Journal of Antimicrobial Chemotherapy (1992) 30, 57-66
Effects of cefaclor, cefetamet and Ro 40-6890 on inflammatory responses of human granolocytes J. Scheffer*, J. KnODer', W. Cullmann* and W. Kdnig*
The effect of three cephalosporins (cefetamet, cefaclor and Ro 40-6890) upon human granulocytes and their ability to modulate the chemiluminescence response, phagocytose, kill bacteria and generate leulcotrienes was studied. In the presence of the cephalosporins there was a significant increase in phagocytosis of Escherichia coli. The bactericidal activity of human granulocytes for several other bacteria was also enhanced. Cefetamet and cefaclor increased the chemiluminescence response of human neutrophils to Pseudomonas aeruginosa and Proteus mirabilis in contrast to Ro 40-68790, which decreased the chemiluminescence response. The cephalosporins decreased the synthesis of leulcotrienes from human neutrophils after stimulation with Escherichia coli and Staphylococcus aureus. These data emphasize the immunomodulatory functions of various cephalosporins on cells involved in host defence.
Introduction
A number of studies have recently shown that antimicrobial agents significantly affect the cells of the immune system. Clindamycin has been shown to cause an increase in the proportion of granulocytes bearing receptors for the Fc portion of IgG and C3b, however the rate of phagocytosis as measured by the chemiluminescence response was slightly decreased (Noess, Hauge, & Solberg, 1989). In contrast, ceftriaxone, a 2aminotniazolyl cephalosporin, did not alter the bactericidal functions of human neutrophils (PMNs). However, low concentrations of ceftriaxone enhanced the susceptibility of several bacterial strains to killing by PMNs (Labro, Babin-Chavaye & Hakim, 1988). Furthermore, Labro el al. (1987) demonstrated that the enhanced bactericidal activity of human neutrophils induced by cefotaxime and cefodizime, two methoxyimino-amino-2-thiazolyl cephalosporins, is linked to the cellular stimulation of oxygendependent and oxygen-independent killing systems, respectively. Moreover, the calcium-ionophore A23187 induced leukotriene generation from PMNs and LMBs was significantly suppressed after preincubation with ciprofloxacin (Knoller et al., 1990a). Incubation of PMNs with ciprofloxacin also induced an enhanced LTB4-receptor expression. Cephalosporins have a relatively broad antibacterial spectrum, are usually resistant to /Mactamases, and rarely show allergic cross reactivity with penicillins: therefore these agents are widely used and are often the drug of choice in infections due to certain Enterobacteriaceae. This study was designed to analyse the effects of three cephalosporins (cefaclor, 57 0305-7453/92/070057+10 $02.00/0
© 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 18, 2015
"Lehrstuhl fir Medizinische Mikrobiologie und Immunologie, Arbeitsgruppe fir Infektabwehrmechanismen, RUHR-Universitdt Bochum, Germany; * Hoffmann-La Roche, Basel, Switzerland
X Scbcffcf tt oL
58
Table L Minimum bactericidal (MBC) and inhibitory (MIC) concentrations
MBC
MIC
MBC
MIC
Ro 40-6890 MBC MIC
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
1000 1000 1000 1000
2
250 250
250
2 7-8
1000 62-5
250
1 1 1 7-8 2
Cefaclor
E.eoli S. aureus K. pneumoniae E. cloacae P. mirabilis
500
Cefetamet
62-5
15-6 15-6 15-6
62-5
20 62-5
15-6 31-3
2-0
4
Materials and methods Bacterial strains The following strains were used: Escherichia coli K12 pANN5211 (hly+); Staphylococcus aureus 121C (hly+); Klebsiella pneumoniae; Enterobacter cloacae; Pseudomonas aeruginosa (hly+); Proteus mirabilis (hly+). Table I summarizes the minimal bactericidal concentrations (MBC) and minimal inhibitory concentrations (MIC) of each agent for each strain. Antibiotics Cefaclor was obtained from Eli Lilly, cefetamet and Ro 40-6890 from Hoffmann La Roche, Basel. Ro 40-6890 is a recently synthesized cephalosporin: (6R, 7R>[(Z)-2-(2AinmcH4-thiazolyl-2^methoxyimino)acet-amido]-3-(azidomethyl)-8-xo-5-thia-1 -azabicylo[4.2.0]oct-2-ene-2-carboxylic acid sodium salt. The compounds were dissolved in distilled water.; a stock solution of 1 g/L was prepared. Preparation of human neutrophils Human polymorphonuclear leucocytes (PMNs) were isolated from 200 mL of heparinized (IS U/mL) blood from three healthy donors, and separated on a Ficoll-metrizoate gradient followed by dextran-sedimentation as described previously (Boyum, 1976). The PMNs were washed twice at 300 g, and yielded more than 95% pure PMNs. The cells were diluted to a final concentration of 2 x 107 cells/mL in PBS.
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 18, 2015
cefetamet and Ro 40-6890) on the acute inflammatory responses involved in the host defence against bacterial infection. Therefore, human PMNs were preincubated with various concentrations of the three cephalosporins and subsequently activated with the calcium-ionophore A23187 or various bacteria as stimuli. Furthermore, various bacterial strains were preincubated with the cephalosporins to assess the extent of bacterial modifications which may affect the immune inflammatory response. The modulation of the chemiluminescence response, of phagocytosis, adherence, bactericidal action and leukotriene formation from PMNs was also studied in detail.
IllflniIH1"*nr7 T
Effects of cefaclor, cefetamet and Ro 40-6890 on inflammatory responses of human granolocytes J. Scheffer*, J. KnODer', W. Cullmann* and W. Kdnig*
The effect of three cephalosporins (cefetamet, cefaclor and Ro 40-6890) upon human granulocytes and their ability to modulate the chemiluminescence response, phagocytose, kill bacteria and generate leulcotrienes was studied. In the presence of the cephalosporins there was a significant increase in phagocytosis of Escherichia coli. The bactericidal activity of human granulocytes for several other bacteria was also enhanced. Cefetamet and cefaclor increased the chemiluminescence response of human neutrophils to Pseudomonas aeruginosa and Proteus mirabilis in contrast to Ro 40-68790, which decreased the chemiluminescence response. The cephalosporins decreased the synthesis of leulcotrienes from human neutrophils after stimulation with Escherichia coli and Staphylococcus aureus. These data emphasize the immunomodulatory functions of various cephalosporins on cells involved in host defence.
Introduction
A number of studies have recently shown that antimicrobial agents significantly affect the cells of the immune system. Clindamycin has been shown to cause an increase in the proportion of granulocytes bearing receptors for the Fc portion of IgG and C3b, however the rate of phagocytosis as measured by the chemiluminescence response was slightly decreased (Noess, Hauge, & Solberg, 1989). In contrast, ceftriaxone, a 2aminotniazolyl cephalosporin, did not alter the bactericidal functions of human neutrophils (PMNs). However, low concentrations of ceftriaxone enhanced the susceptibility of several bacterial strains to killing by PMNs (Labro, Babin-Chavaye & Hakim, 1988). Furthermore, Labro el al. (1987) demonstrated that the enhanced bactericidal activity of human neutrophils induced by cefotaxime and cefodizime, two methoxyimino-amino-2-thiazolyl cephalosporins, is linked to the cellular stimulation of oxygendependent and oxygen-independent killing systems, respectively. Moreover, the calcium-ionophore A23187 induced leukotriene generation from PMNs and LMBs was significantly suppressed after preincubation with ciprofloxacin (Knoller et al., 1990a). Incubation of PMNs with ciprofloxacin also induced an enhanced LTB4-receptor expression. Cephalosporins have a relatively broad antibacterial spectrum, are usually resistant to /Mactamases, and rarely show allergic cross reactivity with penicillins: therefore these agents are widely used and are often the drug of choice in infections due to certain Enterobacteriaceae. This study was designed to analyse the effects of three cephalosporins (cefaclor, 57 0305-7453/92/070057+10 $02.00/0
© 1992 The British Society for Antimicrobial Chemotherapy
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 18, 2015
"Lehrstuhl fir Medizinische Mikrobiologie und Immunologie, Arbeitsgruppe fir Infektabwehrmechanismen, RUHR-Universitdt Bochum, Germany; * Hoffmann-La Roche, Basel, Switzerland
X Scbcffcf tt oL
58
Table L Minimum bactericidal (MBC) and inhibitory (MIC) concentrations
MBC
MIC
MBC
MIC
Ro 40-6890 MBC MIC
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
1000 1000 1000 1000
2
250 250
250
2 7-8
1000 62-5
250
1 1 1 7-8 2
Cefaclor
E.eoli S. aureus K. pneumoniae E. cloacae P. mirabilis
500
Cefetamet
62-5
15-6 15-6 15-6
62-5
20 62-5
15-6 31-3
2-0
4
Materials and methods Bacterial strains The following strains were used: Escherichia coli K12 pANN5211 (hly+); Staphylococcus aureus 121C (hly+); Klebsiella pneumoniae; Enterobacter cloacae; Pseudomonas aeruginosa (hly+); Proteus mirabilis (hly+). Table I summarizes the minimal bactericidal concentrations (MBC) and minimal inhibitory concentrations (MIC) of each agent for each strain. Antibiotics Cefaclor was obtained from Eli Lilly, cefetamet and Ro 40-6890 from Hoffmann La Roche, Basel. Ro 40-6890 is a recently synthesized cephalosporin: (6R, 7R>[(Z)-2-(2AinmcH4-thiazolyl-2^methoxyimino)acet-amido]-3-(azidomethyl)-8-xo-5-thia-1 -azabicylo[4.2.0]oct-2-ene-2-carboxylic acid sodium salt. The compounds were dissolved in distilled water.; a stock solution of 1 g/L was prepared. Preparation of human neutrophils Human polymorphonuclear leucocytes (PMNs) were isolated from 200 mL of heparinized (IS U/mL) blood from three healthy donors, and separated on a Ficoll-metrizoate gradient followed by dextran-sedimentation as described previously (Boyum, 1976). The PMNs were washed twice at 300 g, and yielded more than 95% pure PMNs. The cells were diluted to a final concentration of 2 x 107 cells/mL in PBS.
Downloaded from http://jac.oxfordjournals.org/ at New York University on June 18, 2015
cefetamet and Ro 40-6890) on the acute inflammatory responses involved in the host defence against bacterial infection. Therefore, human PMNs were preincubated with various concentrations of the three cephalosporins and subsequently activated with the calcium-ionophore A23187 or various bacteria as stimuli. Furthermore, various bacterial strains were preincubated with the cephalosporins to assess the extent of bacterial modifications which may affect the immune inflammatory response. The modulation of the chemiluminescence response, of phagocytosis, adherence, bactericidal action and leukotriene formation from PMNs was also studied in detail.
IllflniIH1"*nr7 T
