Departments of 1Physiology and 2Surgery, The Burn and Shock Trauma Institute, Loyola University Medical Center, Maywood, Illinois 60153
Submitted 10 December 2003 ; accepted in final form 4 May 2004
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ABSTRACT |
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catecholamines; sympathetic; cecal ligation and puncture; bacterial endotoxin; -antagonist
Sympathetic activation resulting in the release of catecholamines has been documented in both experimental (13, 14, 39, 40) and clinical (3) studies involving sepsis. However, much of the direct evidence for increased neurotransmitter and hormone release in sepsis has come from experimental paradigms involving spleen, heart, bone marrow, and plasma (13, 14, 39, 40). Our laboratory has previously correlated increased catecholamine release in the bone marrow and enhanced myelopoiesis during sepsis (39). More recently, our laboratory has also demonstrated that bone marrow monocytes developing under the influence of increased sympathetic activation resulting from sepsis are functionally different in terms of their cytokine responses (8). Although sympathetic activation is known to occur in sepsis and may be important for cardiovascular and metabolic compensation, several lines of investigation also suggest that sympathetic activation may mediate immune modulation in sepsis. Maestroni (22, 23) has recently demonstrated that catecholamines modulate the migration and maturation of dendritic cells, which are an important component of both innate and adaptive immune responses and very likely have an important role in the development of sepsis. Equally important is the work of Sanders and coworkers (16, 30, 32) who have presented evidence suggesting that catecholamines may be pivotal in the modulation of Th1 and Th2 cell interactions.
The spleen is one of the lymphoid organs that not only plays an important role in immune regulation following infection but also is highly innervated with sympathetic nerve terminals and, therefore, is an essential target organ for studies involving interactions between the neural and immune systems. Although stimulation of sympathetic nerves to the spleen can mediate the rapid movement of stored red blood cells and leukocytes into the circulation (24, 43), the effect of intense stimulation on innervated immune cells (10) during conditions of sepsis has not yet been elucidated. The potential influence of sympathetic stimulation on immune cell function should be considered in light of the importance of the spleen as an immune organ and its fundamental role in bacterial clearance through antibodies and macrophages, as well as by bacterial endotoxin detoxification (21). Indeed, the significance of the spleen in immune function is highlighted by the incidence of overwhelming postsplenectomy infection, and such occurrences have raised important questions regarding the conditions that warrant splenectomy (12).
In the present study, we have used a murine animal model of surgical trauma and sepsis to assess cytokine-sympathetic neural interactions in splenocyte macrophage cells under closely controlled in vitro conditions. Our goal was to explore macrophage cytokine release by using endotoxin provocation and to define how adrenergic stimulation could modify pro- and anti-inflammatory cytokine interactions, both before and after conditions of injury-induced sepsis. We tested the premise that adrenergic stimulation would enhance anti-inflammatory cytokine release that, in turn, would lead to attenuation of proinflammatory cytokine production. We report that adrenergic stimulation can induce anti-inflammatory cytokine release such as IL-10; however, adrenergic agonist-mediated attenuation of proinflammatory cytokine release may be independent of IL-10.
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MATERIALS AND METHODS |
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Experimental septic peritonitis.
The procedure of cecal ligation and puncture (CLP) with entry of intestinal flora into the peritoneum was used as a model of "polymicrobial sepsis," as originally described by Wichtermann et al. (44). In brief, mice were randomly divided into sham (S), laparotomy (Lap), and CLP groups. All of the animals were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (2.5 mg/kg). Whereas the S group was not surgically manipulated, the Lap and the CLP groups were subjected to a midline ventral laparotomy with exposure and isolation of the cecum. The Lap group was not subjected to any further surgical intervention. In the CLP group, the cecum was tied off 1 cm from the end and punctured with a single hole by using a 21-gauge needle. All animals were resuscitated with 2 ml of sterile saline administered subcutaneously. Surviving animals were euthanized 72 h postinjury, and splenic macrophages were isolated for further studies.
Preparation of splenic macrophage cultures. Splenic macrophages were prepared from each of the three treatment groups 72 h after surgical procedures by placing excised spleens in a plastic culture dish containing HBSS and extracting cells with the use of a rubber-tipped plastic 20-ml syringe plunger. Splenocytes were treated with ACK lysing buffer to remove red cells, washed with additional HBSS, counted, and suspended in RPMI-1640 medium with 10% heat-inactivated FBS at 3 x 106 cells/ml in six-well plates (Costar, Cambridge, MA). After 2-h incubation at 37°C with 5% CO2, nonadherent cells were removed from the culture plate by gentle washing with warm HBSS. Fresh RPMI-1640 with 10% FBS covered the adherent spleen cells, which were then used in cell treatment protocols, as described below.
Cell treatment protocols.
1) To determine the cytokine responses, freshly prepared splenic macrophages were treated with bacterial endotoxin (LPS) from E. coli (Difco, Detroit, MI) at 200 ng/ml for 18 h, and conditioned media were collected and stored at 80°C until analysis. 2) The influence of IL-10 on LPS-mediated cytokine response was determined by 2-h incubation with IL-10 (10 ng/ml), with or without IL-10 antibody (PharMingen, San Diego, CA), followed by LPS for 18 h. 3) The influence of epinephrine on LPS-mediated cytokine response was determined by 2-h incubation with variable concentrations of epinephrine (106 to 1012 M; Sigma Chemical, St. Louis, MO), with or without adrenergic antagonists (Atenolol, selective 1, or ICI 118,551, selective
2 antagonist; Sigma Chemical) followed by LPS for 18 h. 4) To determine the ability of epinephrine to stimulate the release of IL-10 in the absence of endotoxin, macrophages from different treatment groups were incubated with epinephrine (106 M) for 2, 4, 8, 24, or 48 h. 5) The influence of epinephrine to indirectly influence LPS-mediated cytokine response by stimulating IL-10 release was determined by 2-h incubation with epinephrine (106 M; Sigma Chemical), with or without IL-10 antibody (PharMingen), followed by LPS for 18 h. 6) To test whether epinephrine or IL-10 inhibits TNF-
production through cAMP-stimulated protein kinase A activation, splenic macrophages were treated with or without the protein kinase A inhibitor H-89 (106 M; Sigma Chemical) for 1 h, followed by either epinephrine or IL-10 for 2 h, after which LPS was added and maintained for 18 h.
Total cellular protein was assayed by bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL), and the cytokine levels were standardized to the cellular protein concentration.
Cytokine determination.
IL-6, IL-10, and TNF- levels in conditioned media were determined by standard ELISA techniques (BioSource, Camarillo, CA). All cytokine concentrations are reported as picograms per milligram of protein.
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RESULTS |
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Endotoxin-evoked cytokine responses of splenocyte-derived macrophage cells: modulation by epinephrine and IL-10.
The effect of polymicrobial sepsis on splenic macrophage cytokine production and its modulation by epinephrine was investigated in mice 72 h following CLP. Figure 1 describes the influence of S, Lap, and CLP treatments on LPS-stimulated TNF- production. TNF-
responses in cells taken from CLP-treated mice were reduced to 70% of that observed in S and Lap-treated animals (P < 0.05). To determine whether adrenergic agonists or IL-10 modulated TNF-
production, splenic macrophages were pretreated with either epinephrine or IL-10 for 2 h before stimulation with LPS. Exogenous addition of both epinephrine and IL-10 resulted in a significant reduction in TNF-
release (P < 0.05), but, at this single concentration, IL-10 treatment resulted in greater reduction than epinephrine. The reduction of TNF-
release with epinephrine appeared to be greater in the CLP group (23% of that with LPS alone) than that in S (34%) and Lap (35%).
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DISCUSSION |
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Although our observed lack of effect of IL-10 to attenuate proinflammatory cytokine release is in contrast to previous works, suggesting their physiological actions (37, 38), the critical in vivo concentration determining this effect is not known. It can be argued that our ex vivo culture conditions may not accurately represent the in vivo milieu. A much higher concentration of IL-10 could be potentially achieved in the microenvironment that includes both release and site of action. Therefore, the physiological action of IL-10 within the spleen during sepsis remains a possibility. Interestingly, both IL-6 and TNF- production in response to LPS were significantly attenuated in CLP, whereas IL-10 levels were augmented. These findings are consistent with the concept of a progression from proinflammatory to antiinflammatory cytokine responses in developing sepsis (19, 28).
In contrast to our findings, Schwacha et al. (34) have reported ex vivo LPS-stimulated proinflammatroy cytokine responses by splenic macrophages to be exaggerated 7 days after burn injury. These responses, however, involve conditions of thermal injury without infection and may represent a different sequela of responses (34). These authors have attributed the heightened proinflammatory cytokine responses to decreased IL-10 receptor expression, and, therefore, it is important to relate these findings to our data. Although we did not measure them in the present study, a decrease in functional macrophage IL-10 receptors following CLP might explain the lack of IL-10 influence on LPS-stimulated proinflammatory cytokine response. However, similar findings of the lack of IL-10 influence in the uninjured control group (S) seem to make this possibility less likely.
Our pharmacological findings demonstrate that adrenergic inhibition of LPS-mediated cytokine release could be reversed in a dose-dependent manner with selective 2-adrenergic blockade, thus suggesting a clear mechanism for the observed action of epinephrine. Furthermore, our epinephrine dose-response data suggest that inhibition of LPS-stimulated cytokine release occurs at plasma concentrations approximating those reported for mice and at much lower concentrations than would be expected to occur in polymicrobial sepsis based on previous studies in conscious rats (5, 18).
To appreciate the significance of our results, we must view our findings within the framework of other studies involving adrenergic modulation of endotoxin-stimulated TNF-, IL-6, and IL-10 release in both human and animal tissues. Monocytes have been demonstrated to release IL-10 in response to adrenergic stimulation (45), and this response is greatly augmented in the presence of bacterial endotoxin (37, 38). TNF-
and IL-6 release in whole blood, presumably by monocytic cells, has been shown to be inhibited by catecholamines (4, 41, 42), and similar results were found with the use of a monocytic cell line (35). Although catecholamines have been reported to suppress endotoxin-mediated TNF-
release in liver tissue and Kupfer cells,
-adrenergic stimulation in these preparations has been reported to increase IL-6 release (20). Similar to our present findings,
-adrenergic inhibition of IL-6 has also been reported in spleen tissue (36); however, in the kidney and the skin,
-adrenergic stimulation has been reported to increase IL-6 (15, 25, 26). Collectively, these findings suggest that adrenergic modulation of cytokine release appears to be tissue specific, but how such regulation may be altered during conditions of sepsis is largely unexplored.
Bergmann et al. (4) have examined the adrenergic modulation of endotoxin-induced cytokine release in human whole blood taken from septic patients. Like our findings, they report attenuation of TNF- and IL-6 production in sepsis; however, the adrenergic suppression of this release is maintained. They also demonstrate increased IL-10 release under conditions of severe sepsis, but, in contrast to our findings, IL-10 release was not increased by epinephrine. Furthermore, prolonged septic shock ablated the ability of epinephrine to inhibit IL-6 release and markedly reduced the epinephrine attenuation of TNF-
. Direct comparisons between these clinically based findings and our results using experimental sepsis, although important, are complicated by differences in the cell source of cytokines, the severity of sepsis, and the possible effect of therapeutically administered catecholamines in the clinical group. However, these findings do suggest the potential importance of catecholamines to modulate cytokine release in the development of sepsis and septic shock, and additional experimental paradigms would seem well suited to extend the clinical observations under controlled conditions.
Although our data demonstrate that epinephrine and IL-10 have a common result, the suppression of TNF- and IL-6 release, the cell signaling mechanisms involved appear to be quite contrasting. Our results clearly show that, whereas adrenergic stimulation-mediated suppression involves protein kinase A and thus cAMP production, IL-10 suppression of TNF-
does not. Furthermore, previous studies have demonstrated that dibutyryl cAMP inhibits TNF-
synthesis and that this involves the cAMP response element binding protein and interference of the NF-
B nuclear signaling pathway (7, 27, 29). In contrast, cellular mechanisms by which IL-10 has been shown to inhibit TNF-
production are more diverse. These include the absolute requirement for STAT-3 transcription factor (31), inhibition of gene translation through blockade of p38 mitogen-activated protein kinase activation (17), as well as posttranslational mechanisms involving the destabilization of TNF-
mRNA (6).
In summary, our work suggests that increased adrenergic activation could play a dual role in sepsis. While on the one hand it may meet the well-recognized rheological and metabolic needs in sepsis, on the other it may signal an anti-inflammatory phase by ameliorating proinflammatory cytokine production and stimulate anti-inflammatory cytokine production. How catecholamines in sepsis among other biological parameters may alter the balance between pro- and anti-inflammatory cytokines may hold the key to patient outcome.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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