Journal of Antimicrobial Chemotherapy (1990) 25, 613-620
In-vitro enhancement of leukotriene B 4 receptor expression on human neutrophils by cefadroxil J. KnODer, J. Brom, W. ScMnfeld and W. KSnig
The influence of cefadroxil on LTB4-receptor expression of polymorphonudear leucocytes (PMNs) was studied. Furthermore, the effect of cefadroxil on the leukotriene generation from PMNs and the lymphocyte, monocyte and basophil (LMB) containing cell fraction as well as on the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets was analysed. Antibiotic concentrations ranged from 50 to 5 £ig/10T cells. Analysis of the generated leukotrienes was performed by high performance liquid chromatography (HPLQ. Significant augmentation of the LTB«-receptor expression in human PMNs (range 190%220%) was observed at concentrations of SO and 25/ig/lO1 cells. The caltiumionophore A23187 induced LTB4 generation from PMNs as well as 12-HETE synthesis from platelets was not significantly modulated in the presence of cefadroxil. Preincubation of the human LMB fraction led to slight suppression of the ionophore induced LTB4 generation up to 20%. Introduction Cefadroxil is a semisynthctic, broad-spectrum cephalosporin for oral administration. It is active against group A /?-haemolytic streptococci, Streptococcus pneumoniae, staphylococci (including coagulase-positive, coagulase-negative, and pcnicillinase-producing
strains), Escherichia coli, Klebsiella spp., and Proteus mirabilis. In the past intensive research has been directed towards the influence of antibiotics on the function of granulocytes and monocytes which represent the first line of defence against bacterial infections (Manzella & Clark, 1983; Ohnishi et al., 1984; Duncker & UUmann, 1986). The generation of chemiluminescence during phagocytosis (Oleske et al., 1983) as well as the fMLP induced chemiluminescence (Briheim & Dahlgren, 1987) have been used as assay systems to investigate the influence of antimicrobial agents on phagocy tic cells. Leukotriene B4 (LTB4) which is generated via 5-lipoxygenase from arachidonic acid is a highly potent chemoattractant for polymorphonuclear leucocytes (PMNs). Furthermore, it is a potent stimulus in vitro: adherence, degranulation, superoxide production, and release of lysosomal enzymes were studied (O'Flaherty et al., 1986). 12-Hydroxyeicosatetraenoic acid (12-HETE) is also a chemotactic factor, which, however, is less potent than LTB4. It was the purpose of the present study to analyse the effect of cefadroxil on the specific binding of LTB4 to human polymorphonuclear leucocytes. Furthermore, the generation of leukotrienes from PMNs and from the lymphocyte, monocyte and 1
03O5- 7453/9O/04O613+08 $0100/0
613 © 1990 The British Society for Antimicrobial Chemotherapy
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Lehrstuhl fur Medizinische Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr Universitdt Bochum, FRG
614
J. KnSIkr et aL
basophil (LMB) containing cell fraction as well as the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets were analysed. Materials and methods
Materials
Preparation of peripheral human granulocytes and monocytes Heparinized venous blood (200 ml) was separated according to the method of Boyum (1976) by Ficoll-metrizoate gradient centrifugation. After centrifugation the lymphocyte, monocyte and basophil (LMB) containing cell fraction was washed twice in RPMI 1640 medium before use. The content of basophils was estimated using the Alcian blue staining technique (Gilbert & Ornstein, 1975). The unseparated LMB fraction was used in the experiments. Monocytes were identified according to their content of esterases (Tucker, Pierre & Jordan, 1977). The monocytes ranged from 1922%, lymphocytes from 74-78%, and the basophils were less than 4%. The erythrocytes within the granulocyte containing fraction were removed by dextran sedimentation and subsequent lysis (0-3% NaCl for 2 min on ice). The purity of the granulocyte fraction amounted to 99%. Preparation of platelets Nine volumes of human blood from healthy volunteers were mixed with 1 vol 0-9% NaCl containing 1-5% EDTA. Platelet-rich plasma (PRP) was separated by centrifugation at 200 g for 20 min; 2 vol of PRP and 1 vol of 01 M EDTA were mixed and the platelets sedimented at 2500 g for 20 min. The isolated platelets were suspended in 0-1 M EDTA and centrifuged for a further 20 min at 2500 g and finally resuspended in PBS containing 0-1% BSA (1 x 10s platelets/500 ft\ total incubation mixture). LTB4 binding assay The binding studies were performed as described by O'Flaherty, Kosfeld & Nishihira (1986) and Brom et al. (1988a). The assays were carried out using 96 well filtration plates with 5 ftm pore size-polyvinylidene fluoride membranes (Millipore, Eschborn, FRG). Each well contained 2, 3nM 3H-LTB4 (specific activity: 0-74-2-22 TBq/mmol) and 125/ig bovine serum albumin. After preincubation with cefadroxil or PBS the assay was started by the addition of 4 x 10* preincubated PMNs/200/d PBS. After 45 min at 4°C the incubation was terminated by rapid filtration; the filters were transferred into scintillation vials and the radioactivity was measured by liquid scintillation counting. All measurements were carried out in triplicate with variation coefficients between 7% and 12%. Specific binding was expressed as total binding
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on June 21, 2015
(14,15-3H>LTB4 (specific activity l-2TBq/mmol) was supplied by NEN (Dreieich, FRG). Synthetic standards of LTQ, LTD4, LTE^ 20-COOH-, 20-OH-LTB4 and LTB4 were kindly provided by Dr Rokach (Merck Frosst, Pointe Claire, Canada). Fine chemicals were purchased from Sigma Chemical Co. (Munich, FRG). Additional chemicals used were obtained from Merck (Darmstadt, FRG), Baker (Deventer, Netherlands) and Riedel-de-Haen (Seelze, FRG).
Nentrophfl function and cefadroxfl
615
minus nonspecific binding; nonspecific binding was determined in the presence of 220 HM unlabelled LTB4. Scatchard plot analysis was carried out using various concentrations of (3H>LTB4. The binding data were analysed according to Rosenthal (1967). Buffer The phosphate-buffered saline (PBS) comprised Na2HPO4 x 2H2O/3 mM KH2PO4 adjusted to pH 7-40.
120mM
NaCl/lOmM
Sample preparation for HPLC
Stimulation of the cells In the presence of calcium (1 mM) and magnesium (0-5 mM) the granulocytes and 'LMBs' (1 x 107) were stimulated with the calcium-ionophore A23187 (Sigma, Munich, FRG) at a concentration of 7-3 /M for 20 min at 37°C. Platelets were incubated with 1-5 /At calcium-ionophore in the presence of 1 mM CaCl2 and 0-5 mM MgCl2 for 20 min. All cell suspensions were preincubated with cefadroxil at various concentrations or PBS (control) for 15 min. HPLC-analysis (Kndller et al., 1988) Reversed phase-high performance liquid chromatography (RP-HPLC) was carried out with a CM 4000 G-pump and a variable wavelength detector SMD 4000 (LDC Milton Roy Hasselroth, FRG), a Waters Intelligent Sample Processor (Wisp model 310 B) (Waters, Konigsstein/Taunus, FRG), a Nelson Analytical Interface (Series 760) and Nelson Analytical Software Revision 3.6 (AS Analysensysteme Wuppertal, FRG). Solvent A was a mixture of phosphate buffer (17 mM K2HPO4) containing 0-05% EDTA, adjusted to pH 5-0 with phosphoric acid), acetonitrile and methanol (50/30/ 20,v/v). Leukotrienes were separated at a flow rate of 1 ml/min on a 200 x 4-6 mm column filled with Nucleosil 5C18 maintained at 40°C. The effluent was monitored at 280 nm at 0-005 AUFS (absorbance units full scale). The injection volume was 200 /il. Leukotrienes were quantified using peak areas. Standard curves of the individual substances were obtained with five different concentrations (2-500 ng). For example, the correlation coefficient for LTC4 was 0-978 and 0-998 for the a)-oxidation products, respectively. The coefficient of variation was between 1-2% and 2-8%. Possible variations of the elution pattern were corrected by injection of a standard solution after 20 samples. This solvent allows the separation of the cysteinyl-leukotrienes together with their 11-trans-isomers, LTB4, 5S, 12S-LTB4 and the non-enzymatically generated LTB4 isomers as well as the ©-oxidation products. Under these conditions, the HETEs were not eluted from the column.
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on June 21, 2015
After incubation the cells were centrifuged at 600 g. The supernatant was dissolved in 3 ml of acetonitrile/methanol (50/50, v/v), lyophilized, resuspended in 600 /J methanol/ water (30/70, v/v), overlayered with argon and stored at -70°C until the HPLC analyses were performed.
616
J. KnSfler et aL 300
50
25 10 Cefodroxll (/xg/lxlO calls)
PBS
Figure 1. Specific binding of CH)LTB4 (data were presented in % of the PBS control = 100%) after preincubation with cefadroxil (100-10/ig/107 cells - 390-19-7 fat). Each value represents the mean±s.D. of three independent experimenti. **P < 0-01; *P < 0-05.
Statistics All data were calculated as mean ± standard deviation. The significance was evaluated with Student's f-test for independent means, P < 0-05 was considered significant. Results Figure 1 shows the effect of cefadroxil on LTB 4 receptor expression. Incubation of P M N s with cefadroxil (10-100/ig/10 7 cells) led to an augmentation of LTB 4 receptor expression. Enhancement of the LTB 4 binding sites ranged between 220% (PBS = 100%) at a concentration of 25 fig and 190% at 50 ug of cefadroxil. Higher concentrations of cefadroxil (e.g. 100/xg/10 7 P M N s ) decreased the capacity of P M N s to bind 3 HLTB 4 by 30% while lower concentrations (10 ug and 5 /ig/10 7 cells) are within the range of the PBS control.
Table L Scatchard analysis for the LTB4 receptors after preincubation of the cells with 25 ug of cefadroxil High affinity receptors
Low affinity receptors
100% n.d.
100% 171%
PBS cefadroxil n.d., Not detectable.
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100
Nentropidl function and ceftdroxfl
617
120
25 10 Ofodroxll (pg/lxlO calif)
PBS
2. Preincubation of human granulocytes (1 x 107) with cefadrozil (100-5 /ig/10' cells) foDowed by stimulation with the calcium-ionophore (7-3 AIM). Each value represents the mean±sj>. of three independent experiment!. 100% value for calcium-ionophore: 400±75ng.
To characterize the affinity state of the LTB4 receptors a Scatchard analysis was performed (Table I). Analysis of the data reveals that the LTB4-receptors exist as two subsets with different ligand affinities. The respective binding parameters suggest 2-2 ± 0.9 x 103receptorsper cell and a K^ of 2-6 ± 1-4 nM for the high affinity subset and 8-25 ±5.3 x 104 receptors per cell and a K^ of 15-3 ± 8.7 nM for the low affinity subset. Table I shows a Scatchard analysis for the binding of (3H>LTB4 to PMNs. Stimulation of the cells with 25 /*g of cefadroxil for 15 min at 37°C resulted in a shift from the high affinity state, that is not detectable after incubation with 25 /jg of cefadroxil, to the low affinity state of the LTB4 receptor. As is the apparent from Figure 2 prdncubation with the same concentrations of cefadroxil did not alter the calcium-ionophore induced generation of leukotrienes from polymorphonuclear leucocytes compared with the control (preincubation with PBS). The values represent the total amounts of LTB4 generated (sum of LTB4 and 20-OHLTB4 and 20-COOH-LTBJ indicating synthesis as well as metabolism of LTB4. LTB4amounts obtained in the absence of cefadroxil (PBS control) were calculated as 100%; the amounts generated in the presence of cefadroxil were calculated as percentage of the 100% control. Similarly, the opsonized zymosan induced leukotriene generation from PMNs was not significantly affected after preincubation with cefadroxil (data not shown). Preincubation of the lymphocyte, monocyte and basophil (LMB) containing cell fraction with cefadroxil led to a slight suppression of leukotriene generation. As is shown in Figure 3 this suppression is not dose dependent and amounts to 10% and 20%. Preincubation of human platelets with various concentrations of cefadroxil caused no alterations in the synthesis of 12-HETE. As is apparent from Figure 4 cefadroxil at the concentration range from 50/jg to 5/ig/10* platelets did not affect the generation of the hydroxylated eicosatetraenoic acid compared with the PBS control.
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50
618
J. KnODer et aL 120
25 10 Csfadroxil (/tg/lxlO c«ll»)
PBS
Figure 3. Preincubation of the lymphocyte, monocyte and basophil (LMB) containing cell fraction (107) with cefadroxil (100-5 /ig/107 celli) followed by stimulation with the calcium-ionophore (7-3 /of). Each value repretents the mean±s.D. of three independent experiments. *P < 0-05. 100% value for calcium-ionophore: 50±20ng.
Discussion Phagocyte functions of neutrophils play an important role in host defence mechanisms against microbial infection. Since antibiotics show biological effects other than antibacterial activity, there have been many reports as to the influence of PMNs concerning e.g. respiratory burst (Briheim & Dahlgren, 1987) and chemotaxis (Oleske et al., 1983).
120
100
25 10 Cefadroxil (pg/lxlO will)
PBS
Figure 4. Preincubation of human platelets (1 x 10*) with cefadroxil (lOO-S/jg/107 cells) followed by stimulation with the calcium-ionophore (1-5 fat). Each value represents the mcan±s.D. of three independent experiments. 100% value for calcium-ionophore: 1000±200ng.
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100
Netrtropfafl function and ceftdroxfl
619
References Boyum, A. (1976). Isolation of lymphocytes, granulocytes and macrophagcs. Scandinavian Journal of Immunology 5, Suppl., 9-14. Briheim, G. & Dahlgren, C. (1987). Influence of antibiotics on fonnylmethionyl-leucylphenylalanine-induced leukocyte chemiluminescence. Antimicrobial Agents and Chemotherapy 31, 763-7. Brom, J., K6Uer, M., Schonfeld, W., Knoller, J., Erbs, G., Muller, F. E. et al. (1988a). Decreased expression of leukotriene B« receptor sites on polymorphonuclear granulocytes of severely burned patients. Prostagkmdins Leukotrienes and Essential Fatty Acids 34, 153-9.
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on June 21, 2015
The third generation cephalosporins enhance the bactericidal activity of PMN (Labro, Babin-Chevaye & Hakim, 1986; Labro et al., 1987) as well as affect the lymphocyte cell growth in vitro (Leyhausen et al., 1984). Besides this proliferation assay (CH)-thymidine incorporation into lymphocytes), the luminol-enhanced chemiluminescence was frequently chosen to investigate the influence of antimicrobial agents on polymorphonuclear leucocytes (Briheim & Dahlgren, 1987). Furthermore, the stimulusdependent leukotriene generation from PMNs is an effective assay to study the effects of various substances on PMN functions. Our results demonstrate that cefadroxil did not exert adverse effects on the generation of LTB4 from polymorphonuclear leucocytes. The capability of PMNs to respond to LTB4 may be enhanced in the presence of cefadroxil owing to the augmentation of the LTB4 receptor expression. These results indicate that the responsiveness of these cells to generate a chemotactic potential after activation with immunological as well as nonimmunological stimuli is considerably improved. It is well known that LTB4 is one of the most potent chemotactic agents for polymorphonuclear leucocytes and thus is an essential component for host defence. PMNs respond (Goldman & Goetzl, 1984) and metabolize LTB4 via specific receptor sites (Brom, Schonfeld & Konig, 19886). Binding to the high-affinity-receptor results in chemotactic migration of the cells, while binding to the low-affinity receptor leads to the release of granular enzymes. The action of LTB4 as a mediator for the specific immune response as well as in intercellular signalling between lymphocytes and monocytes was also confirmed by thorough studies (Farrar & Humes, 1985). Cefadroxil showed little or no influence on LTB4-generation from the lymphocyte, monocyte and basophil (LMB) containing cell fraction as well as on 12-HETE synthesis from human platelets within the therapeutic range. It is not known how cefadroxil causes the slight inhibition of LTB4 generation when the LMB fraction was studied since toxic concentrations were not used. Our results are in accordance with other studies that describe the influence of /?lactam antibiotics on phagocytic cells (Duncker & Ullmann, 1986). The chemiluminescence reaction of phagocytosing human neutrophil granulocytes was not affected in the presence of third generation cephalosporins with the exception of ceftriaxone; the latter compound increased the chemiluminescence reaction. Furthermore, if antimicrobial agents are to be administered it is important to consider not only the efficiency against bacteria but also the effect of these substances on phagocytic cells and the host-defence system. This aspect is of obvious importance for the therapy of immunocomprised patients, where impaired phagocytic functions limit the therapeutic administration and availability of some antibiotics.
620
J. KnODer et aL
{Received 14 June 1989; accepted 14 October 1989)
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on June 21, 2015
Brom, J., Schonfeld, W. & Kdnig, W. (19886). Metabolism of leukotriene B« by activated human polymorphonuclear granulocytes. Immunology 64, 509-18. Ouncker, D. & UUmann, U. (1986). Influence of various antimicrobial agents on the chemiluminescence of phagocytosing human granulocytes. Chemotherapy 32, 18-24. Farrar, W. L. & Humes, J. L. (1985). The role of arachidonic acid metabolism in the activities of interleukin 1 and 2. Journal of Immunology 135, 1153-9. Gilbert, H. S. & Ornstein, L. (1975). Basophil counting with a new staining method using akaan blue. Blood 46, 279-86. Goldman, D. W. & Goetd, E. J. (1984). Heterogeneity of human polymorphonuclear leucocyte receptors for leukotriene B4. Identification of a subset of high affinity receptors that transduce the chemotactic response. Journal of Experimental Medicine 159, 1027-41. Kndller, J., SchSnfeld, W., Kdller, M., Hensler, T. & Konig, W. (1988). Arachidonic acid metabolities from polymorphonuclear leukocytes of healthy donors, severely burned patients and children with cystic fibrosis—routine monitoring by high-performance liquid chromatography. Journal of Chromatography ATI, 199-208. Labro, M. T., Amit, N., Babin-Chevaye, C. & Hakim, J. (1987). Ccfodizime (HR221) potentiation of human neutrophil oxygen-independent bactericidal activity. Journal of Antimicrobial Chemotherapy 19, 331-41. Labro, M. T., Babin-Chevaye, C. & Hakim, J. (1986). Effects of cefotaxime and cefodizime on human granulocyte functions in vitro. Journal of Antimicrobial Chemotherapy 18, 233-7. Leyhausen, G., Seibert, G., Maidhof, A. & Muller, W. E. G. (1984). Differential stimulation of lymphocyte cell growth in vitro by cephalosporins. Antimicrobial Agents and Chemotherapy 26, 752-6. Manilla, J. p. & Clark, J. K. (1983). Effects of moxalactam and cefuroxime on mitogenstimulated human mononuclear leukocytes. Antimicrobial Agents and Chemotherapy 23, 360-3. O'Flaherty, J., Kosfeld, S. & Nishihira, J. (1986). Binding and metabolism of leukotriene B4 by neutrophils and their subccllular organelles. Journal of Cellular Physiology 126, 359-70. Ohnishi, H., Inaba, H., Mochizuki, H. & Kosuzume, H. (1984). Mechanism of action of AC1370 on phagocyte functions. Antimicrobial Agents and Chemotherapy 25, 88-92. Oleske, J. M., de la Cruz, A., Ahdieh, H., Sorvino, D., La Braico, J., Cooper, R. et al. (1983). Effects of antibiotics on polymorphonuclear leukocyte chemiluminescence and chemotaxis. Journal of Antimicrobial Chemotherapy 12, Suppl. C, 35-8. Rosenthal, H. E. (1967). A graphic method for the determination and presentation of binding parameters in a complex system. Analytical Biochemistry 20, 525-32. Tucker, S. B., Pierre, R. V. & Jordon, R. E. (1977). Rapid identification of monocytes in a mixed mononuclear cell preparation. Journal of Immunological Methods 14, 267-9.
In-vitro enhancement of leukotriene B 4 receptor expression on human neutrophils by cefadroxil J. KnODer, J. Brom, W. ScMnfeld and W. KSnig
The influence of cefadroxil on LTB4-receptor expression of polymorphonudear leucocytes (PMNs) was studied. Furthermore, the effect of cefadroxil on the leukotriene generation from PMNs and the lymphocyte, monocyte and basophil (LMB) containing cell fraction as well as on the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets was analysed. Antibiotic concentrations ranged from 50 to 5 £ig/10T cells. Analysis of the generated leukotrienes was performed by high performance liquid chromatography (HPLQ. Significant augmentation of the LTB«-receptor expression in human PMNs (range 190%220%) was observed at concentrations of SO and 25/ig/lO1 cells. The caltiumionophore A23187 induced LTB4 generation from PMNs as well as 12-HETE synthesis from platelets was not significantly modulated in the presence of cefadroxil. Preincubation of the human LMB fraction led to slight suppression of the ionophore induced LTB4 generation up to 20%. Introduction Cefadroxil is a semisynthctic, broad-spectrum cephalosporin for oral administration. It is active against group A /?-haemolytic streptococci, Streptococcus pneumoniae, staphylococci (including coagulase-positive, coagulase-negative, and pcnicillinase-producing
strains), Escherichia coli, Klebsiella spp., and Proteus mirabilis. In the past intensive research has been directed towards the influence of antibiotics on the function of granulocytes and monocytes which represent the first line of defence against bacterial infections (Manzella & Clark, 1983; Ohnishi et al., 1984; Duncker & UUmann, 1986). The generation of chemiluminescence during phagocytosis (Oleske et al., 1983) as well as the fMLP induced chemiluminescence (Briheim & Dahlgren, 1987) have been used as assay systems to investigate the influence of antimicrobial agents on phagocy tic cells. Leukotriene B4 (LTB4) which is generated via 5-lipoxygenase from arachidonic acid is a highly potent chemoattractant for polymorphonuclear leucocytes (PMNs). Furthermore, it is a potent stimulus in vitro: adherence, degranulation, superoxide production, and release of lysosomal enzymes were studied (O'Flaherty et al., 1986). 12-Hydroxyeicosatetraenoic acid (12-HETE) is also a chemotactic factor, which, however, is less potent than LTB4. It was the purpose of the present study to analyse the effect of cefadroxil on the specific binding of LTB4 to human polymorphonuclear leucocytes. Furthermore, the generation of leukotrienes from PMNs and from the lymphocyte, monocyte and 1
03O5- 7453/9O/04O613+08 $0100/0
613 © 1990 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on June 21, 2015
Lehrstuhl fur Medizinische Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr Universitdt Bochum, FRG
614
J. KnSIkr et aL
basophil (LMB) containing cell fraction as well as the synthesis of 12-hydroxyeicosatetraenoic acid (12-HETE) from human platelets were analysed. Materials and methods
Materials
Preparation of peripheral human granulocytes and monocytes Heparinized venous blood (200 ml) was separated according to the method of Boyum (1976) by Ficoll-metrizoate gradient centrifugation. After centrifugation the lymphocyte, monocyte and basophil (LMB) containing cell fraction was washed twice in RPMI 1640 medium before use. The content of basophils was estimated using the Alcian blue staining technique (Gilbert & Ornstein, 1975). The unseparated LMB fraction was used in the experiments. Monocytes were identified according to their content of esterases (Tucker, Pierre & Jordan, 1977). The monocytes ranged from 1922%, lymphocytes from 74-78%, and the basophils were less than 4%. The erythrocytes within the granulocyte containing fraction were removed by dextran sedimentation and subsequent lysis (0-3% NaCl for 2 min on ice). The purity of the granulocyte fraction amounted to 99%. Preparation of platelets Nine volumes of human blood from healthy volunteers were mixed with 1 vol 0-9% NaCl containing 1-5% EDTA. Platelet-rich plasma (PRP) was separated by centrifugation at 200 g for 20 min; 2 vol of PRP and 1 vol of 01 M EDTA were mixed and the platelets sedimented at 2500 g for 20 min. The isolated platelets were suspended in 0-1 M EDTA and centrifuged for a further 20 min at 2500 g and finally resuspended in PBS containing 0-1% BSA (1 x 10s platelets/500 ft\ total incubation mixture). LTB4 binding assay The binding studies were performed as described by O'Flaherty, Kosfeld & Nishihira (1986) and Brom et al. (1988a). The assays were carried out using 96 well filtration plates with 5 ftm pore size-polyvinylidene fluoride membranes (Millipore, Eschborn, FRG). Each well contained 2, 3nM 3H-LTB4 (specific activity: 0-74-2-22 TBq/mmol) and 125/ig bovine serum albumin. After preincubation with cefadroxil or PBS the assay was started by the addition of 4 x 10* preincubated PMNs/200/d PBS. After 45 min at 4°C the incubation was terminated by rapid filtration; the filters were transferred into scintillation vials and the radioactivity was measured by liquid scintillation counting. All measurements were carried out in triplicate with variation coefficients between 7% and 12%. Specific binding was expressed as total binding
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on June 21, 2015
(14,15-3H>LTB4 (specific activity l-2TBq/mmol) was supplied by NEN (Dreieich, FRG). Synthetic standards of LTQ, LTD4, LTE^ 20-COOH-, 20-OH-LTB4 and LTB4 were kindly provided by Dr Rokach (Merck Frosst, Pointe Claire, Canada). Fine chemicals were purchased from Sigma Chemical Co. (Munich, FRG). Additional chemicals used were obtained from Merck (Darmstadt, FRG), Baker (Deventer, Netherlands) and Riedel-de-Haen (Seelze, FRG).
Nentrophfl function and cefadroxfl
615
minus nonspecific binding; nonspecific binding was determined in the presence of 220 HM unlabelled LTB4. Scatchard plot analysis was carried out using various concentrations of (3H>LTB4. The binding data were analysed according to Rosenthal (1967). Buffer The phosphate-buffered saline (PBS) comprised Na2HPO4 x 2H2O/3 mM KH2PO4 adjusted to pH 7-40.
120mM
NaCl/lOmM
Sample preparation for HPLC
Stimulation of the cells In the presence of calcium (1 mM) and magnesium (0-5 mM) the granulocytes and 'LMBs' (1 x 107) were stimulated with the calcium-ionophore A23187 (Sigma, Munich, FRG) at a concentration of 7-3 /M for 20 min at 37°C. Platelets were incubated with 1-5 /At calcium-ionophore in the presence of 1 mM CaCl2 and 0-5 mM MgCl2 for 20 min. All cell suspensions were preincubated with cefadroxil at various concentrations or PBS (control) for 15 min. HPLC-analysis (Kndller et al., 1988) Reversed phase-high performance liquid chromatography (RP-HPLC) was carried out with a CM 4000 G-pump and a variable wavelength detector SMD 4000 (LDC Milton Roy Hasselroth, FRG), a Waters Intelligent Sample Processor (Wisp model 310 B) (Waters, Konigsstein/Taunus, FRG), a Nelson Analytical Interface (Series 760) and Nelson Analytical Software Revision 3.6 (AS Analysensysteme Wuppertal, FRG). Solvent A was a mixture of phosphate buffer (17 mM K2HPO4) containing 0-05% EDTA, adjusted to pH 5-0 with phosphoric acid), acetonitrile and methanol (50/30/ 20,v/v). Leukotrienes were separated at a flow rate of 1 ml/min on a 200 x 4-6 mm column filled with Nucleosil 5C18 maintained at 40°C. The effluent was monitored at 280 nm at 0-005 AUFS (absorbance units full scale). The injection volume was 200 /il. Leukotrienes were quantified using peak areas. Standard curves of the individual substances were obtained with five different concentrations (2-500 ng). For example, the correlation coefficient for LTC4 was 0-978 and 0-998 for the a)-oxidation products, respectively. The coefficient of variation was between 1-2% and 2-8%. Possible variations of the elution pattern were corrected by injection of a standard solution after 20 samples. This solvent allows the separation of the cysteinyl-leukotrienes together with their 11-trans-isomers, LTB4, 5S, 12S-LTB4 and the non-enzymatically generated LTB4 isomers as well as the ©-oxidation products. Under these conditions, the HETEs were not eluted from the column.
Downloaded from http://jac.oxfordjournals.org/ at Dalhousie University on June 21, 2015
After incubation the cells were centrifuged at 600 g. The supernatant was dissolved in 3 ml of acetonitrile/methanol (50/50, v/v), lyophilized, resuspended in 600 /J methanol/ water (30/70, v/v), overlayered with argon and stored at -70°C until the HPLC analyses were performed.
616
J. KnSfler et aL 300
50
25 10 Cefodroxll (/xg/lxlO calls)
PBS
Figure 1. Specific binding of CH)LTB4 (data were presented in % of the PBS control = 100%) after preincubation with cefadroxil (100-10/ig/107 cells - 390-19-7 fat). Each value represents the mean±s.D. of three independent experimenti. **P < 0-01; *P < 0-05.
Statistics All data were calculated as mean ± standard deviation. The significance was evaluated with Student's f-test for independent means, P < 0-05 was considered significant. Results Figure 1 shows the effect of cefadroxil on LTB 4 receptor expression. Incubation of P M N s with cefadroxil (10-100/ig/10 7 cells) led to an augmentation of LTB 4 receptor expression. Enhancement of the LTB 4 binding sites ranged between 220% (PBS = 100%) at a concentration of 25 fig and 190% at 50 ug of cefadroxil. Higher concentrations of cefadroxil (e.g. 100/xg/10 7 P M N s ) decreased the capacity of P M N s to bind 3 HLTB 4 by 30% while lower concentrations (10 ug and 5 /ig/10 7 cells) are within the range of the PBS control.
Table L Scatchard analysis for the LTB4 receptors after preincubation of the cells with 25 ug of cefadroxil High affinity receptors
Low affinity receptors
100% n.d.
100% 171%
PBS cefadroxil n.d., Not detectable.
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100
Nentropidl function and ceftdroxfl
617
120
25 10 Ofodroxll (pg/lxlO calif)
PBS
2. Preincubation of human granulocytes (1 x 107) with cefadrozil (100-5 /ig/10' cells) foDowed by stimulation with the calcium-ionophore (7-3 AIM). Each value represents the mean±sj>. of three independent experiment!. 100% value for calcium-ionophore: 400±75ng.
To characterize the affinity state of the LTB4 receptors a Scatchard analysis was performed (Table I). Analysis of the data reveals that the LTB4-receptors exist as two subsets with different ligand affinities. The respective binding parameters suggest 2-2 ± 0.9 x 103receptorsper cell and a K^ of 2-6 ± 1-4 nM for the high affinity subset and 8-25 ±5.3 x 104 receptors per cell and a K^ of 15-3 ± 8.7 nM for the low affinity subset. Table I shows a Scatchard analysis for the binding of (3H>LTB4 to PMNs. Stimulation of the cells with 25 /*g of cefadroxil for 15 min at 37°C resulted in a shift from the high affinity state, that is not detectable after incubation with 25 /jg of cefadroxil, to the low affinity state of the LTB4 receptor. As is the apparent from Figure 2 prdncubation with the same concentrations of cefadroxil did not alter the calcium-ionophore induced generation of leukotrienes from polymorphonuclear leucocytes compared with the control (preincubation with PBS). The values represent the total amounts of LTB4 generated (sum of LTB4 and 20-OHLTB4 and 20-COOH-LTBJ indicating synthesis as well as metabolism of LTB4. LTB4amounts obtained in the absence of cefadroxil (PBS control) were calculated as 100%; the amounts generated in the presence of cefadroxil were calculated as percentage of the 100% control. Similarly, the opsonized zymosan induced leukotriene generation from PMNs was not significantly affected after preincubation with cefadroxil (data not shown). Preincubation of the lymphocyte, monocyte and basophil (LMB) containing cell fraction with cefadroxil led to a slight suppression of leukotriene generation. As is shown in Figure 3 this suppression is not dose dependent and amounts to 10% and 20%. Preincubation of human platelets with various concentrations of cefadroxil caused no alterations in the synthesis of 12-HETE. As is apparent from Figure 4 cefadroxil at the concentration range from 50/jg to 5/ig/10* platelets did not affect the generation of the hydroxylated eicosatetraenoic acid compared with the PBS control.
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618
J. KnODer et aL 120
25 10 Csfadroxil (/tg/lxlO c«ll»)
PBS
Figure 3. Preincubation of the lymphocyte, monocyte and basophil (LMB) containing cell fraction (107) with cefadroxil (100-5 /ig/107 celli) followed by stimulation with the calcium-ionophore (7-3 /of). Each value repretents the mean±s.D. of three independent experiments. *P < 0-05. 100% value for calcium-ionophore: 50±20ng.
Discussion Phagocyte functions of neutrophils play an important role in host defence mechanisms against microbial infection. Since antibiotics show biological effects other than antibacterial activity, there have been many reports as to the influence of PMNs concerning e.g. respiratory burst (Briheim & Dahlgren, 1987) and chemotaxis (Oleske et al., 1983).
120
100
25 10 Cefadroxil (pg/lxlO will)
PBS
Figure 4. Preincubation of human platelets (1 x 10*) with cefadroxil (lOO-S/jg/107 cells) followed by stimulation with the calcium-ionophore (1-5 fat). Each value represents the mcan±s.D. of three independent experiments. 100% value for calcium-ionophore: 1000±200ng.
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Netrtropfafl function and ceftdroxfl
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References Boyum, A. (1976). Isolation of lymphocytes, granulocytes and macrophagcs. Scandinavian Journal of Immunology 5, Suppl., 9-14. Briheim, G. & Dahlgren, C. (1987). Influence of antibiotics on fonnylmethionyl-leucylphenylalanine-induced leukocyte chemiluminescence. Antimicrobial Agents and Chemotherapy 31, 763-7. Brom, J., K6Uer, M., Schonfeld, W., Knoller, J., Erbs, G., Muller, F. E. et al. (1988a). Decreased expression of leukotriene B« receptor sites on polymorphonuclear granulocytes of severely burned patients. Prostagkmdins Leukotrienes and Essential Fatty Acids 34, 153-9.
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The third generation cephalosporins enhance the bactericidal activity of PMN (Labro, Babin-Chevaye & Hakim, 1986; Labro et al., 1987) as well as affect the lymphocyte cell growth in vitro (Leyhausen et al., 1984). Besides this proliferation assay (CH)-thymidine incorporation into lymphocytes), the luminol-enhanced chemiluminescence was frequently chosen to investigate the influence of antimicrobial agents on polymorphonuclear leucocytes (Briheim & Dahlgren, 1987). Furthermore, the stimulusdependent leukotriene generation from PMNs is an effective assay to study the effects of various substances on PMN functions. Our results demonstrate that cefadroxil did not exert adverse effects on the generation of LTB4 from polymorphonuclear leucocytes. The capability of PMNs to respond to LTB4 may be enhanced in the presence of cefadroxil owing to the augmentation of the LTB4 receptor expression. These results indicate that the responsiveness of these cells to generate a chemotactic potential after activation with immunological as well as nonimmunological stimuli is considerably improved. It is well known that LTB4 is one of the most potent chemotactic agents for polymorphonuclear leucocytes and thus is an essential component for host defence. PMNs respond (Goldman & Goetzl, 1984) and metabolize LTB4 via specific receptor sites (Brom, Schonfeld & Konig, 19886). Binding to the high-affinity-receptor results in chemotactic migration of the cells, while binding to the low-affinity receptor leads to the release of granular enzymes. The action of LTB4 as a mediator for the specific immune response as well as in intercellular signalling between lymphocytes and monocytes was also confirmed by thorough studies (Farrar & Humes, 1985). Cefadroxil showed little or no influence on LTB4-generation from the lymphocyte, monocyte and basophil (LMB) containing cell fraction as well as on 12-HETE synthesis from human platelets within the therapeutic range. It is not known how cefadroxil causes the slight inhibition of LTB4 generation when the LMB fraction was studied since toxic concentrations were not used. Our results are in accordance with other studies that describe the influence of /?lactam antibiotics on phagocytic cells (Duncker & Ullmann, 1986). The chemiluminescence reaction of phagocytosing human neutrophil granulocytes was not affected in the presence of third generation cephalosporins with the exception of ceftriaxone; the latter compound increased the chemiluminescence reaction. Furthermore, if antimicrobial agents are to be administered it is important to consider not only the efficiency against bacteria but also the effect of these substances on phagocytic cells and the host-defence system. This aspect is of obvious importance for the therapy of immunocomprised patients, where impaired phagocytic functions limit the therapeutic administration and availability of some antibiotics.
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{Received 14 June 1989; accepted 14 October 1989)
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Brom, J., Schonfeld, W. & Kdnig, W. (19886). Metabolism of leukotriene B« by activated human polymorphonuclear granulocytes. Immunology 64, 509-18. Ouncker, D. & UUmann, U. (1986). Influence of various antimicrobial agents on the chemiluminescence of phagocytosing human granulocytes. Chemotherapy 32, 18-24. Farrar, W. L. & Humes, J. L. (1985). The role of arachidonic acid metabolism in the activities of interleukin 1 and 2. Journal of Immunology 135, 1153-9. Gilbert, H. S. & Ornstein, L. (1975). Basophil counting with a new staining method using akaan blue. Blood 46, 279-86. Goldman, D. W. & Goetd, E. J. (1984). Heterogeneity of human polymorphonuclear leucocyte receptors for leukotriene B4. Identification of a subset of high affinity receptors that transduce the chemotactic response. Journal of Experimental Medicine 159, 1027-41. Kndller, J., SchSnfeld, W., Kdller, M., Hensler, T. & Konig, W. (1988). Arachidonic acid metabolities from polymorphonuclear leukocytes of healthy donors, severely burned patients and children with cystic fibrosis—routine monitoring by high-performance liquid chromatography. Journal of Chromatography ATI, 199-208. Labro, M. T., Amit, N., Babin-Chevaye, C. & Hakim, J. (1987). Ccfodizime (HR221) potentiation of human neutrophil oxygen-independent bactericidal activity. Journal of Antimicrobial Chemotherapy 19, 331-41. Labro, M. T., Babin-Chevaye, C. & Hakim, J. (1986). Effects of cefotaxime and cefodizime on human granulocyte functions in vitro. Journal of Antimicrobial Chemotherapy 18, 233-7. Leyhausen, G., Seibert, G., Maidhof, A. & Muller, W. E. G. (1984). Differential stimulation of lymphocyte cell growth in vitro by cephalosporins. Antimicrobial Agents and Chemotherapy 26, 752-6. Manilla, J. p. & Clark, J. K. (1983). Effects of moxalactam and cefuroxime on mitogenstimulated human mononuclear leukocytes. Antimicrobial Agents and Chemotherapy 23, 360-3. O'Flaherty, J., Kosfeld, S. & Nishihira, J. (1986). Binding and metabolism of leukotriene B4 by neutrophils and their subccllular organelles. Journal of Cellular Physiology 126, 359-70. Ohnishi, H., Inaba, H., Mochizuki, H. & Kosuzume, H. (1984). Mechanism of action of AC1370 on phagocyte functions. Antimicrobial Agents and Chemotherapy 25, 88-92. Oleske, J. M., de la Cruz, A., Ahdieh, H., Sorvino, D., La Braico, J., Cooper, R. et al. (1983). Effects of antibiotics on polymorphonuclear leukocyte chemiluminescence and chemotaxis. Journal of Antimicrobial Chemotherapy 12, Suppl. C, 35-8. Rosenthal, H. E. (1967). A graphic method for the determination and presentation of binding parameters in a complex system. Analytical Biochemistry 20, 525-32. Tucker, S. B., Pierre, R. V. & Jordon, R. E. (1977). Rapid identification of monocytes in a mixed mononuclear cell preparation. Journal of Immunological Methods 14, 267-9.
