dextran T70 0-25%, BSA 0-1%) was added to eliminate the unbound ligand (10 min). Supernatant (2500 g, 10 min, 6°Q was counted in a gamma counter (Hewlett-Packard). The test was linear in the range of 106—3 x 107 polymorphonuclear leucocytes. Unstimulated cells were used as control. Unstimulated neutrophils were sonicated in the presence of 2-5 mg/107 cells to obtain the total amount of each of the three proteins tested. Degranulation (%) was expressed as ((test protein in supernatant from stimulated cells)—(test protein in supernatant from unstimulated cells)) x 100/(test protein in supernatant from unstimulated sonicated cells).
686
P. Van der Anwera et aL
Table I. Degranulation of polymorphonudcar leucocytes induced by fMLP and calcium ionophore A23187: influence of vancomycin and teicoplanin. Degranulation (% of total content) expressed as mean ± SEM Control («)
Teicoplanin
Vancomycin 100
fMLP /J-glucuronidase (16) 27-6 ±2-9 28-2 ±2-3 elastase (8) 49-8 ±7-5 391 ±6-9 vitamin B12-binding protein (9) 29-7 ±4-3 30-3 ±3-9
100
27-6 ± 3 4
21-3±2-2"
23-1 ±1-9*
45-7 ±6-6
38-9 ±71
44-9 ±5-7
291 ±5-6
29-8 ±3-3
31-O±3-4
18-7 ±4-7*
15-7 ±2-9*
14O±31*
460 ±7-9
26-7±51*
24-2 ±3-9*
5O0±31
42-7 ±4-9
43-5 ±6-8
'P < (M)5 (Student paired test, two tailed, w control). */ > 0-5 (JM) is a better inducer of primary and tertiary granule release. Therefore, any interaction with one of the steps leading to degranulation would be detected. In addition, elastase is considered to be the major mediator of endothelial injury caused by stimulated neutrophils and probably this applies to most tissues (Henson & Johnson 1987). We have shown that high concentrations (seldom achieved in clinical use) of teicoplanin and vancomycin may impair the degranulation of polymorphonuclear leucocytes. Both receptor-dependent and receptor-independent mechanisms were affected. Inhibition seemed to be specific to one of the granules investigated (/?-glucuronidase containing granules) since degranulation of azurophilic (as explored by elastase) and
Downloaded from http://jac.oxfordjournals.org/ at UQ Library on June 13, 2015
A23187 /?-g]ucuronidase (4) 17-7 ±4-7* 20-35 ±3-5 elastase (4) 45-5 ±10-0 34-2 ±3-8 vitamin B12-binding protein (4) 53-5 ±3-3 46-2 ±5-5
50mg/l
50
Degradation of PMNs
687
Acknowledgements We acknowledge Mrs V. Duchateau and A. van Praet for their technical assistance. This study was supported by a grant from Merrell-Dow Lepetit. References Carlone, N. A. Cuffini, A. M., Ferrero, M., Tullio, V. & Avetta, G. (1989). Cellular uptake, and intracellular bactericidal activity of teicoplanin in human macrophages. Journal of Antimicrobial Chemotherapy 23, 849-59. Carper, H. T., Sullivan, G. W. & Mandell, G. L. (1987). Teicoplanin, vancomycin,rifampicin:invivo and in-vitro studies with Staphylococcus aureus. Journal of Antimicrobial Chemotherapy 19, 659-62. Fietta, A., Bersani, C, De Rose, V., Grassi, F. M. & Grassi, G. G. (1986). The effect of teicoplanin on leukocylic activity and intraleukocytk micro-organisms. Journal of Hospital Infection 7, Suppl. A, 57-63. Hand, W. L., Hand, D. L. & King-Thompson, N. L. (1990). Antibiotic inhibition of the respiratory burst response in human polymorphonuclear leukocytes. Antimicrobial Agents and Chemotherapy 34, 863-70. Henson, P. M. & Johnston, R. B. (1987). Tissue injury and inflammation. Oxidants, proteinases, and cationic proteins. Journal of Clinical Investigation. 79, 669-74.
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specific granules (as explored by vitamin B12 binding protein) were not affected, with the exception of the slight inhibition by teicoplanin of elastase degranulation induced by A23187. Inactivation of the two agonists by the glycopeptides was ruled out as well as the interaction with the assay methods of ^-glucuronidase and elastase. Interaction with microtubules can be excluded, since teicoplanin did not affect the secretion of vitamin B12 binding protein. Interaction with microfiJaments can be excluded, since teicoplanin did not affect random migration and chemotaxis induced by fMLP and zymosan-activated serum (Van der Auwera & Husson, 1989). In the study by Schumacher-Perdreau et al. (1989), teicoplanin (250 mg/1), but not vancomycin, inhibited 44-0-45-3 % of lysozyme and /?-glucuronidase release by neutrophils stimulated by fMLP and 21-9% of /J-glucuronidase release after stimulation with opsonized zymosan. This is in contrast to our results, in which the release of vitamin B12-binding protein (a marker of specific granules, like lysozyme) was not impaired by teicoplanin. This discrepancy might be due to the very high and clinically irrelevant concentration tested by Schumacher-Perdreau et al. (1989). The inhibition of 0-glucuronidase-containing granules might be a consequence of the binding of the glycopeptides to a cellular site, presumably the plasma or the granule membrane of the neutrophil. This binding site has not been identified yet, since radiolabelled vancomycin and teicoplanin (with high specific activity) are not available. Phagolysosomes are probably not a significant binding site since intracellular killing of S. aureus was not, or only modestly, increased (Fietta et al., 1986; Carper et al., 1987; Pascual et al. 1987; Carlone et al., 1989). Subcellular fractionation (Tulkens, 1985) would probably identify the binding site, although the available assay methods of these antimicrobials are not sensitive enough. Intracellular metabolism with generation of a toxic metabolite is also a possibility as yet unexplored. The clinical consequences of the inhibition of degranulation by the glycopeptides (at most 25% inhibition) are probably limited since the concentration at which inhibition was observed was fairly high, and above clinically achievable concentrations with the recommended doses.
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{Received 9 June 1990; accepted 10 July 1990)
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Labro, M. T., cl Bcnna, J. & Babin-Chcvaye, C. (1989). Comparison of the in-vitro effect of several macrolides on the oxidative burst of human neutrophils. Journal of Antimicrobial Chemotherapy 24, 561-72. Metcalf, J. A., Gallin, J. I., Nauseef, W. M. & Root, R. K. (1986). Laboratory Manual of Neutrophii Function. Raven Press, New York. Pascual, A., Tsukayama, D., Kovarik, J., Gekker, G. & Peterson, P. (1987). Uptake and activity of rifapentine in human peritoneal macrophages and polymorphonuclear leukocytes. European Journal of Clinical Microbiology 6, 152-7. Schumacher-Perdreau, F., Schell-Frederick, E., Peters, G. & Pulverer, G. (1987). Effects of glycopeptide and lipopcptide antibiotics on granulocyte function in vitro. In The Influence of Antibiotics on the Host-Parasite Relationship III (Gillessen, G., Opferkuch, W., Peters, G. & Pulverer, G., Eds), pp. 144-51. Springer-Verlag, Berlin. Snyderman, R. & Verghese, M. W. (1987). Leukocyte activation by chemoattractant receptors: roles of a guanine nuclcotide regulatory protein and polyphosphoinositide metabolism. Reviews of Infectious Diseases 9, Suppl. 5, S562-9. Spitznagel, J. K. & Shafer, W. M. (1985). Neutrophii killing of bacteria by oxygen-independent mechanisms: a historical summary. Reviews of Infectious Diseases 7, 398-403. Steadman, R., Topley, N., Jenner, D. E., Davies, M. & Williams, J. D. (1988). Type 1 fimbriate Escherichia coli stimulates a unique pattern of degranulation by human polymorphonuclear leukocytes. Infection and Immunity 56, 815-22. Tulkens, P. M. (1985). The design of antibiotics capable of an intracellular action: aims, potentialities and problems. In Drug Targeting (Bun, P. & Gumma, A., Eds), pp. 179-94. Elsevier Science Publishers, Amsterdam. Van der Auwera, P. & Husson, M. (1989). Influence of antibiotics on motility and adherence of human neutrophils studied in vitro. Drugs Under Experimental and Clinical Research 15, 211-8. Van der Auwera, P., Husson, M. & Fruhling, J. (1987). Influence of various antibiotics on phagocytosis of Staphylococcus aweus by human polymorphonuclear leucocytes. Journal of Antimicrobial Chemotherapy 20, 399-404. Van der Auwera, P., Matsumoto, T. & Husson, M. (1988). Intraphagocytic penetration of antibiotics. Journal of Antimicrobial Chemotherapy 22, 185-92. Van der Auwera, P. & Meunier, F. (1989). In-vitro effects of cilofungin (LY 121019), amphotericin B and amphotericin B-deoxycholate on human polymorphonuclear leucocytes. Journal of Antimicrobial Chemotherapy 24, 747-63. Van der Auwera, P., Petrikkos, G., Husson, M. & Klastersky, J. (1986). Influence of various antibiotics on superoxide generation by normal human neutrophils. Archives Internationales de Physiologie et de Biochimie 94, S23-8. Van der Auwera, P., Petrikkos, G., Matsumoto, T. & Husson, M. (1988). Influence of LY146032 on human polymorphonuclear leucocytes in vitro. Journal of Antimicrobial Chemotherapy 21, 57-63. Verrilli, M., Breaux, S., Dickey, J. & Maderazo, E. G. (1987). Characterization of the inhibitory effects of teicoplanin on the bactericidal activity of human granulocytes. In Program and Abstracts of the Twenty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, New York, NY. 1987. Abstract 880, p. 250. American Society for Microbiology, Washington, DC.
