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Unidad de Investigación, Laboratorio de Farmacología Clínica y Experimental, Hospital Universitario Virgen de Valme,1 Departamento de Farmacología, Radiología y Pediatría, Facultad de Medicina, Universidad de Sevilla, Seville, Spain2
Received 13 March 2005/ Returned for modification 18 April 2005/ Accepted 25 June 2005
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ABSTRACT |
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INTRODUCTION |
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The systemic host responses to gram-negative and gram-positive bacterial infections involve similar clinical symptoms, including leukocytosis and fever (19, 44). Leukocytes, mainly neutrophils, are essential for resistance to bacterial infections, as they are the main source of proinflammatory mediators (25, 32). These cells express specific receptors for immunogenic bacterial cell wall constituents released during infection (1, 37).
Lipopolysaccharide (LPS) is believed to be the most important marker for gram-negative bacteria, whereas peptidoglycan by-products such as muramyl dipeptides (MDP) serve as salient stimuli from gram-positive bacteria (9, 19, 20, 44). Although gram-negative bacterial cell walls also contain peptidoglycan, its concentration is far greater in the walls of gram-positive bacteria (20, 44). Muramyl peptides are key elements of the immune response to gram-positive bacteria, which lack LPS. Presently, much work is focused on LPS effects, but many immunocompromised patients die of gram-positive infections (10). Immune cells activated by binding of these substances release many inflammatory mediators, including eicosanoids, cytokines, and chemokines, that activate other immune cells and can also serve to signal the brain.
In humans and experimental animals, MDP and LPS cause an induction of the acute-phase response, which is characterized by fever and the production of cytokines in vitro and in vivo (9, 20, 30), with cells and animals being more sensitive to LPS than to MDP (42). These studies indicate that the patterns of production of endogenous mediators involved in the primary nonspecific acute host response may be dependent upon the specificity of the microbial pathogens and the host recognition pathways invoked. Like for LPS, it is assumed that MDP produces fever via cytokines and prostaglandin E2 induction (20, 44). The mechanisms underlying the febrile response to LPS and MDP in leukopenic animals were not elucidated in this study. This point remains to be resolved, although it is presently under investigation in our laboratory.
Although there are discrepancies regarding the involvement of specific receptors in recognition of gram-negative and gram-positive cell wall components (1, 38, 39, 43), LPS and MDP elicit similar fevers and apparently similar sickness behaviors. Much evidence now indicates that LPS and MDP activate different cell surface receptors and that the stimulation of these different receptors can lead to different pathways and profiles of proinflammatory mediators (13, 17, 18, 29). More recently, evidence has accumulated to indicate that, in contrast to the case for LPS, the response to MDP is CD14 independent and is recognized by nucleotide-binding oligomerization domain 2 but not by Toll-like receptor 2 (TLR2) or TLR4 MD-2 complex (14, 37).
Circulating proinflammatory mediators such as interleukin-6 (IL-6), IL-8, macrophage inflammatory protein-2 (MIP-2), C-reactive protein (CRP), and, more recently, procalcitonin (PCT) have been suggested to be predictive for a systemic microbial bloodstream infection in neutropenic hosts (4, 8, 11, 12, 24, 35, 36). However, the early cellular response to gram-negative and gram-positive bacteria is still unclear, and little is known about how the profile of these mediators during severe leukopenia might be affected by different microbial stimuli.
The present study was established to compare the induction of fever and proinflammatory mediators (IL-6, IL-10, MIP-2, CRP, and PCT) in normal and leukopenic rats following an injection of LPS or MDP as a model to discriminate between gram-negative and gram-positive infections. For this purpose, we used an experimental model that resembles the pathophysiologic events that may occur in patients receiving cyclophosphamide, an alkylating agent with cytotoxic and immunosuppressive activities used in the treatment of malignancies and as a combatant of transplant rejection.
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MATERIALS AND METHODS |
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Drug solutions. Cyclophosphamide, purified lipopolysaccharide from Escherichia coli (serotype 0111:B4), and MDP (N-acetyl-muramyl-L-alanyl-D-isoglutamine) were obtained from Sigma Chemical Co. (St. Louis, MO). The vehicle for all solutions was sterile, pyrogen-free 0.9% saline (PFS) (Fresenius Kabi, Barcelona, Spain). The same lot and stock solutions were used for all experiments. All drug solutions were prepared in pyrogen-free glassware, passed through 0.22-µm-pore-size Millipore bacterial filters, warmed to 37°C, and briefly sonicated before i.p. administration. None of the MDP and cyclophosphamide solutions used in this study induced gelation in the Limulus amebocyte lysate assay, so any contamination with endotoxin was below the level of 25 pg/ml.
Body temperature measurement. Changes in core body temperature were measured every 5 min using remote radiobiotelemetry (Vitalview Series 4000 system; Mini Mitter, Sunriver, OR) with an accuracy of ±0.1°C. Briefly, anesthetized animals (100 mg/kg ketamine and 5 mg/kg xylazine, both administered by i.p. injection), were implanted i.p. with precalibrated temperature-sensitive radio transmitters (model PDT-4000 E-Mitters; Mini Mitter). Each animal was housed in a separate cage and allowed to recover for 1 week. The regular day-night body temperature rhythm was monitored for another week before the experiment. Animals remained in their home cages during the experiment, while the frequencies emitted by the thermistors were monitored remotely. These frequencies were then converted to temperature (°C) values based on each transmitter's calibration data. For the analysis and graphical documentation, temperature data from adequate time intervals of 15 min were used.
Induction of leukopenia. Chemotherapy-induced profound leukopenia in rats was achieved as described previously (24, 26). In brief, rats were rendered leukopenic by i.p. administration of cyclophosphamide at 150 and 50 mg/kg, 3 days and 1 day, respectively, before injection or any further treatment. This protocol resulted in leukocyte counts of <0.1 x 109/liter on the day of drug injection (day 0), mainly due to a very low number of neutrophils. Also, the numbers of lymphocytes and monocytes are strongly reduced and contribute to the profound leukopenia observed in this experimental model (see Table 1). Animals were monitored for evidence of overt illness, activity, and body temperature after the treatments. No animals died following cyclophosphamide administration.
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Fever induction. Fever was induced by i.p. administration of a single dose of LPS (100 µg/kg) or MDP (100 µg/kg), as previously described (23, 30, 33, 41). Control animals received an equivalent volume of PFS as vehicle (1 ml/kg). The order of PFS and pyrogen injections was randomized. All LPS and MDP treatments were carried out 24 h after the second injection of cyclophosphamide, when leukocyte counts are very low (see Table 1). To avoid the development of toxin tolerance, each animal was injected with LPS or MDP only once. All injections were done at the same time of day (0700 h), so that the circadian rhythms of the animals were identical across studies. The changes in core body temperatures were monitored for 12 h posttreatment. For hematological studies and the quantitative determination of inflammatory mediators, additional groups of normal and leukopenic rats were used. Blood samples were collected from rats anesthetized as described above via cardiac puncture (under terminal anesthesia) at selected times after drug injection for later measurement of cell counts and serum mediators.
Hematological parameters. For hematological studies, whole blood for cell counts was collected into sterile tubes containing K3EDTA, and cells were counted by an automatic hematological analyzer equipped with veterinary software (Cell Dyne 3500; Abbot, Allentown, PA), which uses a laser beam and measures the light scattered by the cells to give the total and differential leukocyte counts. Total peripheral leukocytes, lymphocytes, monocytes, and neutrophils counts were determined at 1, 2, 4, 8, and 12 h after LPS, MDP, or PFS administration for both normal and cyclophosphamide-treated rats.
Inflammatory mediators. For measurement of IL-6, IL-10, MIP-2, CRP, and PCT production in serum, blood was collected into sterile tubes containing trisodium citrate and a proteinase inhibitor mixture (125 mmol/ml citrate, 5000 mmol/ml EDTA, 6000 mmol/ml N-ethylmaleimide, and 500 kIU/ml aprotinin). The blood samples were immediately centrifuged at 2,000 x g for 10 min at 4°C and stored at -80°C until the day of analysis (within 1 month). Quantitative determination of rat cytokines in serum was done by enzyme-linked immunosorbent assays, using specific kits for IL-6 (R&D Systems Europe, Abingdon, United Kingdom), IL-10 (IBL, Hamburg, Germany), and MIP-2 (BioSource International, Camarillo, CA). The lower detection limits were 10 pg/ml for IL-6, 5 pg/ml for IL-10, and 1 pg/ml for MIP-2. The serum PCT concentrations were measured using a chemiluminescent immunoassay kit (LUMItest PCT; B.R.A.H.M.S. Diagnostica GMBH, Berlin, Germany) with a lower detection limit of 0.1 ng/ml. CRP in serum was measured by a particle-enhanced immunoturbidometric method, using a Cobas Integra 700 (Roche Diagnostic System, Basel, Switzerland). All samples were analyzed in duplicate and were assayed at optimal concentrations, according to the manufacturer's instructions.
Data analysis. Data are presented as means ± standard errors of the means (SEM) and were analyzed using SigmaStat software (SPSS Inc., Chicago, IL). In graphs of the thermal and cytokine responses, the mean changes were plotted over time. Integrated areas under the curve (AUC), from 0 to 12 h posttreatment, were calculated by trapezoid analysis and were used to compare the differences in the responses of each rat to pyrogen and PFS injections. Data were analyzed by one-way analysis of variance (ANOVA). In cases where the comparisons using ANOVA were shown to be significant, a further test using Student-Newman-Keuls post hoc analyses was performed to compare the effect of each treatment. A P value of <0.05 was considered significant.
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RESULTS |
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60 cells/ml; P < 0.05), reduced numbers of lymphocytes (8 ± 1 cells/ml) and monocytes (10 ± 1 cells/ml) also contributed to the decreased total cell count (P < 0.05). Some characteristic toxic effects of immunosuppressive chemotherapy were noted; e.g., the leukopenic rats appeared more inactive and may have lost some weight. Figure 1 shows the effects of i.p. administration of 100 µg/kg LPS or 100 µg/kg MDP on the total count of white blood cells for immunocompetent and leukopenic rats. All immunocompetent rats had a transient leukopenia at 1 h after MDP or LPS administration (P < 0.05). This leukopenia was followed by leukocytosis due to a marked recovery in neutrophils to a level that exceeded the normal level at 4 or 8 h after MDP or LPS administration, respectively (data not shown). At 12 h, total peripheral leukocyte counts were similar to preinjection values (time zero) (P > 0.05). However, in contrast to the case for immunocompetent rats, the same doses of either LPS or MDP did not modify the leukocyte populations deteriorated from a normal range due to cyclophosphamide during all experimental periods (P > 0.05).
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Effects of LPS and MDP on body temperature. To test for the development of a pyrogenic response to LPS or MDP, normal rats and rats rendered leukopenic with cyclophosphamide were injected i.p. with 100 µg of either LPS or MDP per kg (Fig. 2). Importantly, the mean body temperature did not differ among the experimental groups, and all animals exhibited a similar basal body temperature that ranged between 36.9 and 37.3°C at the time of injection. The body temperature in the PFS-treated control groups remained stable throughout the study and was not different among groups (P > 0.05). Intraperitoneal injection of either LPS or MDP caused fever in both normal and leukopenic rats (Fig. 2A and B). Fifteen minutes after challenge with 100 µg LPS or 100 µg MDP, body temperatures in immunocompetent rats started to rise, and they reached the peak level at 6 to 8 h in normal rats (Fig. 2A). The return to the preinjection level was gradual but was essentially completed by 12 h after challenge (Fig. 2A). In contrast, the fevers induced by LPS or MDP in leukopenic rats remained elevated up to 12 h following the injection and were significantly higher than those of the immunocompetent animals throughout (P < 0.05) (Fig. 2B). Thus, although the onset latencies of both groups of rats were not significantly different, the postfebrile recovery after LPS and MDP administration in leukopenic animals was not completed after 12 h after pyrogenic stimuli, and there was a significant enhancement in the fever (Fig. 2C). By 96 h postinjection, body temperature declined to or near to preinjection values (data not shown). As shown in Fig. 2C, injection of LPS or MDP in leukopenic rats resulted in a significant increase in core body temperature, which was significantly higher than that of the immunocompetent group (Fig. 2C). Furthermore, the febrile response evoked by LPS was significantly higher than that evoked by an equivalent dose of MDP in both groups of rats (Fig. 2C) (P < 0.05).
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The comparative study using analysis of integrated areas under the curve during the early phase of LPS- or MDP-induced fever showed significant differences between immunocompetent and leukopenic rats (Fig. 5). Baseline levels of IL-6, CRP, and PCT in serum were significantly higher in leukopenic rats. In contrast, baseline levels of MIP-2 were significantly higher in immunocompetent animals, and those of IL-10 were not detectable in either group of rats. Injection of LPS or MDP evoked a significant decrease in IL-10, MIP-2, and PCT levels in leukopenic rats compared to immunocompetent rats (P < 0.05) (Fig. 5). By contrast, IL-6 production was significantly higher in leukopenic rats than in immunocompetent rats after LPS administration (P < 0.05) but not after MDP administration (P > 0.05). Both groups of rats that received MDP showed lesser increases in serum IL-6 (Fig. 5A), IL-10 (Fig. 5B), and MIP-2 (Fig. 5C) production than were observed in rats that received a similar dose of LPS. Finally, PCT and CRP failed to discriminate between bacterial stimuli in both groups of animals (Fig. 5D and E).
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DISCUSSION |
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Gram-negative and gram-positive organisms have been considered the most important causes of infection in leukopenic patients who undergo chemotherapy. It is therefore not surprising that bacterial constituents that are present on the outside of the cell and therefore readily accessible to detection have been selected by the immune system as indicators of bacterial presence and potent inducers of a host response. During a bacterial infection in neutropenic patients, it is of vital importance to the infected host that the presence of pathogenic bacteria is detected as soon as possible, since these patients present a high morbidity and this may lead to mortality if the infection is treated inappropriately or too late. The present knowledge regarding endogenous inflammatory mediators in leukopenic patients is limited but indicates that the host response to microbial infection is mediated by the release of some pyrogenic mediators into the bloodstream (see the introduction).
Studies with immunocompromised patients have shown that these patients are capable of producing high serum concentrations of several mediators during severe systemic bacterial infection (4, 11, 21). In febrile neutropenic patients, elevated circulating IL-6 and IL-8 also predicted microbial infection (8, 12). In some reports, significant differences in the serum levels of different inflammatory mediators between gram-negative and gram-positive infections were described. Several investigators found differences between neutropenic patients with and without bacteremia and/or differences between gram-negative and gram-positive bacteremia. The latter observations, however, are difficult to interpret. Many patients with other causes of fever are omitted from such an analysis, but they need to be considered in order to calculate true sensitivities and specificities.
A key event in the initiation of fever in response to acute bacterial infection is the induction and release of endogenous pyrogens (i.e., IL-1, IL-6, IL-8, RANTES, and MIPs) by polymorphonuclear and mononuclear leukocytes into the bloodstream (19, 23, 32, 40, 41, 44). Normal leukocytes contain no detectable pyrogen and do not produce any during in vitro incubation. After stimulation, however, pyrogen production begins within a few hours and continues at a steady rate for 12 h or more (5). The key event is a rapid activation of humoral cascade systems and of leukocytes that support the inflammatory reaction. These cells are thought to be an essential target for gram-positive and gram-negative bacteria, as they are the main source of inflammatory mediators involved in the host acute response to these organisms (32). We found that leukopenic rats exhibit higher fevers after systemic injection of LPS and MDP than immunocompetent rats, despite lower increase in circulating mediators. Moreover, we found that, independently of the immune status, rats that received similar doses of LPS showed higher fevers and greater increases in serum cytokine levels than those observed after MDP challenge. Interestingly, febrile responses actually subsided before the disappearance of elevated levels of serum cytokines after injection of LPS and MDP in leukopenic rats. These results are consistent with the idea that even when cytokines are detected in the blood, their timing does not always correlate with acute-phase activation after administration of immune stimuli (19).
Concerning the role of administration of cyclophosphamide, it has previously been shown that this immunosuppressive drug induces a nonspecific leukopenia (fewer than 100 cells/ml for 6 to 7 days) (24, 26) and a selective depletion of the lymphoid tissue (2, 42). When such treated patients become afflicted with a bacterial infection, they develop high fevers. These results suggest that a lesser contribution of leukocytes may be important to the exaggerated fever that occurs in leukopenic patients. However, in addition to peripheral blood leukocytes, the tissue resident macrophages are required for host defense against acute bacterial infection. In this context, production of cytokines at the tissue level, rather than in circulation, could be an alternative pathway for the induction of the signal leading to fever in leukopenic animals.
Because the liver is the principal organ for LPS clearance and its resident macrophages, the Kupffer cells, are a major source of pro- and anti-inflammatory cytokines, it has been suggested that these cells could be central to production of fever by LPS (44). However, the activity of Kupffer cells is also depressed by cyclophosphamide in mice and rats (2, 22), and temporary blockade of these cells with gadolinium chloride, a substance known to eliminate Kupffer cells (31), did not prevent the febrile response to i.p. LPS in leukopenic rats (E. Tavares, unpublished data). These results indicated that other cells types (fibroblasts, endothelial cells, glial cells, etc) involved in the acute inflammatory response are major sites of the action of bacterial toxins in immunosuppressed rats.
The present results indicate that cytokines such as IL-6, IL-10, and MIP-2 are regulated or released independently of the leukocyte count. How the absence of leukocytes leads to an intensified rather than a reduced febrile response to LPS and MDP and an associated decreased production of circulating inflammatory mediators is still unknowable. That is, the reduction of circulating leukocytes by cyclophosphamide caused significantly enhanced fevers (in both magnitude and duration), and this effect was associated with a significant reduction of circulating inflammatory cytokines compared to those in immunocompetent animals. The present findings confirm previous experimental and clinical studies that show that the production of fever was not dependent on the presence of leukocytes in the peripheral circulation (3, 24, 28). Furthermore, they suggest that other, yet-to-be identified mediators as possible mechanisms of immune signaling are involved in the development of the acute-phase response to gram-negative and gram-positive infections. Together, these findings suggest that in immunosuppressed animals leukocytes may play a "negative" role in the development of a fever induced by exogenous pyrogens. However, further studies on the pathophysiology of fever and the contribution of early markers regulated or released independently of the leukocyte count are necessary to verify this hypothesis.
Our findings suggesting that the patterns of early production of proinflammatory mediators may be dependent upon the specificity of the microbial pathogens are in agreement with recent studies (13). The differences in the cytokine pattern and the febrile responses to LPS and MDP may correspond to the different cell surface receptors stimulated by these bacterial pyrogens, with leukopenic animals being more sensitive to LPS than to MDP, which is in agreement with previous in vitro and in vivo studies (43). Functional characterization of TLRs has established that innate immunity is a skillful system that detects invasion of microbial pathogens. Recognition of microbial components by TLRs initiates signal transduction pathways, which triggers expression of genes. These gene products control innate immune responses and further instruct development of antigen-specific acquired immunity (37). In agreement with these findings, the present results indicate that the patterns of production of endogenous mediators involved in the primary nonspecific acute host response may be dependent upon the specificity of the microbial pathogens and the host recognition pathways invoked.
The present findings also illustrate the potential differences in the patterns of whole-blood leukocytes in response to two disparate, but common, bacterial stimulants. Taking all these data together, it is improbable that the pyrogenic effects of LPS and MDP seen clinically in leukopenic patients are directly and exclusively due to stimulation of pyrogen production by leukocytes. Overall, serum IL-6, IL-10, and MIP-2 levels are able to discriminate between gram-negative and gram-positive infection at start of fever in both immunocompetent and leukopenic rats with acceptable specificity. Furthermore, they suggest that the exaggerated fever response occurring after LPS injection in leukopenic animals is partially mediated via IL-6. On the other hand, CRP and PCT are not sensitive markers in distinguishing these types of infections. However, although we used an experimental rat model that resembles the pathophysiologic events that may occur in leukopenic patients, the findings reported here cannot necessarily be extrapolated to the response to different gram-negative or gram-positive pathogens or their constituents in immunosuppressed patients. The mechanisms underlying the febrile response to LPS and MDP in leukopenic animals were not elucidated in this study. This point remains to be resolved, although it is presently under investigation in our laboratory.
The data presented here extend the argument that LPS and MDP induce differential fever responses and cytokine profiles in normal and immunodeficient animals. Finally, these results indicate that the determination of the serum pattern of specific inflammatory markers at the onset of fever may be used as an early diagnostic tool to differentiate gram-negative from gram-positive infections in both immunocompetent and leukopenic hosts. A rapid bedside test may thus help guide the choice of specific antibiotic therapies, especially for neutropenic patients, even before culture results are available.
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ACKNOWLEDGMENTS |
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This study was supported by Fundación Valme, Junta de Andalucía, and by a grant from the Spanish Fondo de Investigaciones Sanitarias (FIS 00-1066).
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FOOTNOTES |
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REFERENCES |
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| Antimicrob. Agents Chemother. | Clin. Microbiol. Rev. | Infect. Immun. |
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Tc) were determined in separate groups of immunocompetent (A) and leukopenic (B) rats during 12 h after i.p. injection of either PFS (
), 100 µg/kg LPS (•), or 100 µg/kg MDP (
). The mean baselines of absolute body temperatures at time zero for the groups were not significantly different (36.9 to 37.3°C). Integrated thermal responses (C) (AUC) of immunocompetent and leukopenic rats after administration of LPS (black bars), MDP (hatched bars), or PFS (white bars) were calculated for the whole 12-h recording period from the data shown in panels A and B (10 to 12 rats under each condition). Data are expressed as means ± SEM. 
and *, statistically significant differences (P < 0.05) between LPS- and MDP-treated rats and normal rats, respectively. 
, significantly different (P < 0.05) from corresponding values between immunocompetent and leukopenic rats (ANOVA, Student-Newman-Keuls post hoc test).
