Introduction

The physiological importance of the adrenal gland pertains to the release of epinephrine and glucocorticoids in response to cognitive stress, recognized as the ‘fight or flight’ response. Such responses involving the hypothalamopituitary–adrenal axis begin with the hypothalamic release of corticotrophin-releasing hormone (CRH), which mediates the release of adrenocorticotrophic hormone (ACTH) from the pituitary, which in turn stimulates the synthesis and release of cortisol from the adrenal cortex. Hypothalamic stimulation also initiates the release of epinephrine from the adrenal medulla, and that of the neurotransmitter norepinephrine from adrenal medulla and the adrenergic nerve terminals. The action of these hormones and neurotransmitters is traditionally thought to serve in a compensatory manner, facilitating heightened mental awareness along with metabolic and cardiovascular activity that supports rapid increases in muscular work.

Thermal injury, like other forms of trauma, as well as infectious challenge, is a non-cognitive stimulus but also results in an elevated hormone/neurotransmitter milieu similar in magnitude to that of the cognitive ‘fight or flight’ response.^1,2 However, there are important characteristics of the injury response that contrast with the fight or flight response. These include prolonged hormone/neurotransmitter elevation, the absence of increased muscle work limiting metabolic demand, and the presence of massive tissue injury. Additional hormone/neurotransmitter responses may also be evoked by surgical debridement of burn wounds and skin grafting procedures. The second surge of stress hormones complicates the severe metabolic derangements and compromised immune capacity, which is characteristic of the burn course during the initial 7–10 days following injury.

Cognitive and non-cognitive events that increase corticosteroid and catecholamine levels represent a stress response that may or may not involve pathology. Traumatic injury, however, clearly initiates a stress response, with a magnitude proportional to the severity of injury. Under these conditions, hormone/neurotransmitter release seems to promote survival. Although such a benefit may be difficult to see in human injury, experimental animal models have provided some insights. Animals that lack stress hormones or suffer from impaired release or by pharmacological block often die of an otherwise survivable event. Similarly, overwhelming traumatic injury in man may result in stress hormone release that is detrimental to survival. Whereas this may also be difficult to document in burn patients, animal studies have demonstrated that exogenous administration of large amounts of stress hormones is detrimental.

Two historical perspectives are important to consider regarding stress and the trauma of burn injury. First is the concept described by Cuthbertson,³ where the initial response to thermal injury is considered an ‘ebb’ phase characterized by reduced metabolism and tissue perfusion. Within days there is a transition to a ‘flow’ phase typified by increased resting energy expenditure and hypermetabolism, with supportive cardiovascular function. Changes in endocrine hormone levels are important for these acute catabolic alterations. The concepts of ‘stress’ and release of ‘stress hormones’ widely used today were clarified by the classic work of Seyle.⁴ His notion of stress responses include an initial ‘alarm reaction’ of fairly short duration with high levels of stress hormones, followed by a prolonged ‘resistance phase’ during which there is compensation to maintain homeostasis during continued stress. Seyle’s final stage of ‘exhaustion’ is where compensation cannot be maintained and death rapidly follows. The acute initial period of high stress hormone release encompasses both Cuthbertson’s ‘ebb’ phase and Seyle’s ‘alarm reaction,’ and the ‘flow phase’³ has similar features to the ‘resistance phase.’⁴ These compensatory changes promote increased energy expenditure and support cardiovascular function. However, in patients with severe injury the same compensatory changes result in depletion of energy reserves, extensive muscle wasting, and immune suppression, all of which are hallmarks of post-burn sequelae. This compensatory pattern is described as a hypermetabolic state, with patients displaying elevated resting metabolic rates for several months following injury.^5–7 The extent of the hypermetabolic response is dependent upon the extent and depth of burn injury, septic complications, and surgical interventions. Nonetheless, questions related to the magnitude of stress hormone responses and beneficial versus detrimental actions in the recovery from severe thermal injury remain unanswered.

During the last 50 years great advances have been made in the care of burn patients and with various treatment modalities attempting to exploit some of the concepts described above. Examples include rapid fluid resuscitation to stabilize cardiovascular function and reduce the stimulus for continued sympathetic drive; nutritional support to meet metabolic demands and to support homeostasis during healing; elevated environmental temperatures and occlusive dressings to lessen the metabolic demands, reduce metabolic rate and cardiac output, and to optimize wound healing. Aside from the regulation of metabolic and cardiovascular function, neuroimmune interactions may be important in mediating the marked alterations in immune function that often follow severe injury. In this chapter we have chosen to present the adrenomedullary–neurotransmitter activation and actions as separate from the adrenocortical activation and actions to clarify specific responses, as we currently understand them. To this end we will review the magnitude and time course of the stress hormone/neurotransmitter responses to thermal injury as well as how the action of these substances may be integrated. How these responses may be beneficial or detrimental during the course of recovery will also be addressed. Given the range of pharmacologic antagonists and agonists available to the clinician, taking a mechanistic approach to understanding adrenal hormones and neurotransmitter involvement in the pathophysiology of burn injury is critical to further advance the successful treatment of these patients.

Sympathetic influences on immune function

Over the last 20 years experimental evidence describing the interactions between neural and immune cell systems has expanded greatly. This has important implications in our understanding of the pathobiology of thermal injury, as in vivo sympathetic activation as well as compromised immune function both occur following thermal injury. In addition to endogenous release of catecholamines, burn patients are often treated with adrenergic pressor agents; they may be on a therapeutic regimen of β-adrenergic antagonist prior to injury, or hypermetabolism may be controlled in pediatric burn patients with β-adrenergic antagonists. Thus, adrenergic modulation following thermal injury may involve therapeutic as well as endogenous mediators and may have an important impact on immune system function.

For the activation of sympathetic nerves to influence immune responses, evidence of sympathetic innervation in peripheral lymphoid organs is essential. Existing anatomical evidence is based on immunohistochemical techniques to visualize tyrosine hydroxylase, the rate-limiting step in the biosynthesis of norepinephrine. These studies clearly indicate a substantial innervation of all primary (thymus and bone marrow) and secondary (spleen and lymph nodes) lymphoid organs.^59–63 Furthermore, they also show sympathetic innervations in the immune cell compartment of the spleen (the white pulp), the periarterial lymphoid sheath, marginal zone and marginal sinus areas, as well as in the splenic capsule and trabeculae.^64–67 Sympathetic nerve terminals have been described in direct apposition to T cells, interdigitating dendritic cells and B cells.⁶⁵ The proximity of nerve terminals to immune cells may be critical in achieving the necessary local concentrations of neurotransmitters at the neuroimmune junctions to modulate immune functions. In fact, the neuroimmune junction is estimated at 6 nm,⁶⁸ compared to 20 nm in typical CNS junctions, indicating that sufficient neurotransmitter concentration could be released across these small junctions to affect resident immune cells.

Anatomical evidence of sympathetic innervations of the immune system is complemented by evidence for nerve-stimulated release of norepinephrine in both spleen and bone marrow.⁶⁹ Whereas evidence in spleen has been recognized for many years and has been assessed in a variety of ways, norepinephrine release in bone marrow has only been recently described, using norepinephrine turnover techniques based on radiotracer methods involving in vivo experimental paradigms.⁶⁹ In contrast, exocytosis of norepinephrine from lymph nodes has not been demonstrated. To complete the criteria for the physiologic importance of functional innervations, nerve-stimulated release of norepinephrine within lymphoid organs must increase at the appropriate time to influence the immune response in conjunction with demonstration of norepinephrine-mediated immune modulations.

Lymphocytes, including activated and resting B cells, naïve CD4⁺ T cells, T-helper (Th1) cell clones, and newly generated Th1 cells, express β adrenergic receptors, but they are not expressed in newly generated Th2 cells.^70–72 Furthermore, there is significant evidence that norepinephrine can modulate the function of CD4⁺T cells, which in turn can modulate antibody production of B cells.⁷³ In addition, norepinephrine can directly influence B-cell antibody production depending on the time of exposure following activation.⁷⁴ The physiologic importance of these in vitro findings is supported by a series of in vivo experiments involving severe combined immunodeficient (scid) mice depleted of norepinephrine prior to reconstitution with antigen-specific Th2 and B cells. These experiments demonstrate that norepinephrine is necessary for maintaining a normal level of antibody production in vivo.⁷⁰ Furthermore, other recent whole animal experiments, also involving scid mice, provide evidence that the immune response itself stimulates the release of norepinephrine from adrenergic nerve terminals in bone marrow and spleen, which in turn can influence antibody production by B cells.⁷⁵ However, in a prospective intent-to-treat study, β-blockade in 20 paediatric burn patients significantly reduced serum TNFα and IL-1β.⁷⁶ Although these findings fall far short of direct application to immune cell function following thermal injury, they suggest the potential of sympathetic activation in mediating immune responses.

In addition to neural influences on T- and B-cell function, there are direct effects on myeloid cell function, particularly with respect to lipopolysaccharide (LPS)-stimulated cytokine production. The most striking examples of neural influences on macrophage function were demonstrated by the work of Spengler et al.,⁷⁷ who concluded that α-adrenergic stimulation increases TNF-α release, whereas β-adrenergic stimulation reduces such release in response to LPS. They provided further evidence to suggest that extracellular stores of catecholamines in macrophages are capable of modulating TNF-α release. Furthermore, sympathetic inhibition of LPS-induced TNF-α release has been suggested to occur in whole animal preparations, although adrenergic actions on macrophages were indirect.^32,78–83 More direct evidence of adrenergic inhibition of LPS-stimulated TNF-α production has involved whole blood.^83–88 In our recent documentation, blocking β-adrenergic receptors soon after injury partially reversed LPS-stimulated TNF-α potential of circulating monocytes lost during the course of burn injury and sepsis.⁸⁹ Apart from adrenergic inhibition of LPS-stimulated TNF-α release in isolated macrophages,^90–93 similar inhibition of LPS-stimulated TNF-α production has also been demonstrated in human mast cells,⁹⁴ microglial cells,⁹⁵ astrocytes,⁹⁶ and cytotoxic T lymphocytes.⁹⁷ In contrast to adrenergic stimulation of TNF-α release, experiments with isolated atria,^98,99 myenteric plexus,¹⁰⁰ and brain tissue¹⁰¹ have demonstrated that TNF-α can negatively affect the release of norepinephrine.

Adrenergic influences on the expression and release of interleukin (IL)-6 have been suggested by a number of studies demonstrating increases in plasma IL-6 in response to direct or indirect stimulation.^102–104 Adrenergic enhancement of IL-6 responses to LPS has also been demonstrated in vivo^79,81 as well as ex vivo⁹³ and in isolated cell systems.^105,106 Catecholamines in combination with IL-1β stimulate IL-6 release from rat C6 glioma cells, and vasoactive intestinal polypeptide (VIP) has been reported to synergize with norepinephrine to induce IL-6 release in astrocytes.^107,108 In addition, adrenergic agonists have been shown to mediate IL-6 release in brown adipocytes, pituicytes, hepatocytes, astrocytes, and thymic epithelial cells.^107–113

In contrast, Nakamura et al.⁹⁶ reported that catecholamines reduced the IL-6 response to LPS, and van der Poll et al.⁸⁷ demonstrated that norepinephrine inhibits the LPS-induced IL-6 response in whole blood. Other evidence for adrenergic suppression of IL-6 responses has been suggested by the work of Straub.^114–116 Using isolated splenic tissue preparation, electrically stimulated release of norepinephrine appeared to inhibit IL-6 production induced by LPS or bacteria. These authors suggest that adrenergic inhibition of IL-6 is reduced under conditions simulating infection, where cytokine mediation of the inflammatory response is compensatory in eradicating the bacterial load. It is apparent from these studies that catecholamines can exert a negative or a positive influence on IL-6. Given these different modulatory functions, the role(s) catecholamines play in the pathophysiology of burn injury remain unexplored.

Although the exact mechanisms of the negative modulation of proinflammatory cytokines by catecholamines are poorly understood, it may be achieved through the ability of catecholamines to induce the anti-inflammatory cytokine IL-10.^{81,84,117,118} Whole animal studies involving assessment of circulating levels of IL-10⁸¹ as well as studies of human whole blood and mononuclear cells stimulated with LPS in the presence of adrenergic agonists^83,84,118 support this premise. In addition, experimental neurotrauma results in increased IL-10 consequent upon endogenous adrenergic stimulation in the absence of LPS or other evidence of infectious challenge.¹¹⁹ The only experimental evidence suggesting an attenuation of IL-10 with adrenergic stimulation involved a macrophage cell line (RAW 264.7).¹²⁰

Evidence that elevations of IL-10 can be blocked with inhibition of protein kinase A (PKA)^118,121 is consistent with adrenergic mediation of changes in TNF-α and IL-6, and suggest that activation of PKA is important in effecting these adrenergic modulations of cytokine release. More specifically, the recent work of Platzer et al.¹¹⁷ suggests that catecholamines in monocytic cells directly stimulate the IL-10 promoter/enhancer, and provide evidence that a cAMP response element was the major target of the cAMP/PKA pathway. In contrast, two independent studies from our laboratory report evidence that, although adrenergic stimulation increases IL-10 release from macrophages, release of TNF-α and IL-6 is inhibited by direct adrenergic stimulation not secondary to IL-10.^122,123 These studies involved both peritoneal-elicited and bone marrow progenitor-derived macrophages under normal conditions as well as following cecal ligation and puncture injury. Epinephrine attenuated TNF-α but increased IL-10; however, addition of anti-IL-10 antibody did not prevent epinephrine’s ability to block TNF-α reduction. Further experiments demonstrated the action of epinephrine to inhibit LPS-stimulated release of proinflammatory cytokines to be mediated by β₂-adrenergic receptors. The dominance of direct adrenergic inhibition of LPS-mediated proinflammatory cytokines was maintained during conditions of sepsis, although such conditions elevated endogenous levels of IL-10.

Adrenergic stimulation of bacterial growth

Since the identification of mammalian hormone and neurotransmitter receptors in bacterial cells, there has been considerable interest in defining a role for such signaling molecules in bacterial cells. As a consequence, support has emerged for the concept that release of norepinephrine within intestinal tissue promotes the growth of bacteria in the gut.^124–126 Initial experiments demonstrated the growth-promoting action of catecholamines in vitro using several different bacterial species, and provided evidence that these compounds were not acting as nutritional substrates. Since adrenergic blocking agents did not block the growth-stimulating effects of norepinephrine, adrenergic receptors do not appear to be involved.^127,128 Further observations suggest that norepinephrine may act within 8 hours to induce bacterial growth, during which time stimulation of growth factors can promote bacterial growth.^129,130 Norepinephrine-stimulated bacterial growth has also been shown to produce Shiga-like enterotoxins from enterohemorrhagic strains of E. coli. Furthermore, norepinephrine promotes the expression of K99⁺ pilus adhesin, a virulence factor known to play a critical role in the attachment of these bacteria to the intestinal wall, which initiates the infective process.^131,132 Although these studies use very high concentrations of norepinephrine compared to the observed plasma concentrations following thermal injury, bacteria in vivo may be exposed to high norepinephrine concentrations if they are in close proximity to the nerve terminal synapse. A related concern is the lack of information regarding the actual norepinephrine concentration in the culture media throughout the incubation period. Whereas rapidly growing bacterial cultures may generate an acid environment in which catecholamines are quite stable, initial growth conditions containing low bacterial counts and minimal nutrients may promote rapid deterioration of norepinephrine. However, high initial norepinephrine concentrations in culture may counteract such unfavorable conditions but in turn would provide misleading dose–response information.

The extensive sympathetic innervations of the gut and associated structures has been recognized for many years, with well-defined nerve terminals located primarily along blood vessels but without evidence of neurotransmitter release into the intestinal lumen. Furthermore, there is considerable evidence that, once released from nerve terminals, most norepinephrine is taken back into the same terminals by reuptake mechanisms, metabolized into a non-active form, or diffused through tissues to reach blood vessels to become part of the circulation.¹³³ Therefore, even though intestinal bacterial growth has the potential to be enhanced by the neurotransmitter norepinephrine, transport of the norepinephrine into the intestinal lumen would seem problematic. However, as massive catecholamine release is such a consistent component of burn patients, especially those with superimposed sepsis, the hypothesis that bacterial growth can be enhanced by norepinephrine is very appealing.

This concept is strengthened by studies demonstrating that cecal bacterial growth increases dramatically following massive in vivo release of norepinephrine, and that passage of bacteria through the gut enhances their growth response to norepinephrine. In the first case,¹²⁶ mice were treated with 6-hydroxydopamine, a neurotoxin that displaces norepinephrine from adrenergic nerve terminals, causing a transient but massive sympathetic reaction. At 24 hours post treatment cecal bacterial growth was elevated 3–4 degrees of magnitude compared to vehicle-treated controls, but bacterial growth returned to control levels by 14 days. In the second study¹³¹ an attenuated strain of Salmonella typhimurium was administered to rhesus monkeys, whereupon isolated fecal bacterial cultures from these animals displayed increased in vitro growth responses to norepinephrine. To elucidate whether this hypothesis has a role in the pathophysiology of thermal injury with sepsis, future studies must build on experimental paradigms of thermal injury to demonstrate that endogenous norepinephrine enhances bacterial growth leading to sepsis.

Evidence for norepinephrine regulation of myelopoiesis in experimental thermal injury with sepsis

Patients with severe burn trauma often display significant impairment in cell-mediated immunity involving defective neutrophil chemotaxis, phagocytosis, and superoxide production.^134–137 Patients with sepsis and a systemic inflammatory response may also present with monocytosis and neutropenia.^138,139 Whereas neutropenia and defective neutrophil functions may compromise host defense, monocytosis has the potential to fuel excessive cytokine production through increased availability of circulating and tissue monocyte/macrophages. For the past 10 years our laboratory has been investigating bone marrow following thermal injury with sepsis to understand the mechanisms that govern leukocyte production, and how they might contribute to the observed defects in leukocyte function. The potential for sympathetic activation to modulate myelopoiesis following thermal injury and sepsis is supported by previous studies where adrenergic stimulation has been shown to participate in the regulation and control of hematopoiesis.^140,141 Maestroni¹⁴¹ has not only provided evidence for the presence of adrenergic receptors on bone marrow immune cells, but also that adrenergic agonists stimulate lymphopoiesis while attenuating myelopoiesis under normal, non-injury conditions. These findings are further strengthened by animal experiments where adrenergic agents have been shown to modulate both lympho- and myelopoiesis.^142–145 Another important factor supporting the possible adrenergic regulation of myelopoiesis following thermal injury is that sympathetic activation can occur directly within the bone marrow compartment, with nerve-stimulated release of norepinephrine in close proximity to developing immune cells. We have documented a significant increase in murine bone marrow norepinephrine release in response to either cold exposure or bacteria through the use of traditional pulse-chase experiments.⁶⁹ Furthermore, we have extended these measurements to demonstrate increased bone marrow norepinephrine release in response to thermal injury with sepsis in our murine model.¹⁴⁶

These findings suggest that sympathetic activation has the capacity to drive events within the bone marrow, and adrenergic-mediated expansion of leukocyte production could conceivably contribute to perturbed inflammatory responses with immune challenge following thermal injury with sepsis. Experimental evidence suggesting that adrenergic stimulation inhibits myelopoiesis under normal conditions¹⁴¹ but is shifted following injury to enhanced monocyte development¹⁴⁶ is a most interesting phenomenon. Whether adrenergic stimulation within the bone marrow functions in a compensatory or a decompensatory way toward the host following thermal injury is also interesting to consider. Is adrenergic stimulation involved in the immunosuppression of patients with severe burns through functional alterations in circulating and tissue leukocytes?^147–153 These and other important questions are likely to involve events that occur within the bone marrow, as it serves as a major source of new leukocytes both in the circulation and in tissues following thermal injury with sepsis. Our laboratory is beginning to confirm that perturbed bone marrow progenitor development could lead to the production of hyporesponsive monocytes and dendritic cells following thermal injury and sepsis.¹⁵⁴

Alterations in bone marrow hematopoietic progenitor cells has been the focus of our work and has involved the murine model of thermal injury (15% TBSA) and sepsis via Pseudomonas aeruginosa applied directly to the wound site to establish sepsis. Following the demonstration of a shift in bone marrow myeloid commitment toward monocytopoiesis and away from granulocytopoiesis in thermal injury and sepsis,^155,156 we began to focus on the potential significance of the increased nerve-stimulated release of norepinephrine within the bone marrow under these same experimental conditions. We tested the premise that neural stimulation was modulating myeloid lineage function by manipulating the peripheral stores of norepinephrine prior to injury. This was achieved by administering 6-hydroxydopamine (6-OHDA) before subjecting the animals to thermal injury and sepsis. Femoral bone marrow cells from mice with reduced norepinephrine content demonstrated a significant decrease in monocytopoietic potential compared to mice with intact norepinephrine stores.¹⁴⁶ In addition, reduction of peripheral norepinephrine content prior to the injury protocol resulted in a significant survival benefit compared to animals with intact norepinephrine content.

The influence of norepinephrine on bone marrow monocyte progenitor differentiation following thermal injury with sepsis was assessed by cell surface expression patterns of ER-MP12 and ER-MP20. Whereas ER-MP12 antigen is expressed in early monocyte progenitors and represents predominantly CFU-M, progressively more ER-MP20 antigen is expressed from the CFU-M stage onwards but disappears in mature monocytes.¹⁵⁷ By following the distribution pattern of the expression of these two antigens on bone marrow cells, the phenotypic separation and identification of bone marrow monocyte precursors have been demonstrated.¹⁵⁸ Taken together, results from ER-MP12 and ER-MP20 expression patterns in burn-septic animals with intact and depleted norepinephrine stores suggest that monocyte maturation pathways may be greatly influenced by the presence of norepinephrine, and that stimulation of such pathways may be involved in the pathobiology of thermal injury with sepsis.

Cohen et al. have also examined very early progenitors not committed to the myeloid lineage that express the CD117 antigen.¹⁵⁹

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