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Laboratory of Neuroendocrinoimmunology, Department of Internal Medicine I, University Hospital Regensburg
Institute of Medical Microbiology and Hygiene, University of Regensburg, Regensburg
Institute of Medical Microbiology, Immunology, and Hygiene, Technical University of Munich, Munich, Germany
Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus
The sympathetic nervous system is intensely activated during bacteremia, but its immediate influence on the bacterial tissue burden remains unclear. We demonstrate that prior ablation of the sympathetic nervous system decreases this dissemination of Pseudomonas aeruginosa or Escherichia coli through a mechanism of increased secretion of peritoneal tumor necrosis factor, improved phagocytic response of peritoneal cells, and increased influx of monocytes into the peritoneal cavity. When gram-positive Staphylococcus aureus strains were used, sympathectomy increased the bacterial tissue burden, which was caused by a reduction in corticosterone tonus, and decreased both interleukin-4 secretion from peritoneal cells and the influx of lymphocytes into the peritoneal cavity. In both models, the peritoneal wall was the critical border for systemic infection. These results show the dual role of the sympathetic nervous system in sepsis. It can be favorable or unfavorable, depending on the innate immune effector mechanisms necessary to overcome infection.
There is now agreement that sepsis and the systemic inflammatory response syndrome are accompanied by a dysregulation of the inflammatory response. During the onset of sepsis, the inflammatory system becomes hyperactive, evoking a strong anti-inflammatory feedback response by the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). SNS tone rapidly increases after the experimental injection or infusion of bacteria [1, 2] and, similarly, during bacteremia in humans [3]. One of the important cytokines in this respect is interleukin (IL)1 [4]. The question arises whether this uniform SNS response is always advantageous.
It was dogma in the 1980s that the "sympathetic storm" dampens immune responses [5, 6]. This suggestion was based on the immunosuppressive activity of norepinephrine in cell-culture experiments, such as the inhibition of tumor necrosis factor (TNF) [7, 8], interferon (IFN) [9], or IL-12 [10] secretion. However, in recent years, the immunosuppressive role of the SNS has been called into question [1115]. During bacteremia, the favorable or unfavorable role of the SNS may largely depend on immune mechanisms necessary to eliminate a particular infectious agent. If the SNS inhibits an important bacteriostatic factor, SNS ablation will be advantageous for the host. However, if the SNS stimulates a bacteriostatic factor, its inhibition will lead to a higher bacterial tissue burden in tissues. Recent studies have shown that ablation of the SNS decreased the bacterial tissue burden in the spleen of experimental animals after challenge with gram-negative bacteria [16, 17]. Similarly, activation of SNS by experimental stress led to an increased tissue burden of gram-negative bacteria [18]. We aimed to investigate the immunological mechanisms involved and organs affected, in addition to the spleen, in experimental infections with gram-negative and -positive bacteria.
MATERIALS AND METHODS
Mice.
Female outbred Naval Medical Research Institute (NMRI) mice (age, 810 weeks; weight, 2634 g; Charles River) were used in most experiments. In addition, the following female mice of the same age and weight were used: C3H/HeN, C3H/HeJ, Toll-like receptor (TLR) 2 wild type (wt; background, C57BL/6), and TLR2 knockout (background, mixed 129SV × C57BL/6). TLR2-knockout mice were generated by replacing a portion of the tlr2 gene with the neomycin resistance gene [19]. These mice do not produce the TLR2 protein and are functionally deficient in TLR2-mediated signaling [19]. Housing and procedures involving experimental animals were approved by the institutional animal care committee and by the Bavarian government (Regierung der Oberpfalz 6252531.121/01).
Sympathetic denervation.
Chemical sympathectomy was performed in NMRI mice by use of 6OHDA (Sigma). Preliminary sympathectomy experiments were performed by use of saporin-coupled antibodies against dopamin-hydroxylase, which yielded results (with Pseudomonas aeruginosa) similar to those obtained with 6OHDA. Therefore, we used 6OHDA in all subsequent experiments. 6OHDA was dissolved in sterile saline and injected intraperitoneally (ip) at 200 mg/kg of body weight 5 days before the injection of bacteria. Control mice received the respective vehicle. Five days after sympathectomy, mice were injected ip with bacteria (see below).
Strains of bacteria and determination of bacterial tissue burden.
For injection, bacteria were grown to the midlogarithmic phase in Luria broth (LB) or LB with ampicillin (for Escherichia coligreen fluorescent protein ). After harvest, the cell density was estimated from the absorbance at 620 nm by use of a calibration curve. NMRI mice were injected ip with the following bacteria: 107 cfu of P. aeruginosa, 108 cfu of E. coli, 108 cfu of Staphylococcus aureus with toxic shock syndrome toxin1 (51 TSST-1), or 108 cfu of S. aureus without TSST-1 (ATCC 6538). In preliminary experiments, these amounts led to a significant accumulation of bacteria in the spleen (test organ). P. aeruginosa and E. coli were isolated from specimens sent to the Institute of Medical Microbiology; these represent wt isolates. S. aureus ATCC 6538 and S. aureus harboring TSST-1 were obtained from the strain collection of the Institute of Medical Microbiology. E. coliGFP was obtained by transformation of a clinical isolate of E. coli with the high-copy plasmid pCU18-GFP, which carries a modified gfp gene [20]. To determine bacterial growth in tissue, mice were killed 7 h after the initiation of infection, by asphyxiation with CO2; organs were removed, weighed, washed with sterile saline, and homogenized in 1 mL of sterile saline. The number of colony-forming units was determined by plating 10-fold serial dilutions of the sample in sterile saline. Bacterial colony-forming units were counted manually 1236 h later, depending on the strain.
Histologic analysis of the spleen and peritoneal wall.
The spleen and peritoneal wall were removed 7 h after the initiation of infection and embedded in Tissue-Tek (Sakura Finetek Europe) and quickly frozen by floating on liquid nitrogen. The tissue samples were cut into 46-m-thick sections and placed on precoated slides (SuperFrost Plus; Menzel-Glser). To visualize nuclei in spleen sections, fixed samples were incubated with 4,6-diamidino-2-phenylindole hydrochloride (Sigma). Peritoneal wall sections were subjected to standard hematoxylin-eosin staining.
Growth of bacteria in the presence of norepinephrine.
Bacteria were grown in LB for 24 h, and norepinephrine was added in concentrations of 10-410-8 mol/L. To prevent the oxidation of norepinephrine, LB was enriched with 0.57 mmol/L ascorbic acid (Sigma). A bacterial growth curve was obtained by recording the optical density at 620 nm every 2 h for 12 h.
Cytokine release from splenocytes and determination of cytokines.
Single-cell suspensions were obtained by homogenizing spleens on a sterile cell strainer (BD Bioscience). A total of 5 × 105 splenocytes in medium (RPMI 1640, 25 mmol/L NaHCO3, 5% fetal calf serum, and 30 mol/L mercaptoethanol; Sigma) were seeded per microplate well and incubated for 12 h at 37°C together with 0, 1, 10, 100, and 1000 cfu of P. aeruginosa or S. aureus (ATCC 5638)/mL; 10 cfu/mL yielded reproducible data with the smallest coefficient of variation. This amount was then used in all experiments shown (the use of more bacteria resulted in bacterial overgrowth and the destruction of cells). Supernatant was collected, sodium azide was added to stop bacterial growth, and cytokines were determined by use of antibody pairs (OptEIA; Pharmingen BD Bioscience). Intra- and interassay coefficients of variation were <10% for all cytokines. In experiments with isoproterenol (Sigma), splenocytes were incubated together with bacteria and the -adrenergic agonist at 10-6 mol/L (optimum concentration).
Superfusion of pieces of peritoneal wall.
Superfusion was performed as described for spleen slices [16, 21]. One hour after the injection of P. aeruginosa into control and sympathectomized mice, 8 circular pieces of peritoneal tissue (defined by a 4-mm circular punch; Stiefel) were carefully washed, transferred to superfusion chambers (80 L volume), and superfused at 37°C with sterile culture medium (see above) with antibiotics (100 IU/mL penicillin, 100 g/mL streptomycin [both from Sigma], and 8 g/mL ciprofloxacin ). Superfusion was performed for 1 h at 37°C at a flow rate of 25 L/min. Superfusate was collected, sodium azide was added to stop bacterial growth, and the presence of TNF was determined by ELISA (OptEIA) on the same day, to avoid having to freeze and thaw the material.
Enzyme-linked immunospot (ELISPOT) assay for TNF and IL-4.
We used an ELISPOT assay (Pharmingen BD Bioscience) for the detection of TNF and IL-4. Control or sympathectomized NMRI mice were injected with 107 cfu of P. aeruginosa or 108 cfu of S. aureus and killed 1 h later by CO2 asphyxiation, and peritoneal cells were removed by lavage. After 3 washes with cold PBS, 105 peritoneal cells in culture medium with antibiotics were seeded into 1 ELISPOT assay well for 12 (for TNF) or 24 (for IL-4) h. The ELISPOT assay was developed, and, after drying, well bottoms were excised and mounted on a sheet of paper. The paper was then scanned digitally (Office Jet G85; Hewlett Packard) and analyzed by use of Visitron Image Analyzing Software (Metavue version 5.0R1; Visitron).
Phagocytosis of P. aeruginosa.
Twelve hours after 1 mL of sterile saline was injected into the peritoneal cavity of control and sympathectomized NMRI mice (stimulus to mobilize cells), mice were killed by CO2 asphyxiation, and peritoneal cells were removed by lavage and washed. P. aeruginosa in the midlogarithmic growth phase was stained with PKH26 (Sigma), and 108 cfu/mL was incubated together with 106 peritoneal cells/mL for 30 min at 37°C in culture medium without antibiotics. After incubation, cells were washed 3 times with cold PBS, to remove extracellular bacteria. For the identification of macrophages, cells were incubated with Alexa Fluor 647coupled monoclonal antibody (MAb) directed against F4/80 (Caltag) for 30 min at room temperature. Fluorescence-activated cell-sorting analysis was performed by use of COULTER EPICS XL and XL-MCL devices (Beckman Coulter).
Cell influx into the peritoneal cavity.
Twelve hours after 1 mL of sterile saline was injected into the peritoneal cavity of control and sympathectomized NMRI mice, mice were killed by CO2 asphyxiation, and peritoneal cells were removed by lavage. A standard differential blood count was performed on ADIVA 120 (Bayer).
Measurement of corticosterone.
Mice were killed by CO2 asphyxiation, and blood was collected from the left ventricle after opening of the thorax. Corticosterone in the serum was measured by RIA (IBL).
RU486 experiments.
For experiments with the glucocorticoid receptor antagonist RU486 (Roussel Uclaf), NMRI mice were injected subcutaneously with 25 mg of RU486/kg of body weight on 4 consecutive days. On the morning of the fifth day, mice were injected with S. aureus, organs were removed 7 h later, and the bacterial tissue burden was determined as described above.
Experiments with and without corticosterone.
Mock-treated or sympathectomized mice received either vehicle or corticosterone (1 mg/kg of body weight) daily for 3 days. Splenocytes were then harvested, and 5 × 106 cells/mL were cultured together with 10 cfu of S. aureus/mL, as described above. Supernatant was removed after 24 h, sodium azide was added to stop bacterial growth, and the presence of IL-4 was determined by ELISA (OptEIA; Pharmingen BD Bioscience).
Statistical analysis.
All data are given as box plots with the 5th, 25th, 50th (median), 75th, and 95th percentiles. To compare medians, the Mann-Whitney rank test for unpaired data was used (SPSS version 11.5; SPSS). P < .05 was the level of significance.
RESULTS
Influence of the SNS on bacterial tissue burden.
Ablation of the peripheral SNS resulted in a marked reduction in bacterial tissue burden with P. aeruginosa or E. coli in the lungs, spleen, and liver (figure 1A and 1B). In an exemplary experiment, we were able to demonstrate that sympathectomy by intramuscular injection of 6OHDA or saporin-coupled antidopamine-hydroxylase antibodies yielded a similar reduction of bacterial tissue burden (data not shown). To visualize the influence of sympathectomy on the bacterial tissue burden, GFP-transfected E. coli were used to infect control or sympathectomized mice, and the spleens were removed after 7 h. Ablation of the SNS caused a clear reduction in visible E. coliGFP (figure 1C and 1D). This reduction was evident in the nonT lymphocyte areas of the spleen (figure 1C and 1D). Counting of colony-forming units showed the same result (data not shown).
Similar experiments were performed with the 2 gram-positive strains of S. aureus with 51 TSST-1 and without TSST-1. Ablation of the SNS increased bacterial tissue burden in the investigated organs (figure 2A and 2B). The effect was particularly evident in the lungs, livers, and spleens of S. aureusinfected mice (figure 2A). For further studies with gram-negative and -positive bacteria, only P. aeruginosa and S. aureus without TSST-1 were used.
Recent studies demonstrated that norepinephrine, the most important neurotransmitter of the SNS, can support the growth of gram-negative bacteria [22]. In our study, norepinephrine did not stimulate or inhibit the growth of gram-negative or -positive bacteria used (data not shown). Thus, a direct influence of norepinephrine on bacterial tissue burden was unlikely.
Critical cytokines in gram-negative and -positive sepsis.
During the coevolution of bacteria and mice, it might be expected that bacteriostatic programs were evolutionarily conserved. To find such factors, we studied cytokine release from splenocytes after coincubation with P. aeruginosa or S. aureus. Splenocytes stimulated with P. aeruginosa released significantly more TNF, IL-6, and IFN- than did those stimulated with S. aureus (figure 3A). In contrast, splenocytes stimulated with S. aureus secreted larger amounts of IL-4, but not of IL-10, than did splenocytes stimulated with P. aeruginosa (figure 3B). IL-1 was assayed but was not detectable (data not shown).
These results prompted us to conduct experiments with well-defined neutralizing MAbs against TNF (100 g of anti-TNF/mouse; clone V1qH8 [23]), IFN- (100 g of anti-IFN-/mouse; clone R46A2 HC-3 [24]), the IL-1 receptor-1 (800 g of antiIL-1R1/mouse; clone Reg21 [25]), and IL-4 (100 g of antiIL-4/mouse; clone 11B11 [26]). When anti-TNFpretreated mice were challenged ip with P. aeruginosa, a significantly higher bacterial tissue burden was observed 7 h after infection (figure 3C). This was not observed for antiIL-1R1 or antiIFN- pretreatment (data not shown). Similar results were obtained when antiIL-4pretreated mice were infected with S. aureus (figure 3D). However, antiIL-4 had to be administered daily for 3 days before infection, because short-term pretreatment had no effect (data not shown). When sympathectomized mice were challenged with S. aureus and then given IL-4 or corticosterone treatment for 3 days before death, the bacterial tissue burden decreased to the control level (table 1).
These data demonstrated that TNF and IL-4 (with or without corticosterone) were critical factors in determining elimination of gram-negative P. aeruginosa or gram-positive S. aureus, respectively. These 2 cytokines are differentially regulated by the SNS: norepinephrine inhibits the secretion of TNF by macrophages and stimulates IL-4 production by peripheral blood mononuclear cells [7, 8, 27]. The latter effect may be mediated by norepinephrine-induced inhibition of Th1 cytokines, such as IFN- [9]. This prompted us to investigate the impact of sympathectomy on the production of these critical cytokines.
Increase in peritoneal TNF release, peritoneal phagocytosis of bacteria, and monocyte/neutrophil influx into the peritoneal cavity by ablation of the SNS.
TNF release from peritoneal cells from NMRI mice was measured by use of a superfusion system. Sympathectomy caused increased release in mice infected with P. aeruginosa for 1 h (figure 4A). Also, as shown in ELISPOT assays, peritoneal cells from these mice secreted significantly more TNF than did those from control mice (figure 4B). Because the -adrenergic receptor agonist isoproterenol inhibited the secretion of TNF, IL-6, and IFN- by normal spleen cells (figure 4C), we suggest that the suppressive effect was due to -adrenoceptor ligation.
Because TNF is a strong stimulator of macrophage and neutrophil phagocytosis [28, 29], we studied phagocytosis of fluorescently labeled P. aeruginosa by peritoneal macrophages (figure 4D). Sympathectomy led to increased phagocytosis. Taken together, these results indicate that the SNS significantly inhibits TNF release and TNF-dependent mechanisms.
It was described recently that sympathectomy affected leukocyte influx into the peritoneal cavity [30]. In accordance with that finding, table 2 shows that ablation of the SNS increased influx of monocytes and, as a trend, also of neutrophils.
Glucocorticoids, which are secreted during sympathetic activation, can stimulate IL-4 secretion [31, 32] and IL-4mediated antibody production [33]. Thus, corticosterone tonus may be another important defense factor against staphylococci. Ablation of the SNS significantly reduced serum corticosterone concentrations on the morning of day 5 before the injection of live bacteria but not 7 h after infection (figure 5B), probably because adrenal corticosterone secretion may be directly stimulated by circulating proinflammatory cytokines [34]. When NMRI mice were treated for 4 days with the glucocorticoid receptor antagonist RU486 and then infected with S. aureus, the bacterial tissue burden was significantly increased (figure 5C). Thus, the SNS, together with corticosterone, seems to play an important role in controlling infection with S. aureus. This was also obvious in corticosterone-treated sympathectomized mice (table 1).
To investigate a possible cooperative effect of corticosterone and the SNS on IL-4 secretion, NMRI mice were sympathectomized and either treated with corticosterone (1 g/g body weight) for 3 days before the removal of splenocytes or left untreated. Without corticosterone pretreatment, sympathectomy did not reduce IL-4 production on stimulation with bacteria (figure 5D). With corticosterone pretreatment, this effect was greatly enhanced, because IL-4 production was significantly increased in control mice, compared with that in sympathectomized mice (figure 5D). This indicates that the production of IL-4 depends on both the SNS and an adequate antecedent corticosterone tonus.
The production of IL-4 may depend additionally on the presence of lymphocytes in the peritoneal cavity. Table 2 shows that ablation of the SNS or neutralization of IL-4 reduced the influx of lymphocytes (table 2), which indicates that the decreased secretion of IL-4 observed in sympathectomized mice may also depend on reduced lymphocyte numbers.
DISCUSSION
Only limited information is available about the role that the sympathetic branch of the autonomic nervous system plays in gram-negative and -positive sepsis. Here we demonstrate that the SNS plays different roles in bacterial sepsis caused by P. aeruginosa or S. aureus, which largely depend on different bacteriostatic pathways.
In sepsis caused by gram-negative or -positive bacteria, the HPA axis and SNS are strongly stimulated (figure 7) [13]. Their activation decreased peritoneal TNF secretion, phagocytosis of bacteria, and an influx of monocytes and neutrophils into the peritoneal cavity (figure 7)activities necessary for the defense against gram-negative bacteria, such as P. aeruginosa or E. coli. In this respect, it has been demonstrated that TNF is a decisive factor in bacterial peritonitis [23]. We therefore hypothesize that infection-induced stimulation of the SNS has an unfavorable effect on clearance of these gram-negative bacteria. We have demonstrated that prior ablation of the SNS markedly reduced the bacterial tissue burden, which corroborates 2 recent studies in mice [16, 17]. Another study, which used beagles with a partial peritoneal sympathetic blockade, described increased TNF concentrations in serum after an ip application of E. coli [35], which supports our results in peritoneal cells. We conclude from our results that it is likely that the peritoneal wall is an important barrier in transmitting effects of the SNS to the local defense system. There, the SNS suppresses TNF secretion, and the peritoneal release of sympathetic neurotransmitters inhibits phagocytosis by local cells. Furthermore, sympathectomy increased the influx of defending cells into the peritoneal cavity. In conclusion, ablation of the SNS supports bacteriostatic mechanisms against these tested gram-negative organisms in the peritoneal wall and cavity and leads to markedly reduced dissemination of these bacteria to the spleen, lungs, and liver.
The role of the SNS is supportive in peritonitis caused by S. aureus because it stimulates 3 important defense mechanisms. (1) SNS stimulates IL-4 secretion from peritoneal cells, which is necessary for the inhibition of S. aureus dissemination (figure 7): 2 other studies have demonstrated that protein A and lipoteichoic acid from staphylococci preferentially induced Th2 cytokines, such as IL-4 [36, 37]. Another study in mice also demonstrated the importance of IL-4 in host resistance during infection with S. aureus [38]. Furthermore, IL-4 is an activator of neutrophils [39, 40]. (2) The SNS provides an adequate corticosterone tonus, which isby stimulating IL-4 productionanother bacteriostatic factor (figure 7). Glucocorticoid-stimulated IL-4 secretion and IL-4mediated antibody production have been demonstrated elsewhere [3133]. (3) SNS maintains the lymphocyte influx into the peritoneal cavity (figure 7). As a consequence, ablation of the SNS is unfavorable and leads to an increased dissemination of S. aureus into the spleen, lungs, and liver.
Acknowledgments
We thank Elke Lorenz and Melanie Grünbeck, for excellent technical assistance.
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