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Effects of aztreonam on natural immunity in mice
International Journal of Antimicrobial Agents 13 (1999) 41 – 46 www.elsevier.com/locate/isc
Original article
Effects of aztreonam on natural immunity in mice Elena Ortega a, Manuel A. de Pablo a, Aurelia Ma, Gallego a, Carmen Alvarez a, Pedro L. Pancorbo a, Alfonso Ruiz-Bravo b, Gerardo Alvarez de Cienfuegos a,* a
Department of Health Sciences, Uni6ersity of Jae´n, Paraje Las Lagunillas S/N, 23071 Jae´n, Spain b Department of Microbiology, Uni6ersity of Granada, 18071 Granada, Spain Received 22 April 1999; accepted 18 June 1999
Abstract The influence of the dose and the duration of treatment with aztreonam, a monocyclic b-lactam antibiotic, on the natural immune response of mice has been investigated. The results show the effects induced by the antibiotic on several immune parameters were affected by the duration of treatment. Thus, treatment with 28 mg/kg per day of aztreonam over 14 days increased every immune parameter tested, while treatment with 57 mg/kg per day of aztreonam for 7 days only enhanced the natural killer (NK) activity of splenocytes. Since aztreonam does not apparently impair the innate immune response, it might be a suitable therapy for the treatment of patients who are immunosuppressed. © 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: b-Lactams; Aztreonam; Natural immunity; Cytokines; Chemiluminescence; Natural killer activity
1. Introduction Antibacterial drugs are extremely useful for controlling infectious diseases, but an important factor in obtaining complete recovery with antibacterial therapy is often the efficient function of the host defense system [1]. Recently, there has been increasing interest in the relationship between antibiotics and the immune response; the significance of antibiotic therapy in reducing the number and the virulence of bacteria is obvious, but the antibiotic may be ineffective when the immune system be no longer functional. Thus, antibiotic choice should not only be influenced by the susceptibility of the pathogen, but also by any potential effect on the host defense [2]. Antibiotics may affect the immune response in many ways [3,4], and such effects may be clinically relevant when such an impairment occurs [5]. The concept of biological response modifiers or, more exactly, immune response modifiers applied to * Corresponding author. Tel.: +34-953-212-160; fax: +34-953212-141. E-mail address: [email protected] (G.A. de Cienfuegos)
antibacterial agents is recent and is a subject of growing research [6,7]. Thus, the understanding of possible interactions between antibiotics and the immune system is of great importance to the clinical approach to the total process of therapy [8,9]. It has been shown that the state of the nonspecific host defense system is important for the outcome of antimicrobial chemotherapy, especially when antibiotics are administered to patients who are susceptible to infection because of defects in phagocytic function or granulocytopenia. It has been established that tobramycin, azlocillin [10], tetracyclines, sulphonamides and trimethoprim [11] impair phagocytic function, whereas some b-lactam antibiotics [12,13], clindamycin and chloramphenicol [14] have no effect on this immune parameter. b-Lactams are widely used in immunocompromised patients and are generally considered not to have an effect on immune cell function [15]. It has been previously reported that imipenem/cilastatin does not impair several innate immune responses when it is administered to mice at 1 or 2 g/70 kg per day (doses that are usually employed for human therapy) [16].
0924-8579/99/$ - see front matter © 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 9 9 ) 0 0 1 0 5 - 3
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E. Ortega et al. / International Journal of Antimicrobial Agents 13 (1999) 41–46
Aztreonam is a monobactam or monocyclic b-lactam antibiotic produced by many naturally occurring soil inhabitants. It is poorly absorbed from the gastro-intestinal tract and so is administered by deep intramuscular injection or intravenously [17]. Since it is not ototoxic or nephrotoxic, aztreonam is used as an alternative to aminoglycosides for the treatment of infections caused by susceptible Gram-negative aerobic organisms [18]; the restricted antimicrobial spectrum of aztreonam has also been proposed as an advantage as this would allow targeting of a Gram-negative pathogen with minimal disruption of the (largely anaerobic) intestinal flora [19]. In this study, the influence of the dose and the duration of the treatment with aztreonam given by intraperitoneal route, on several innate immune functions in mice was investigated (peritoneal cell counts, oxidative activity and interleukin (IL)-1 production by phagocytic cells and natural killer (NK) activity of splenic mononuclear leukocytes), in order to examine the effects of the different antibiotic treatments on both cellular and humoral innate immune responses.
2. Materials and methods
2.1. Animals Six- to 8-week-old male BALB/c mice were used for the experiments. They were maintained under pathogen-free conditions, with free access to food and water.
2.2. Antibiotic regimen Aztreonam was provided by Squibb (Madrid, Spain). The antibiotic, diluted in sterile phosphate-buffered saline (PBS; Sigma, St. Louis, MO), was given by intraperitoneal (i.p.) injection to mice (seven animals per group) at 24 h intervals for 7 (or 14) consecutive days. The doses were calculated using the same per kg body weight basis as in man: 57, 28, 14 and 7 mg/kg per day for 7-days therapy, and 28 mg/kg per day for 14-days therapy. These dosages roughly correspond to 4, 2, 1 and 0.5 g for 7-days therapy, and 2 g for 14-days therapy, respectively, for a 70-kg man. Control mice received equal volumes of PBS for 7 or 14 days, and immune parameters were tested the day after the last injection.
2.3. Peritoneal cell counts In order to obtain peritoneal cell counts (PC) counts, mice were sacrificed and injected i.p. with 3 ml of Hank’s balanced salt solution (HBSS; Sigma). The peritoneal fluids were aspirated and washed twice by centrifugation at 200× g and 4°C for 5 min with HBSS.
Viable cells were counted by trypan blue exclusion, and results were expressed as the number of viable PCs per mouse.
2.4. Preparation of peritoneal macrophage suspensions PCs were resuspended in HBSS without phenol red (HBSS; Sigma) for chemiluminescence (CL) assays, or in complete medium RPMI 1640 (Sigma) supplemented with 10% heat-inactivated calf serum (Flow Laboratories, Irvine, Scotland), 50 mM 2-mercaptoethanol (Sigma), 1% penicillin G/streptomicin solution (Sigma), 1% L-glutamine (Sigma), 1% sodium pyruvate (Sigma), 5% sodium bicarbonate (Sigma) and 1% Hepes (Flow Laboratories), for IL-1 producing cultures.
2.5. CL assay This assay was modified from the technique described by Moeller-Larsen et al. [20]. PCs were collected in HBSS without phenol red and adjusted to a concentration of 105 viable cells/ml. The samples were prepared in Eppendorf tubes and consisted of 100 ml of adjusted cellular suspension activated by addition of 20 ml of an opsonized dark-adapted zymosan A suspension at a concentration of 50 mg/ml. Seventy ml of luminol (5-amino-2,3-dehydro-1,4-phthalazinedione) in PBS at a concentration of 100 mg/ml were added to each sample and the CL mixtures were finally incubated for 2 min at 37°C and dispensed in scintillation vials. The production of reactive oxygen intermediates by peritoneal cells was measured by photon emission (LS1801, Beckman Instruments, Irvine, CA) for 0.1 min sequentially, over a 60 min period.
2.6. IL-1 assay The thymocyte costimulation bioassay was used for the measurement of soluble IL-1 using the technique described by Muegge and Durum [21]. PCs were resuspended in complete medium and adjusted to a concentration of 2× 106 viable cells/ml. One ml of these cell suspensions was added to 35-mm-diameter plastic Petri dishes, along with 1 ml of Escherichia coli lipopolysaccharide (LPS; Sigma) at a concentration of 20 mg/ml. The dishes were incubated for 24 h at 37°C in 5% CO2, and finally supernatants were collected and stored at − 80°C until the quantitative assay. This was carried out using thymuses aseptically removed from 6- to 12-week-old mice. The thymuses were homogenized in sterile HBSS. Thymocytes were washed twice by centrifugation and finally adjusted to a concentration of 107 viable cells/ml in complete medium with 10 mg/ml phytohemagglutinin (PHA; Sigma). IL-1 test samples were serially diluted in complete medium without PHA and 96-well flat-bottom microtiter plates received 100 ml
E. Ortega et al. / International Journal of Antimicrobial Agents 13 (1999) 41–46
of thymocyte suspension and 100 ml of diluted sample per well. The plates were incubated for 48 h at 37°C in 5% CO2, and cellular proliferation was measured by colorimetric reading of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction, as described by Mosmann [22].
2.7. NK acti6ity assay Mice were sacrificed and spleens were aseptically removed and homogenized in sterile HBSS. Mononuclear splenic cells were isolated according to Boyum [23], in a Ficoll-Hypaque density gradient. The mononuclear cell band obtained was collected, washed twice by centrifugation, and finally resuspended and adjusted to a concentration of 6×105 viable cells/ml with complete medium. NK activity was tested according to the technique described by Dinota et al. [24], based on the inhibition of target cell clone growth in plasma clot semisolid medium. A suspension of the NK-susceptible YAC-1 cell line, adjusted to a concentration of 105 viable cells/ml in complete medium, was used as target population. Two hundred ml effector cell suspension were incubated with 200 ml of target cell suspension at 37°C for 4 h in the presence of 5% CO2. Control experiments were carried out by using YAC-1 cells alone. Next, 20 ml of cell mixture were added to 35-mm-diameter Petri dishes, along with 1 ml of plasma clot medium (25% pool of human plasma +75% complete medium). Finally, 40 ml of calcium chloride (55 mg/ml) were added directly to the petri dishes in order to obtain the plasma clot. After incubation at 37°C in a 5% CO2 atmosphere over 96 h, clonal scoring was performed by using an inverted microscope. Only clones with more than eight cells were counted and the percentage of cloning inhibition was calculated, estimating control experiment as maximum cell proliferation.
2.8. Statistics The differences between treated and control groups were analyzed by using analysis of variance (ANOVA) test. A P value of less than 0.05 was considered significant.
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Table 1 Effect of aztreonam treatment on peritoneal cell (PC) counts Dosage of aztreonam (mg/kg per day)a
Treatment dura- No. PC/mouse tion (days) (106)b
Control mice 57 28 14 7 Control mice 28
7 7 7 7 7 14 14
4.02 9 1.79 5.88 91.59 3.83 9 0.76 4.33 9 0.13 3.539 0.68 3.10 9 0.25 4.65 91.91
a
Mice in the control group received sterile phosphate-buffered saline (PBS). The assay was performed at 24 h after the last aztreonam or PBS injection. b Data represent the means 9 S.D. for seven mice.
injected to mice play an important role in the effect of the drug on PC counts. Thus, treatment with 57 mg/kg per day for 7 days, as well as therapy with 28 mg/kg per day prolonged for 14 days induced a slight increase in PC counts, compared with that obtained in untreated control mice, whereas treatment with 28, 14 or 7 mg/kg per day for 7 days did not significantly modify PC counts in antibiotic-treated mice, with regard to the control group.
3.2. Effect of aztreonam on the oxidati6e acti6ity of peritoneal cells Because production of reactive oxygen intermediates by phagocytic cells constitutes an important antibacterial mechanism of the natural immune response, the effect of aztreonam on this cellular activity of peritoneal cells, determined by CL was examined. The results of CL assay are shown in Table 2. Treatments with 7, 14, or 28 mg/kg per day injected to mice for 7 days did not significantly modify the cellular activation state of these phagocytic cells. Table 2 Effect of aztreonam treatment on respiratory activation measured by chemiluminescence (CL) Dosage of aztreonam (mg/kg per day)a
Treatment dura- CL values expressed tion (days) as cpm (103)b
3.1. Effect of aztreonam on PC counts
Control mice 57 28 14 7 Control mice 28
7 7 7 7 7 14 14
The PC counts were examined in order to detect a possible inflammatory reaction caused by the aztreonam treatment, that could explain an alteration on the cellular activity of peritoneal leukocytic cells. As shown in Table 1, the dose of the antimicrobial agent
a Mice in the control group received sterile phosphate-buffered saline (PBS). The assay was performed at 24 h after the last aztreonam or PBS injection. b Values represent peaks of the CL responses. Data represent the means 9 S.D. for seven mice. c PB0.001.
3. Results
131.48 928.41 186.65 9 96.54 114.009 58.05 129.72930.11 123.83 9 47.56 121.19 9 16.23 269.31 9 87.02c
E. Ortega et al. / International Journal of Antimicrobial Agents 13 (1999) 41–46
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Table 3 Effect of aztreonam treatment on interleukin-1 (IL-1) production measured by the thymocyte costimulation bioassay Dosage of azteonam (mg/kg per day)a
Treatment duration (days)
Optical density at 570– 630 nm (103)b
Control mice 57 28 14 7 Control mice 28
7 7 7 7 7 14 14
279.009 12.72 271.509 40.40 283.20 9 4.55 256.909 29.20 285.759 9.94 280.949 5.20 399.83 918.79c
a Mice in the control group received sterile phosphate-buffered saline (PBS). The assay was performed at 24 h after the last aztreonam or PBS injection. b Values represent the IL-1-induced thymocyte proliferation. Results represent the means 9 S.D. for seven mice, and of at least three replicates of each sample. c PB0.01.
On the contrary, when mice were injected with 57 mg/kg per day for 7 days, a slight increase in CL values was detected, and when the therapy with 28 mg/kg per day was prolonged for 14 days, the treatment with aztreonam induced an increased level of cellular activation in peritoneal cells. In mice receiving prolonged therapy, CL values were significantly increased (by more than 100%) with regard to the control group (PB 0.001).
3.3. Effect of aztreonam on IL-1 production The production of IL-1 by peritoneal leukocytes from aztreonam-treated mice was tested, in order to examine the effect of this treatment on the cytokine production by innate immune cells. The results summarized in Table 3 show the proliferation of thymocytes co-cultured with samples obtained from supernatants of LPS-stimulated peritoneal macrophages. Results were not significantly influenced by aztreonam treatment, at any dose over 7 days; however, when therapy with 28 mg/kg per day was prolonged for 14 days a remarkable increase of the capacity of IL-1 production by peritoneal macrophages isolated from treated mice, in relation to that observed in the control group was detected.
3.4. Effect of aztreonam on NK acti6ity In order to examine the activation level in NK cells, we tested the cytotoxic activity of splenic mononuclear leukocytes against a YAC-1 cell line. As shown in Table 4, two different effects were obtained in mice treated with aztreonam with different doses. Thus, the NK activity of mononuclear splenic cells was significantly increased in mice treated with 57 mg/kg per day
for 7 days (PB 0.01) and in mice injected with 28 mg/kg per day for 14 days (PB0.001). On the other hand, treatment with 7, 14, or 28 mg/kg per day for 7 days, did not significantly modify the NK activity of mononuclear splenic cells.
4. Discussion Apart from direct antimicrobial activity, the biological properties of antibiotics include interactions and alterations in the cellular or humoral immune responses of patients [25,26]. Antibacterial agents have been recently considered as immune response modifiers and there have been several investigations attempting to describe the immunological profiles of the new antimicrobial agents [4,27]. Since effector cells of the innate immune defense, such as phagocytes and NK cells, play an important role both in the clearance of bacteria and have a role in some clinical aspects of infectious diseases [28], the possible effect of antibiotics on such immune populations may be of great importance. The data presented here show the modification of different parameters of the innate immune response by aztreonam in a murine experimental model. As was described previously when using a imipenem/cilastatin combination [16], the effects of the antibiotic on the different immune functions depend on both the dose and the time of administration. As phagocytic cells are essential for the host defense against infections, the respiratory activity of peritoneal cells has been studied by measuring CL, a powerful analytical tool for establishing the degree of oxidative reactivity in phagocytic cells [20,29]. In the present Table 4 Effect of aztreonam treatment on natural killer (NK) activity of mononuclear splenic cells Dosage (mg/kg per day)a
Treatment duration (days)
Percentage of cloning inhibitionb
Control mice 57 28 14 7 Control mice 28
7 7 7 7 7 14 14
30.15 99.00 41.85 9 5.99c 21.90 97.18 30.20 97.64 38.60 96.98 30.36 98.47 49.50 98.38d
a Mice in the control group received sterile phosphate-buffered saline (PBS). The assay was performed at 24 h after the last aztreonam or PBS injection. b Values represent the percentage of cloning inhibition of the NK-susceptible YAC-1 cell line. Results represent the means 9 S.D. for seven mice, and in duplicate for each sample. c PB0.01. d PB0.001.
E. Ortega et al. / International Journal of Antimicrobial Agents 13 (1999) 41–46
study, increased maximum values of oxidative activity were found in mice treated with 28 mg/kg per day aztreonam, for 14 days, with respect to those obtained in the control group. Likewise, the highest dose (57 mg/kg per day) administered to mice for 7 days increased oxidative activity, as measured by CL assay, of murine peritoneal phagocytic cells induced by zymosan although the difference was not significant. Previous studies have shown that intracellular penetration is a very important factor in the antimicrobial activity of antibiotics. Intracellular organisms may be protected from complete eradication by the immune system as well from the activity of antibiotics that are unable to enter phagocytes [30,31]. Moreover, it has been shown that the activity in vitro of antibiotics that penetrate phagocytic cells against cell-associated bacteria is significantly higher than that of compounds that are excluded from these cells [32]. Other authors have proposed a different interpretation for the intracellular activity of b-lactams such as the triggering of some intracellular reactions through the interaction of the antibiotic with some surface components of bacterial structure [33,34]. b-Lactams do not penetrate PMN, but it has been shown that theydo penetrate monocytes, probably by pinocytosis [35]. Adinolfi et al. [36] demonstrated that aztreonam possesses an intracellular antibacterial activity against E. coli which is greater than its extracellular one. These authors attribute this effect of aztreonam to a positive cooperation of the antibiotic with the O2-independent microbicidal system of macrophages. The results show that, at least in prolonged therapy, aztreonam may also contribute to the eradication of intracellular infectious agents through an increase in the O2-dependent microbicidal systems of murine peritoneal macrophages, as the results from the CL assay suggest. IL-1 is a cytokine produced by macrophages and epithelial cells that induces activation of both macrophages and T-lymphocytes. In this study it was observed that aztreonam exerted similar effects on IL-1 production by peritoneal macrophages as those described for the respiratory activity of this cellular population. Only when mice were treated for a prolonged time (14 days) with the equivalent to the therapeutic dose used in human medicine was a significant increase in the capacity of production of IL-1 by peritoneal macrophages detected. Similar dose-dependent modification of IL-1 production was previously described by Roche et al. [37] using quinolones and an in vitro model with human monocytes. The results agree with those of Roszkowski et al. [9] who showed dose-dependent effects for all antibiotics tested (cefotaxime, amikacin, mezlocillin, piperacillin and clindamycin). This study also agreed with the thesis that even small differences in the molecular formula of
45
antibiotics may have considerable immunopharmacological effects. With respect to NK activity of splenic mononuclear cells, these results were similar to those obtained previously when mice were injected with imipenem/cilastatin [16]. Both doses (57 mg/kg per day) administered to mice for 7 days and therapy with 28 mg/kg per day prolonged for 14 days induced a significant increase in the percentage of cloning inhibition of the NK-susceptible YAC-1 cell line. IL-2 is a cytokine known to act as a growth and differentiation factor for NK cells. Thus, as shown for the combination imipenem/cilastatin [16], the increase in the NK activity of splenic cells could be due to the concurrent increase in IL-2 production by T-cells which is also induced, in aztreonam treatments, by the highest dose employed, as well as by therapy with 28 mg/kg per day prolonged for 14 days (data not shown). As aztreonam does not apparently impair the innate immune response, it should be specially suited for the treatment of patients who are immunosuppressed. Anyhow, it is believed that the experimental design represents a more suitable approximation of the clinical situation than in vitro assays, although further studies are needed regarding the effects of aztreonam on nonspecific as well as on specific immune responses in humans.
Acknowledgements This work was supported by the Plan Andaluz de Investigacio´n (CTS 0105).
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Original article
Effects of aztreonam on natural immunity in mice Elena Ortega a, Manuel A. de Pablo a, Aurelia Ma, Gallego a, Carmen Alvarez a, Pedro L. Pancorbo a, Alfonso Ruiz-Bravo b, Gerardo Alvarez de Cienfuegos a,* a
Department of Health Sciences, Uni6ersity of Jae´n, Paraje Las Lagunillas S/N, 23071 Jae´n, Spain b Department of Microbiology, Uni6ersity of Granada, 18071 Granada, Spain Received 22 April 1999; accepted 18 June 1999
Abstract The influence of the dose and the duration of treatment with aztreonam, a monocyclic b-lactam antibiotic, on the natural immune response of mice has been investigated. The results show the effects induced by the antibiotic on several immune parameters were affected by the duration of treatment. Thus, treatment with 28 mg/kg per day of aztreonam over 14 days increased every immune parameter tested, while treatment with 57 mg/kg per day of aztreonam for 7 days only enhanced the natural killer (NK) activity of splenocytes. Since aztreonam does not apparently impair the innate immune response, it might be a suitable therapy for the treatment of patients who are immunosuppressed. © 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. Keywords: b-Lactams; Aztreonam; Natural immunity; Cytokines; Chemiluminescence; Natural killer activity
1. Introduction Antibacterial drugs are extremely useful for controlling infectious diseases, but an important factor in obtaining complete recovery with antibacterial therapy is often the efficient function of the host defense system [1]. Recently, there has been increasing interest in the relationship between antibiotics and the immune response; the significance of antibiotic therapy in reducing the number and the virulence of bacteria is obvious, but the antibiotic may be ineffective when the immune system be no longer functional. Thus, antibiotic choice should not only be influenced by the susceptibility of the pathogen, but also by any potential effect on the host defense [2]. Antibiotics may affect the immune response in many ways [3,4], and such effects may be clinically relevant when such an impairment occurs [5]. The concept of biological response modifiers or, more exactly, immune response modifiers applied to * Corresponding author. Tel.: +34-953-212-160; fax: +34-953212-141. E-mail address: [email protected] (G.A. de Cienfuegos)
antibacterial agents is recent and is a subject of growing research [6,7]. Thus, the understanding of possible interactions between antibiotics and the immune system is of great importance to the clinical approach to the total process of therapy [8,9]. It has been shown that the state of the nonspecific host defense system is important for the outcome of antimicrobial chemotherapy, especially when antibiotics are administered to patients who are susceptible to infection because of defects in phagocytic function or granulocytopenia. It has been established that tobramycin, azlocillin [10], tetracyclines, sulphonamides and trimethoprim [11] impair phagocytic function, whereas some b-lactam antibiotics [12,13], clindamycin and chloramphenicol [14] have no effect on this immune parameter. b-Lactams are widely used in immunocompromised patients and are generally considered not to have an effect on immune cell function [15]. It has been previously reported that imipenem/cilastatin does not impair several innate immune responses when it is administered to mice at 1 or 2 g/70 kg per day (doses that are usually employed for human therapy) [16].
0924-8579/99/$ - see front matter © 1999 Elsevier Science B.V. and International Society of Chemotherapy. All rights reserved. PII: S 0 9 2 4 - 8 5 7 9 ( 9 9 ) 0 0 1 0 5 - 3
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E. Ortega et al. / International Journal of Antimicrobial Agents 13 (1999) 41–46
Aztreonam is a monobactam or monocyclic b-lactam antibiotic produced by many naturally occurring soil inhabitants. It is poorly absorbed from the gastro-intestinal tract and so is administered by deep intramuscular injection or intravenously [17]. Since it is not ototoxic or nephrotoxic, aztreonam is used as an alternative to aminoglycosides for the treatment of infections caused by susceptible Gram-negative aerobic organisms [18]; the restricted antimicrobial spectrum of aztreonam has also been proposed as an advantage as this would allow targeting of a Gram-negative pathogen with minimal disruption of the (largely anaerobic) intestinal flora [19]. In this study, the influence of the dose and the duration of the treatment with aztreonam given by intraperitoneal route, on several innate immune functions in mice was investigated (peritoneal cell counts, oxidative activity and interleukin (IL)-1 production by phagocytic cells and natural killer (NK) activity of splenic mononuclear leukocytes), in order to examine the effects of the different antibiotic treatments on both cellular and humoral innate immune responses.
2. Materials and methods
2.1. Animals Six- to 8-week-old male BALB/c mice were used for the experiments. They were maintained under pathogen-free conditions, with free access to food and water.
2.2. Antibiotic regimen Aztreonam was provided by Squibb (Madrid, Spain). The antibiotic, diluted in sterile phosphate-buffered saline (PBS; Sigma, St. Louis, MO), was given by intraperitoneal (i.p.) injection to mice (seven animals per group) at 24 h intervals for 7 (or 14) consecutive days. The doses were calculated using the same per kg body weight basis as in man: 57, 28, 14 and 7 mg/kg per day for 7-days therapy, and 28 mg/kg per day for 14-days therapy. These dosages roughly correspond to 4, 2, 1 and 0.5 g for 7-days therapy, and 2 g for 14-days therapy, respectively, for a 70-kg man. Control mice received equal volumes of PBS for 7 or 14 days, and immune parameters were tested the day after the last injection.
2.3. Peritoneal cell counts In order to obtain peritoneal cell counts (PC) counts, mice were sacrificed and injected i.p. with 3 ml of Hank’s balanced salt solution (HBSS; Sigma). The peritoneal fluids were aspirated and washed twice by centrifugation at 200× g and 4°C for 5 min with HBSS.
Viable cells were counted by trypan blue exclusion, and results were expressed as the number of viable PCs per mouse.
2.4. Preparation of peritoneal macrophage suspensions PCs were resuspended in HBSS without phenol red (HBSS; Sigma) for chemiluminescence (CL) assays, or in complete medium RPMI 1640 (Sigma) supplemented with 10% heat-inactivated calf serum (Flow Laboratories, Irvine, Scotland), 50 mM 2-mercaptoethanol (Sigma), 1% penicillin G/streptomicin solution (Sigma), 1% L-glutamine (Sigma), 1% sodium pyruvate (Sigma), 5% sodium bicarbonate (Sigma) and 1% Hepes (Flow Laboratories), for IL-1 producing cultures.
2.5. CL assay This assay was modified from the technique described by Moeller-Larsen et al. [20]. PCs were collected in HBSS without phenol red and adjusted to a concentration of 105 viable cells/ml. The samples were prepared in Eppendorf tubes and consisted of 100 ml of adjusted cellular suspension activated by addition of 20 ml of an opsonized dark-adapted zymosan A suspension at a concentration of 50 mg/ml. Seventy ml of luminol (5-amino-2,3-dehydro-1,4-phthalazinedione) in PBS at a concentration of 100 mg/ml were added to each sample and the CL mixtures were finally incubated for 2 min at 37°C and dispensed in scintillation vials. The production of reactive oxygen intermediates by peritoneal cells was measured by photon emission (LS1801, Beckman Instruments, Irvine, CA) for 0.1 min sequentially, over a 60 min period.
2.6. IL-1 assay The thymocyte costimulation bioassay was used for the measurement of soluble IL-1 using the technique described by Muegge and Durum [21]. PCs were resuspended in complete medium and adjusted to a concentration of 2× 106 viable cells/ml. One ml of these cell suspensions was added to 35-mm-diameter plastic Petri dishes, along with 1 ml of Escherichia coli lipopolysaccharide (LPS; Sigma) at a concentration of 20 mg/ml. The dishes were incubated for 24 h at 37°C in 5% CO2, and finally supernatants were collected and stored at − 80°C until the quantitative assay. This was carried out using thymuses aseptically removed from 6- to 12-week-old mice. The thymuses were homogenized in sterile HBSS. Thymocytes were washed twice by centrifugation and finally adjusted to a concentration of 107 viable cells/ml in complete medium with 10 mg/ml phytohemagglutinin (PHA; Sigma). IL-1 test samples were serially diluted in complete medium without PHA and 96-well flat-bottom microtiter plates received 100 ml
E. Ortega et al. / International Journal of Antimicrobial Agents 13 (1999) 41–46
of thymocyte suspension and 100 ml of diluted sample per well. The plates were incubated for 48 h at 37°C in 5% CO2, and cellular proliferation was measured by colorimetric reading of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction, as described by Mosmann [22].
2.7. NK acti6ity assay Mice were sacrificed and spleens were aseptically removed and homogenized in sterile HBSS. Mononuclear splenic cells were isolated according to Boyum [23], in a Ficoll-Hypaque density gradient. The mononuclear cell band obtained was collected, washed twice by centrifugation, and finally resuspended and adjusted to a concentration of 6×105 viable cells/ml with complete medium. NK activity was tested according to the technique described by Dinota et al. [24], based on the inhibition of target cell clone growth in plasma clot semisolid medium. A suspension of the NK-susceptible YAC-1 cell line, adjusted to a concentration of 105 viable cells/ml in complete medium, was used as target population. Two hundred ml effector cell suspension were incubated with 200 ml of target cell suspension at 37°C for 4 h in the presence of 5% CO2. Control experiments were carried out by using YAC-1 cells alone. Next, 20 ml of cell mixture were added to 35-mm-diameter Petri dishes, along with 1 ml of plasma clot medium (25% pool of human plasma +75% complete medium). Finally, 40 ml of calcium chloride (55 mg/ml) were added directly to the petri dishes in order to obtain the plasma clot. After incubation at 37°C in a 5% CO2 atmosphere over 96 h, clonal scoring was performed by using an inverted microscope. Only clones with more than eight cells were counted and the percentage of cloning inhibition was calculated, estimating control experiment as maximum cell proliferation.
2.8. Statistics The differences between treated and control groups were analyzed by using analysis of variance (ANOVA) test. A P value of less than 0.05 was considered significant.
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Table 1 Effect of aztreonam treatment on peritoneal cell (PC) counts Dosage of aztreonam (mg/kg per day)a
Treatment dura- No. PC/mouse tion (days) (106)b
Control mice 57 28 14 7 Control mice 28
7 7 7 7 7 14 14
4.02 9 1.79 5.88 91.59 3.83 9 0.76 4.33 9 0.13 3.539 0.68 3.10 9 0.25 4.65 91.91
a
Mice in the control group received sterile phosphate-buffered saline (PBS). The assay was performed at 24 h after the last aztreonam or PBS injection. b Data represent the means 9 S.D. for seven mice.
injected to mice play an important role in the effect of the drug on PC counts. Thus, treatment with 57 mg/kg per day for 7 days, as well as therapy with 28 mg/kg per day prolonged for 14 days induced a slight increase in PC counts, compared with that obtained in untreated control mice, whereas treatment with 28, 14 or 7 mg/kg per day for 7 days did not significantly modify PC counts in antibiotic-treated mice, with regard to the control group.
3.2. Effect of aztreonam on the oxidati6e acti6ity of peritoneal cells Because production of reactive oxygen intermediates by phagocytic cells constitutes an important antibacterial mechanism of the natural immune response, the effect of aztreonam on this cellular activity of peritoneal cells, determined by CL was examined. The results of CL assay are shown in Table 2. Treatments with 7, 14, or 28 mg/kg per day injected to mice for 7 days did not significantly modify the cellular activation state of these phagocytic cells. Table 2 Effect of aztreonam treatment on respiratory activation measured by chemiluminescence (CL) Dosage of aztreonam (mg/kg per day)a
Treatment dura- CL values expressed tion (days) as cpm (103)b
3.1. Effect of aztreonam on PC counts
Control mice 57 28 14 7 Control mice 28
7 7 7 7 7 14 14
The PC counts were examined in order to detect a possible inflammatory reaction caused by the aztreonam treatment, that could explain an alteration on the cellular activity of peritoneal leukocytic cells. As shown in Table 1, the dose of the antimicrobial agent
a Mice in the control group received sterile phosphate-buffered saline (PBS). The assay was performed at 24 h after the last aztreonam or PBS injection. b Values represent peaks of the CL responses. Data represent the means 9 S.D. for seven mice. c PB0.001.
3. Results
131.48 928.41 186.65 9 96.54 114.009 58.05 129.72930.11 123.83 9 47.56 121.19 9 16.23 269.31 9 87.02c
E. Ortega et al. / International Journal of Antimicrobial Agents 13 (1999) 41–46
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Table 3 Effect of aztreonam treatment on interleukin-1 (IL-1) production measured by the thymocyte costimulation bioassay Dosage of azteonam (mg/kg per day)a
Treatment duration (days)
Optical density at 570– 630 nm (103)b
Control mice 57 28 14 7 Control mice 28
7 7 7 7 7 14 14
279.009 12.72 271.509 40.40 283.20 9 4.55 256.909 29.20 285.759 9.94 280.949 5.20 399.83 918.79c
a Mice in the control group received sterile phosphate-buffered saline (PBS). The assay was performed at 24 h after the last aztreonam or PBS injection. b Values represent the IL-1-induced thymocyte proliferation. Results represent the means 9 S.D. for seven mice, and of at least three replicates of each sample. c PB0.01.
On the contrary, when mice were injected with 57 mg/kg per day for 7 days, a slight increase in CL values was detected, and when the therapy with 28 mg/kg per day was prolonged for 14 days, the treatment with aztreonam induced an increased level of cellular activation in peritoneal cells. In mice receiving prolonged therapy, CL values were significantly increased (by more than 100%) with regard to the control group (PB 0.001).
3.3. Effect of aztreonam on IL-1 production The production of IL-1 by peritoneal leukocytes from aztreonam-treated mice was tested, in order to examine the effect of this treatment on the cytokine production by innate immune cells. The results summarized in Table 3 show the proliferation of thymocytes co-cultured with samples obtained from supernatants of LPS-stimulated peritoneal macrophages. Results were not significantly influenced by aztreonam treatment, at any dose over 7 days; however, when therapy with 28 mg/kg per day was prolonged for 14 days a remarkable increase of the capacity of IL-1 production by peritoneal macrophages isolated from treated mice, in relation to that observed in the control group was detected.
3.4. Effect of aztreonam on NK acti6ity In order to examine the activation level in NK cells, we tested the cytotoxic activity of splenic mononuclear leukocytes against a YAC-1 cell line. As shown in Table 4, two different effects were obtained in mice treated with aztreonam with different doses. Thus, the NK activity of mononuclear splenic cells was significantly increased in mice treated with 57 mg/kg per day
for 7 days (PB 0.01) and in mice injected with 28 mg/kg per day for 14 days (PB0.001). On the other hand, treatment with 7, 14, or 28 mg/kg per day for 7 days, did not significantly modify the NK activity of mononuclear splenic cells.
4. Discussion Apart from direct antimicrobial activity, the biological properties of antibiotics include interactions and alterations in the cellular or humoral immune responses of patients [25,26]. Antibacterial agents have been recently considered as immune response modifiers and there have been several investigations attempting to describe the immunological profiles of the new antimicrobial agents [4,27]. Since effector cells of the innate immune defense, such as phagocytes and NK cells, play an important role both in the clearance of bacteria and have a role in some clinical aspects of infectious diseases [28], the possible effect of antibiotics on such immune populations may be of great importance. The data presented here show the modification of different parameters of the innate immune response by aztreonam in a murine experimental model. As was described previously when using a imipenem/cilastatin combination [16], the effects of the antibiotic on the different immune functions depend on both the dose and the time of administration. As phagocytic cells are essential for the host defense against infections, the respiratory activity of peritoneal cells has been studied by measuring CL, a powerful analytical tool for establishing the degree of oxidative reactivity in phagocytic cells [20,29]. In the present Table 4 Effect of aztreonam treatment on natural killer (NK) activity of mononuclear splenic cells Dosage (mg/kg per day)a
Treatment duration (days)
Percentage of cloning inhibitionb
Control mice 57 28 14 7 Control mice 28
7 7 7 7 7 14 14
30.15 99.00 41.85 9 5.99c 21.90 97.18 30.20 97.64 38.60 96.98 30.36 98.47 49.50 98.38d
a Mice in the control group received sterile phosphate-buffered saline (PBS). The assay was performed at 24 h after the last aztreonam or PBS injection. b Values represent the percentage of cloning inhibition of the NK-susceptible YAC-1 cell line. Results represent the means 9 S.D. for seven mice, and in duplicate for each sample. c PB0.01. d PB0.001.
E. Ortega et al. / International Journal of Antimicrobial Agents 13 (1999) 41–46
study, increased maximum values of oxidative activity were found in mice treated with 28 mg/kg per day aztreonam, for 14 days, with respect to those obtained in the control group. Likewise, the highest dose (57 mg/kg per day) administered to mice for 7 days increased oxidative activity, as measured by CL assay, of murine peritoneal phagocytic cells induced by zymosan although the difference was not significant. Previous studies have shown that intracellular penetration is a very important factor in the antimicrobial activity of antibiotics. Intracellular organisms may be protected from complete eradication by the immune system as well from the activity of antibiotics that are unable to enter phagocytes [30,31]. Moreover, it has been shown that the activity in vitro of antibiotics that penetrate phagocytic cells against cell-associated bacteria is significantly higher than that of compounds that are excluded from these cells [32]. Other authors have proposed a different interpretation for the intracellular activity of b-lactams such as the triggering of some intracellular reactions through the interaction of the antibiotic with some surface components of bacterial structure [33,34]. b-Lactams do not penetrate PMN, but it has been shown that theydo penetrate monocytes, probably by pinocytosis [35]. Adinolfi et al. [36] demonstrated that aztreonam possesses an intracellular antibacterial activity against E. coli which is greater than its extracellular one. These authors attribute this effect of aztreonam to a positive cooperation of the antibiotic with the O2-independent microbicidal system of macrophages. The results show that, at least in prolonged therapy, aztreonam may also contribute to the eradication of intracellular infectious agents through an increase in the O2-dependent microbicidal systems of murine peritoneal macrophages, as the results from the CL assay suggest. IL-1 is a cytokine produced by macrophages and epithelial cells that induces activation of both macrophages and T-lymphocytes. In this study it was observed that aztreonam exerted similar effects on IL-1 production by peritoneal macrophages as those described for the respiratory activity of this cellular population. Only when mice were treated for a prolonged time (14 days) with the equivalent to the therapeutic dose used in human medicine was a significant increase in the capacity of production of IL-1 by peritoneal macrophages detected. Similar dose-dependent modification of IL-1 production was previously described by Roche et al. [37] using quinolones and an in vitro model with human monocytes. The results agree with those of Roszkowski et al. [9] who showed dose-dependent effects for all antibiotics tested (cefotaxime, amikacin, mezlocillin, piperacillin and clindamycin). This study also agreed with the thesis that even small differences in the molecular formula of
45
antibiotics may have considerable immunopharmacological effects. With respect to NK activity of splenic mononuclear cells, these results were similar to those obtained previously when mice were injected with imipenem/cilastatin [16]. Both doses (57 mg/kg per day) administered to mice for 7 days and therapy with 28 mg/kg per day prolonged for 14 days induced a significant increase in the percentage of cloning inhibition of the NK-susceptible YAC-1 cell line. IL-2 is a cytokine known to act as a growth and differentiation factor for NK cells. Thus, as shown for the combination imipenem/cilastatin [16], the increase in the NK activity of splenic cells could be due to the concurrent increase in IL-2 production by T-cells which is also induced, in aztreonam treatments, by the highest dose employed, as well as by therapy with 28 mg/kg per day prolonged for 14 days (data not shown). As aztreonam does not apparently impair the innate immune response, it should be specially suited for the treatment of patients who are immunosuppressed. Anyhow, it is believed that the experimental design represents a more suitable approximation of the clinical situation than in vitro assays, although further studies are needed regarding the effects of aztreonam on nonspecific as well as on specific immune responses in humans.
Acknowledgements This work was supported by the Plan Andaluz de Investigacio´n (CTS 0105).
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