Cold-Restraint-induced Immune and Biochemical Changes Inhibit Host Resistance to Listeria

C H A P T E R

48 Cold-Restraint-induced Immune and Biochemical Changes Inhibit Host Resistance to Listeria REBECCA T. EMENY AND DAVID A. LAWRENCE

I. INTRODUCTION: NEUROIMMUNE INTERACTIONS AND HOST DEFENSES 1035 II. SHORT-TERM COLD-RESTRAINT TREATMENT: A MODEL FOR STRESS-RELATED IMMUNOSUPPRESSION 1036 III. HOST RESISTANCE TO LISTERIA MONOCYTOGENES INFECTION IS IMPAIRED BY COLD-RESTRAINT TREATMENT 1037 IV. β-ADRENERGIC RECEPTOR EXPRESSION AND SIGNALING IN IMMUNE AND NONIMMUNE CELLS 1042

frequent occurrences in everyday life, namely, psychological or physical stressors and exposure to common infectious agents. Our studies have demonstrated that the psychological stress of inescapable restraint, combined with the physiological stress of cold (4° C) exposure for 1 hour, suppresses host defenses against a low-dose infection with the facultative, intracellular, Gram-positive bacterium Listeria monocytogenes (LM) (17). Studies from our laboratory and others have provided evidence for an immunosuppressive role of the peripheral sympathetic nervous system (SNS) during experimental LM infection. A current research focus is on understanding the β-adrenergic receptor (βAR) mechanisms that regulate host-pathogen interactions under normal or stressful conditions. This chapter reviews the use of the cold-restraint model to investigate stress-induced inhibition of host defenses against a bacterial infection. A summary of evidence for the role of sympathetic mediators of the stress response, particularly the β1and β2-adrenergic receptors (β1AR and β2AR), in immune regulation will be presented, followed by a review of the known and postulated consequences of adrenergic signaling in immune cells. Finally, the importance of understanding cellular and molecular mechanisms that regulate neuroimmune interactions will be addressed, in the context of public health and clinical practice.

I. INTRODUCTION: NEUROIMMUNE INTERACTIONS AND HOST DEFENSES The nervous system and the immune system rival one another in terms of both complexity and adaptive potential. Both systems can influence and regulate themselves and one another; in individuals who experience excessive stress, disrupted neuroimmune homeostasis can cause sickness. A focus of our research is on the underlying mechanisms of neuroimmune interactions, specifically those processes that influence an individual’s susceptibility to infections. The paradigm of short-term cold-restraint stress has been used to assess effects on host defenses of mice infected with a low dose of bacteria. This model attempts to simulate PSYCHONEUROIMMUNOLOGY, 4E VOLUME II

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II. SHORT-TERM COLD-RESTRAINT TREATMENT: A MODEL FOR STRESSRELATED IMMUNOSUPPRESSION In general medical terms, stress is considered to be any condition resulting in perturbation of the body’s homeostasis (Gr, homeo—stable/same + Gr. -stasis— state) (23). The term allostasis (Gr. allos—other/different) is used to define a normally beneficial process that maintains stability under abnormal or “stressful” conditions (122). Physiologic responses to stress include increased heart rate and subsequently elevated blood pressure, increased glycogenolysis in the liver, increased lipolysis in adipose tissue, and relaxation of smooth muscle in the lungs. The crucial basis for the existence (i.e., the raison d’etre) of this evolutionarily programmed process is to mobilize glucose and the oxygen needed to generate ATP, the energy source required to adapt and respond to an environmental stressor. The unregulated or excessive production of stress response mediators that induce these organspecific changes is termed allostatic load, and heightened or prolonged allostatic load can have damaging effects, leading to disease and susceptibility to infection (83, 84). When mice undergo a 1-hour cold-restraint treatment, increased allostatic load (or stress mediators) renders the mice more susceptible to a low-dose bacterial infection (17). The combined psychological and physiological changes elicited by cold-restraint treatment cause a biochemical response characteristic of the fight/flight and freezing/hide reactions observed in dominant and subordinate animals, respectively, when confronted with an environmental stressor (61). For our cold-restraint model, mice are contained in well-ventilated 50-ml conical tubes for 1 hour at 4° C, in the dark. Cold-restraint is a very acute stressor, in that corticosterone (a common indicator of stress) is immediately elevated for a transient period. Historically, cold-restraint treatment has been used to characterize neurogastric interactions, since short-term cold-restraint treatment can cause ulcers in rats (96). Many other stress-inducing models, such as shortterm restraint (2–6 hours), repeat restraint (14 hours daily for 7 consecutive days), forced swimming, social confrontation, and inescapable foot shock, are known to cause immunologic alterations. It is important to note that systemic changes differ for each acute (29) or chronic (100) stressor studied. For example, 40-minute swimming and 1-hour cold treatment (both considered to be physical, metabolic stressors) as well as 1-hour restraint, a psychological stressor, cause an increase in

corticosterone, but only restraint induces significant increases in serum homocysteine (29), a stress-induced cardiovascular risk factor that is also implicated in immune dysfunction (28,116). Therefore, direct comparison of the stress-induced immunologic changes that arise within different stress paradigms is challenging. In the following discussion of stress-induced neuroimmune interactions, we will focus only on consequences of short-term restraint or cold-restraint treatment. For a better understanding of the neuroimmune interactions that accompany cold-restraint inhibition of immunity against LM, a review of the systemic changes induced by cold-restraint treatment will be presented in section III. LM infection is a widely utilized model of host-pathogen interaction that has been extensively studied (129). Section IIIB and Section IIIC will introduce the host defense mechanisms that are known to contribute to a successful anti-LM response, followed by an analysis of the neuroimmune interactions that likely influence host defenses during LM infection.

A. Neuroendocrine and Biochemical Changes Associated with Cold-Restraint Treatment Upon perception of a challenge or threat, initial sensory impulses arising in the cortex are relayed through brain stem nuclei (i.e., the dorsal vagal complex and adrenergic cell groups) and the limbic region to stimulate the paraventricular nucleus of the hypothalamus to produce corticotropin-releasing factor (CRF), a critical mediator of the stress response (7,12,61). Cold-restraint stress has been shown to cause alterations of norepinephrine (NE) levels in limbic regions (92); as a consequence of neurotransmitter signaling, CRF is released and the hypothalamicpituitary-adrenal (HPA) axis and the sympathoadreno-medullary system (SAS) are activated. Glucocorticoids (corticosterone in the mouse and cortisol in humans), the hallmark products of HPA activation, are released by the adrenal cortex, while sympathetic neurons that synapse with cells of the inner adrenal medulla release the catecholamines NE and epinephrine (Epi) directly into the blood stream. Post-ganglionic sympathetic neurons also release NE as a neurotransmitter directly into innervated tissue. Both the primary and secondary lymph organs are innervated by sympathetic neurons (15,36,40,107,123). Our studies show that cold-restraint treatment induces the HPA axis, the SNS, and oxidative stress in

48. Cold-Restraint-induced Immune and Biochemical Changes Inhibit Host Resistance to Listeria

mice. Significant increases in the levels of plasma corticosterone and splenic NE are measured in stresstreated mice, relative to controls (Figure 1A and B). As previously mentioned, this biochemical response is acute, in that corticosterone levels return to baseline by 3–4 hours post-stress treatment (18, 20). In addition to neuroendocrine changes, cold-restraint treatment induces a mild hypothermic state, which likely contributes to the oxidative stress commonly observed in this model (Figure 1C).

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Cellular oxidation is characterized by a loss of free sulfhydryls or thiols (R-SH), due to the conversion of these groups to intra- or inter-chain disulfides (R-S-SR) on or between proteins. Immediately following a 1-hour cold-restraint treatment, total cellular thiols increase and extracellular surface thiols decrease, as measured by the thiol-specific compounds coumarinyl-phenyl maleimide and alexa-488 maleimide, respectively (5,67) (Figure 2). A loss of surface thiols on peripheral blood lymphocytes, particularly those of the natural killer (NK) cell subset, occurs after the coldrestraint treatment (Figure 2A). The marked decreases of extracellular thiols with concomitant increases in total cellular thiols induced by cold-restraint have been observed for blood, but not splenic, lymphocytes (Figure 2B). In support of our findings, others have demonstrated that cold-restraint–induced oxidative stress, reported as increased gastric mucosal myeloperoxidase activity (102), superoxide dismutase activity, and lipid peroxidation (127), resulted in damaged gastric mucosal surfaces and ulceration. Not surprisingly, increased lipid peroxidation in the serum of clinical peptic ulceration and gastric carcinoma patients has been reported (127). Additionally, hepatic glutathione (GSH) levels are decreased following cold-restraint, which indicates an oxidized microenvironment in liver tissue following this stress treatment (120). Oxidative stress is known to specifically affect immune cell function. For example, the activation and proliferation of lymphocytes depend on the redox state of the cellular microenvironment (38,67). Macrophage (Mφ) function is also redox-dependent (2,99,104). An increase in free radicals following 4hour cold restraint was recently shown to be associated with altered Mφ activity, as well as decreased chemotaxis and phagocytosis (51). Furthermore, the intracellular thiol content of activated Mφ in vitro is known to influence the Th1/Th2 generation of cytokines (89).

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FIGURE 1 Cold-restraint treatment (1 hour) is associated with increased HPA (A) and SNS (B) activity and decreased core body temperature (C). Immediately following treatment with cold, restraint, or combined cold restraint for 1 hour, core body temperatures were measured before BALB/c mice (n = 12) were sacrificed, to yield blood that was processed for serum CORT analysis by EIA and spleens that were immediately frozen and stored for HPLC analysis of NE. Data are presented as mean ± Std. Dev. *Significant differences (p < 0.05) between treatment groups were evaluated using one-way ANOVA and Tukey’s post hoc test (R. T. Emeny, J. Hornickel, D. A. Lawrence).

III. HOST RESISTANCE TO LISTERIA MONOCYTOGENES INFECTION IS IMPAIRED BY COLD-RESTRAINT TREATMENT To identify the deleterious effects of cold-restraint stress on host resistance to LM, it is critical that we understand the pathology of an infection with LM. Primary infection with LM activates both innate and adaptive immune mechanisms. Early host responses

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FIGURE 2 Intracellular and cell-surface changes in thiol content measured in peripheral blood lymphocytes and splenocytes obtained from 6–8-week-old male mice. (A) Changes in the quantity of free surface sulfhydryls on blood lymphocyte populations (B-cells, NKs, and T-cells, expressing CD19+, CD3−, and CD3+, respectively) were measured by fluorescent labeling with the impermeable alexa-488 maleimide compound immediately following 1-hour cold-restraint (CR) treatment of BALB/c mice (n = 12 control, n = 14 CR). Similar results were obtained using FVB/NJ mice (data not shown). (B) Numeration of total thiols of serum, white blood cells (wbc), and splenic lymphocytes obtained from FVB/NJ mice (n = 3) were detected after 1-hour cold-restraint treatment using the thiol-specific compound coumarinyl-phenyl maleimide. Data are presented as mean ± Std. Dev. *Significant differences (p < 0.05) between treatment groups were evaluated using one-way ANOVA and Tukey’s post hoc test (R. T. Emeny, J. Hornickel, D. A. Lawrence).

are characterized by the recruitment of neutrophils, macrophages, and NK cells to sites of infection. Once activated, these innate cells produce bactericidal oxidants and immune-modulating cytokines (101). Cytokine production is greatly influenced by the bacterial load (55), in that an increase in LM colonization elicits an inflammatory-stress response and subsequent upregulation of the cytokines IL-1, IL-6, and TNF-α, through a process termed neurogenic inflammation (12). Essential to the non-specific host response to LM is the early recruitment of monocytes to sites of bacterial infection (26). Activated macrophages are thought to be the main effectors of bacterial clearance (60);

however, NK cells and recruited CD8+ T-cells are also involved in mediating successful bacterial clearance, which occurs on about day 3 after the initial infection, the time at which innate responses begin to convert to adaptive immune mechanisms (91,129). Whether an experimental infection is administered intraperitoneal (i.p.) or intravenous (i.v.), the liver is thought to be the organ primarily responsible for LM clearance (87). Since a successful host response against primary LM infection is dependent upon the coordination of both innate and T-cell (but not B-cell) defense mechanisms, it is a useful infectious model for the analysis of neuroimmune effects on host innate and cell-mediated immunity.

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Cold-restraint treatment administered just before a low-dose infection with LM (i.v.) inhibits host resistance (Table 1, Figure 3). Cold-restraint treated BALB/ c mice had significantly increased LM colonization in the liver and spleen on day 3 as compared to control mice (17, 19). The increased bacterial burden was associated with significantly higher levels of IL-6 and TNFα in the spleen, measured on days 1 and 2, and with higher levels of IL-6 and IFN-γ in the serum on day 3 (20). Cytokine mRNA evaluated 1 day after LM infection by RNase protection assay showed significant increases in IL-1β, IL-1α, IL-1Ra, and IL-6 expression in spleens from cold-restraint treated mice, in comparison to those from control mice (20). In general, the absolute numbers of splenic and blood immune cell subsets (B-, T-, and NK cells; Mφ; neutrophils) do not change over the course of initial infection (days 1–3); however, cold-restraint treatment was observed to cause a significant increase on day 1 of activated (CD69+) CD4+ and CD8+ T-cells and non-T (CD3−) cells in the spleen (19). To further evaluate specific immune cell subsets that may be negatively affected by cold-restraint–induced immunosuppression, studies with SCID mice (18) and mice deficient in CD4 T-cells were conducted (19). Neither SCID mice nor CD4−/− mice were immunocompromised by coldrestraint treatment; in fact, anti-LM responses were enhanced overall in these immunodeficient mice as compared to wild-type BALB/c mice. These experiments suggest that CD4+ T-cells, possibly regulatory T-cells, or downstream CD8+ T-cell effectors are involved in stress-induced immunosuppression. TABLE 1 Pharmacologic treatment or genetic deficiency Intrastriatal 6-OHDA Intracerebroventricular 6-OHDA DA β hydroxylase−/− i.p. 6-OHDA i.p. 6-OHDA in IFN-γ−/− i.p. 6-OHDA in IL-10−/− i.p. 6-OHDA Metyrapone Phentolamine Propranolol Nadolol Atenolol ICI118,551 CD4−/− SCID IL-6−/−

However, both CD4−/− and SCID mouse strains have robust innate immunity to compensate for the lack of adaptive cell populations, which suggests that the allostatic load induced by 1-hour cold-restraint is not suppressive of such vigorous innate defenses.

A. Host Resistance to Listeria monocytogenes Infection Is Regulated by Sympathetic Activity Although cold-restraint influences both SNS and HPA activities, pharmacological studies from our laboratory have demonstrated that sympathetic activity mediates the observed stress-induced immunosuppression (Table 1). To delineate the impact of stressinduced SNS activity during an anti-LM response, the neurotoxin 6-OHDA was used to selectively and temporarily ablate dopaminergic nerves (thereby depleting peripheral tissues of catecholamines, since dopamine [DA] is the precursor to NE and Epi). Metyrapone was used to inhibit corticosterone biosynthesis, thus blocking the predominant mediator of HPA activity. Mice treated with 6-OHDA prior to coldrestraint treatment and subsequent LM-infection were protected from the negative effects of cold-restraint treatment, while the administration of metyrapone exacerbated stress-induced immunosuppression (17). In fact, mice that received chemical ablation of their peripheral SNS cleared LM more effectively than did their control litter mates, whether or not they were treated with cold-restraint. This indicates that peripherally released catecholamines are potent

Neuroimmune Mediators of Anti-listerial Host Resistance Stress treatment No stress No stress No stress No stress No stress No stress Cold-restraint Cold-restraint Cold-restraint Cold-restraint Cold-restraint Cold-restraint Cold-restraint Cold-restraint Cold-restraint Cold-restraint

Neuroimmune component disrupted

Anti-listerial response*

Ref.

Systemic catecholamines Systemic catecholamines Systemic catecholamines Peripheral catecholamines IFN-γ synthesis IL-10 synthesis Peripheral catecholamines Corticosterone Peripheral αARs Peripheral and central βARs Peripheral βARs β1ARs β2ARs CD4+ and CD8+ lymphocytes CD4+ and CD8+ lymphocytes IL-6 synthesis

Impaired Impaired Impaired Enhanced Equal to control Equal to control Enhanced Impaired Impaired Enhanced Enhanced Enhanced Impaired Enhanced Enhanced Impaired

(39) (94) (3) (17; 88; 108; 109) (88) (88) (18) (18) (18) (18) (18) (18) (18) (19) (18) (20)

*Outcome of infection as compared to control (i.e., no drug treatment or genetic alteration).

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FIGURE 3 Viable LM numbers in organs of cold-restraint (acute cold-restraint stress, ACRS) and control mice during primary infection. Male BALB/c mice (10–12-week-old; n = 3–7 mice per group) subjected to ACRS (triangles) or undisturbed (circles) were infected with LM (5–6 × 103 CFU/ mouse, i.v.) immediately after 1-hour cold restraint or an undisturbed period. Kinetics of viable LM numbers per spleen (A) and liver (B) were determined at day 0 (7 hours), or day 1, 2, 3, 5, 7, or 9 after infection. Bacteria were counted in serial dilutions of organ homogenates using blood-agar plates. Data are presented as mean ± S.E.M. ANOVA was followed by SNK post hoc analysis to compare different treatment groups; *indicates p < 0.05, and **indicates p < 0.01, when non-stressed control and cold-restraint groups are compared at the same time-point. (From L. Cao and D. A. Lawrence, 2002; with permission from the publisher.)

immunosuppressive agents against anti-LM immunity. Overall, our cold-restraint studies suggest an immunoprotective effect of corticosteroids during an LM-specific inflammatory response, whereas the activation of adrenergic receptors by stress-induced catecholamines has immunosuppressive consequences. For over 3 decades, the influences of the SNS, particularly NE and the adrenergic receptor family, have been researched and have been shown to exert modulatory effects on immune functions, with some detrimental clinical outcomes (121,128). Many of the early

studies on SNS-immune interactions evaluated phenotypic changes in cell populations or antigen-specific responses in vitro after pharmacologic or mechanical sympathectomy (75–78,133). As mentioned, studies in the last 5 years have provided ample evidence for the importance of sympathetic activity in regulation of host susceptibility to infectious organisms, in particular LM. Along with our lab, others have shown that in a normal experimental infection, host defenses against LM are enhanced by treatment with 6-OHDA: the LM colonization of livers and spleens in sympathectomized mice is significantly reduced around days 3–5 of the infection as compared to the colonization in control mice (18,88,108,109). The reduced bacterial load in mice with denervation of peripheral organs was correlated with increased numbers of splenic neutrophils (108) and activated peritoneal Mφs (109) during the first 3 days of an i.p. infection. Conversely, 6-OHDA treatment decreased splenic leukocyte numbers between days 5–7 in LM-infected mice (108), a result similar to that observed in a model of HSV infection in which 6-OHDA was shown to suppress the generation of both primary and secondary virusspecific cytotoxic T lymphocytes (CTLs) (70). It is important to note that all observed effects of 6-OHDA reported in the cited papers were reduced if mice were pre-treated with the catecholamine uptake blocker desipramine, which prevents the destruction of dopaminergic neurons by blockage of 6-OHDA uptake into NE nerve terminals (18,39,88,108,109). Interestingly, the peripheral injection of 6-OHDA does not affect the central nervous system since this molecule cannot pass the blood-brain barrier (133). However, central ablation of dopaminergic neurons by intra-striatal injection of 6-OHDA (39), bilateral injection of 6-OHDA into the lateral ventricles (94), or genetic removal of DA β-hydroxylase (an enzyme required for the production of dopamine [3]) impairs cellular immunity (Table 1). Therefore, blockage of the stimulation of ARs in the brain has immunosuppressive effects, while the peripheral loss of adrenergic signaling is immunoenhancing. How the elimination of catecholamine signaling in the brain functions to suppress peripheral immune activity is as of yet unresolved, but it likely involves inappropriate HPA axis activation (39), and dysregulation of peripheral catecholamines essential for immune homeostasis (94). The positive outcome of sympathectomy on host resistance to LM infection is thought to be a consequence of increased production of inflammatory cytokines, including IL-12, TNF-α, and IFN-γ, given that 6-OHDA treatments increased production of these cytokines by dendritic cells (DC) and splenic cultures. No immunoenhancing effect of 6-OHDA treatment was observed

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48. Cold-Restraint-induced Immune and Biochemical Changes Inhibit Host Resistance to Listeria

B. Cold-Restraint-induced Immunosuppression of Host Immunity against Listeria monocytogenes Is Mediated by b-adrenergic Receptors; Pharmacologic Studies Reveal a Role for b1-adrenergic Receptors in Neuroimmune Interactions The catecholamines NE and Epi bind both αARs and βARs. The AR family is composed of three types of 7 transmembrane, G-protein–coupled receptors, α1ARs, α2ARs, and βARs, and each type has three subtypes, namely, α1A/D, 1B, and 1C; α2A, B, and C; and β1, 2, and 3, respectively. This review focuses on the βAR subtypes; however, information from αARs that is pertinent to the functional biology of ARs in general is included. Pharmacologic studies that used αAR and βAR subtype blocking and stimulating agents identified β1AR as the predominant neurotransmitter signaling receptor through which host resistance to LM is inhibited by the cold-restraint treatment (Table 1) (18). When mice were given phentolamine, an αAR antagonist, before being subjected to LM infection either with or without cold-restraint pre-treatment, their anti-LM responses were diminished. In fact, blockage of NE signaling through αARs made even non-stressed mice sicker than their control littermates. However, mice treated with the non-selective βAR blocker propranolol showed improved host resistance, regardless of stress treatment. Mice treated with propanolol, but not phentolamine, had decreased IL-6 and IFN-γ levels in their sera and spleens on day 3 postinfection; these levels were comparable to the levels observed in non–stress-treated control mice. In splenic samples, IL-1β and TNF-α were decreased by βblockers, but not by α-blockers (18). Further βAR blocking studies using β1AR- and β2AR-specific antagonists were conducted, in order to delineate the effects of catecholamine signaling through two of the three known βARs. Administration of the β2AR-specific blocker ICI118,551 caused increased bacterial burden in both liver and spleen, whereas mice that received the β1-specific antagonist atenolol

had overall improved host defenses against the LM infection. Cold-restraint–treated mice with atenolol pre-treatment had bacterial colonization in liver and spleen almost equal to that in control mice. Therefore, the elimination of catecholamine signaling through β1ARs completely blocked the deleterious effects of cold-restraint treatment. Combined, these data suggest that a key immunomodulatory role for cold-restraint– induced signaling is through β1ARs since β2AR antagonists worsen, while β1AR antagonists improve, host resistance against LM.

C. Immunosuppression of Cold-Restraint Treatment in b2-adrenergic Receptor Deficient but Not b1-adrenergic Receptor Deficient Mice Studies performed with βAR deficient mice (24,110) have substantiated the pharmacologic evidence that signaling of NE and Epi through β1ARs affects host responses to an LM infection differently than does signaling of catecholamines through β2ARs. Our results have shown that the immunosuppressive effect of cold-restraint treatment is abolished in β1AR−/− mice, while β2AR−/− and wild-type mice succumb to LM infection when they have been immunoimpaired by cold-restraint stress pre-treatment (Figure 4). Further studies have demonstrated that on day 3 of a low-dose infection, β1AR−/− mice treated or not treated with cold restraint had lower bacterial loads in their spleen and liver homogenates than those of wild-type (FVB/NJ) control mice (34). In contrast, the splenic and liver LM colonization was increased by approximately 1 log in the cold-restraint–treated FVB/NJ and β2AR−/− mice, compared to the LM colonization of non-stressed control mice. Based on bacterial burden and mortality data, cold-restraint treatment administered before 100 80

% Survival

in studies with IFN-γ−/− and TNFα−/− mice (88). Taken together, these studies support the hypothesis that the presence of NE during an infection diminishes innate mechanisms of host defense, specifically, the recruitment of and cytokine secretion by innate immune cells. In our model of cold-restraint–induced alterations of host-pathogen interactions, the detrimental consequences of catecholamine signaling of cell subtypes, either immune or non-immune, remain uncertain; the possible mechanisms will be discussed later.

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FIGURE 4 The survival of wild-type FVB/NJ and βARdeficient mice (age 6–8 weeks, n = 4–6 per group) following a low-dose LM infection (3 × 103 colony-forming units, i.v.) with or without cold-restraint pre-treatment (R. T. Emeny and D. A. Lawrence).

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bacterial infection was clearly immunosuppresive for both wild-type and β2AR−/− mice, whereas such treatment was not harmful for the β1AR−/− mice. The beneficial effects for the latter may be due to the positive influences of corticosterone in the absence of catecholamine signaling through the β1AR. Additionally, the limitation of β1AR activity may boost immunity. Our observation that β2AR−/− and FVB wild-type mice respond similarly to the LM infection, with or without stress treatment, is supported by recent evidence that β2AR−/− mice do not differ from wild-type mice in their ability to mount contact hypersensitivity responses or T-cell–dependent antibody responses. Nonetheless, β2AR−/− B-cells are unable to mount an antigenspecific antibody response in the presence of IL-4 in vitro (115).

IV. b-ADRENERGIC RECEPTOR EXPRESSION AND SIGNALING IN IMMUNE AND NON-IMMUNE CELLS Subclasses of βARs were first discerned based on the order of pharmacological potencies of three classi-

cal catecholamines—isoproterenol (Iso), Epi, and NE (64). Kidney and adipose tissue were found to express β1ARs with binding affinities of Epi greater than or equal to those of NE, while skeletal muscle, liver, lung, and trachea expressed β2ARs and bound Epi to an extent approximately 10 times greater than for NE (64). Heart tissue has been shown to express both β1AR and β2AR (14). β3AR was found predominantly on adipocytes along with moderate levels of β1AR and with low levels of α1A+DARs (22). Pharmacologic expression studies that used Chinese hamster ovary cells indicated that β3ARs have a higher affinity for NE than for Epi, in contrast to the affinity of β2ARs (124). Because little is known about the β3AR in immune function, this chapter will focus mainly on the β1- and β2ARs. The murine β1AR and β2AR structures are shown in Figure 5. Both receptors are palmitylated, indicating localization in lipid rafts that could, hypothetically, place them in close proximity to T-cell receptors on lymphocytes. Differences occur in both the amino- and carboxy-ends of the proteins that dictate unique activities. The β1AR has a cysteine on the extracellular Nterminus and one potential serine phosphorylation site on its intracellular cytoplasmic end, whereas the N-

-NH3+

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CHO Cysteine Palmitate FIGURE 5 The structures of murine β1AR and β2ARs are depicted. Indicated are the 7transmembrane spanning regions through the lipid bilayer of the cell surface membrane, the extracellular amino-terminal (—NH3+), the intracellular carboxyl-terminal (—COO−), glycosylation sites (—CHO), the location of the amino acid cysteine, and a palmitate moiety (16-carbon saturated fatty acid).

48. Cold-Restraint-induced Immune and Biochemical Changes Inhibit Host Resistance to Listeria

terminus of β2AR is devoid of sulfhydryls and the Cterminus has four possible phosphorylation sites; it is hypothesized that these structural differences affect susceptibility to redox modulations that could affect configuration, and thus binding of ligands and intracellular signaling. As an example, the intracellular tail of human kidney-derived β2AR binds to an Na+/H+ exchanger regulatory factor protein that is involved in the regulation of cellular pH (46). Furthermore, recent studies that used hamster kidney cells transfected with wild-type or chimeric human βARs implicate the Cterminus of the βAR in the regulation of receptor internalization and in lysosomal degradation (71). In these studies, wild-type β1ARs and chimeric β2ARs with β1AR C-termini were resistant to agonist-mediated downregulation. Results of such research not only emphasize functional differences between these two receptors, but also illuminate actions of βAR signaling that are independent of G-protein interactions. The next section will describe the actions of βAR signaling that have been characterized in non-immune cells and in immune cells, with a review of the detection of βARs on immune cell subsets.

A. Non-immune Consequences of b-adrenergic Receptor Signaling Intrinsic to catecholamine binding by cell-surface βARs of all types is the immediate activation of Gprotein coupling to the intracellular effector adenylate cyclase (AC). This enzyme then acts to produce the second messenger cAMP, and to activate downstream molecules, such as protein kinase A (PKA) and extracellular signal-regulated kinase (ERK2). Globally, it is understood that βAR signaling increases cAMP through binding of Gs, which stimulates AC, while αARs decrease cAMP through their association with Gi, which inhibits AC, or Gq, which in turn stimulates phospholipase C (65). Furthermore, the αARs have been shown to increase protein synthesis in a fashion similar to insulin (86), while βAR signaling generally decreases protein synthesis. In myeloid cells (rat myeloid leukemia cell line IPC-81), cardiomyocytes, and adipocytes, the βAR agonist isoproterenol increases phosphorylation of eukaryotic elongation factor 2 (eEF2) (41,48,85). Phosphorylation of eEF2 inhibits its activity that is required for translocation in peptidechain elongation. Therefore, stress-induced catecholamines shut down protein synthesis in order to make ATP available for fight or flight responses such as cardiac contraction. In many tissues, the functions of βAR subtypes have been described. β1AR signaling in the heart increases the strength of contractions, and in the kidney it stimu-

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lates the release of rennin so as to increase blood pressure. Stimulation of β2AR can have vasodilating effects on blood vessels in cardiac and skeletal muscles, and can cause the relaxation of smooth muscles in the GI tract. The β3ARs mediate lipolysis and thermogenesis in adipose tissue (124). The activation of each specific βAR subset may result in either regional or localized intracellular changes. In neurons and in the myocardium, the signaling effects of β1ARs appear to have widespread cellular consequences as a result of PKA activation, whereas signaling by β2ARs appears to be spatially restricted. Immunoprecipitation and electrophysiological studies have shown that the cytoplasmic C-terminus of β2AR co-localizes with the pore-forming subunit of the predominant L-type Ca++ channel, in the rat hippocampus (27). In immune cells, this could hypothetically translate to T-cell receptor (TCR)–linked modulation (local) that affects antigen-specific activation or actin mobilization and diapedesis (regional).

B. Expression of b-adrenergic Receptors on Immune Cell Subsets Because it was known pharmacologically that the synthetic non-specific agonist isoproterenol and the natural ligands NE and Epi stimulated βARs to increase cAMP and AC activity, the first indication of βAR expression on human lymphocytes was demonstrated by catecholamine-induced lymphoblast transformation (44) and AC activity (13,82). Thereafter, radioisotype-labeled ligand-binding studies that used a heterologous mixture of cells purified from human blood provided direct evidence for the presence of βARs on mononuclear cells, polynuclear cells, and lymphocytes (134). In these studies, isoproterenol was shown to be the most potent agonist, followed by Epi and then NE. Studies of both lymphocytes and neurons have shown that Epi binds βARs with a 10-fold greater affinity than NE (4,54,134). The distribution of βARs in immune cell subpopulations is influenced by cell type. The highest receptor densities have been shown by radioligand-binding studies to be on NK cells and B-cells, followed by CD8+ T-cells and monocytes, while the lowest numbers were measured on T helper cells (Th) (11,54,74,81,100,130). Approximate receptor densities averaged between 1000 and 2000 receptors/cell, depending upon the cell type examined, but regardless of whether intact cells or isolated membranes were measured (14). Not only does receptor expression vary for each lymphocyte subset, but receptor affinities also differ, in that NKs, CTLs, and monocytes appear to have greater βAR coupling capacities than do Th and B-cells, with regard to cAMP production (56,80). For example, subsets of

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human lymphocytes were shown to bind the radioligand [125I]-cyanopindolol (ICYP) differentially, with the binding affinity of T suppressor cells (Ts; Leu2+, 9.3−) > T cytolytic cells (Tc; Leu2+, 9.3+) > Th cells (Leu3+) (54). The number of binding sites on each T lymphocyte subset (Ts∼2900, Tc∼1800, Th∼750), the affinity of agonist for βAR, and the biological activity (as measured by cAMP production or AC activity) appear to correlate (53,54,68,134). Similar to the differentiation between β1AR and β2AR expression on cells of other tissue types, the expression of these receptors on lymphocyte subsets has been determined largely by the pharmacologic activities of non-specific or specific agonists and antagonists. For example, β2ARs have been thought to be the primary βAR on rat peritoneal macrophages since a non-specific βAR antagonist (bufetolol) was shown to block Epi-induced cAMP production, while the selective β1AR antagonist failed to do so (49). Similarly, because Epi demonstrates greater binding affinities towards the βARs on T lymphocyte subsets than NE, it was concluded that β2ARs are the majority of βARs present (54). Recently, RT-PCR analysis has partially substantiated this, by demonstrating expression of β2AR mRNA by Th0 and Th1 clones, but not Th2 clones, implicating the receptor in differential regulation of Th1 and Th2 functions (105,113,115). Early radioligand binding studies demonstrated the presence of β2ARs, but not β1ARs, on NK cells (81,130). Competitive radioligand-binding assays also demonstrated that a 20-minute infusion of Epi (but not NE) into healthy volunteers decreased the expression of β2AR and α1AR on the CD16+ peripheral lymphocyte populations (which includes all FcRγIII+ cell subsets), and increased β2AR densities on the CD4+ and CD8+ peripheral blood leukocyte subsets (52). More recently, however, RT-PCR analysis identified mRNA expression of β1ARs and β2ARs as well as α1AAR on murine Langerhans cells (117), and β1-, β2-, α1-, α2A-, and α2CARs on bone marrow–derived dendritic cells (BMDCs, CD11c+) (79). Freshly isolated human peripheral blood monocytes express mRNA for β2AR and α2ARs, but stimulation with dexamethasone or the β2AR-specific agonist terbutaline can induce expression of α1B- and α1DARs (112). The expression and activity of a particular βAR on lymphoid and myeloid cells is likely to be complex, regulated by the microenvironment of the cell as well as by the surface expression and activity of other βARs (22), αARs (52), GCs (1,112) and prostaglandins (8) (to name only a few among perhaps many other molecules that can affect βAR function). An indication of the potential complexities has been demonstrated by analyses of mRNA expression in brown adipocytes.

Although mature adipocytes express β1-, β3-, α1A-, and α1DARs, mRNA expression of ARs is altered by NE such that β1ARs increase, β3ARs decrease, and β2ARs are transiently expressed (9). Additionally, NEmediated glucose uptake appears to function normally in β3AR−/− mice, which indicates that β1AR, perhaps β2AR, and α1AR signaling can compensate for β3AR deficiencies in these mice (22). The degree to which lymphocytes can be activated by catecholamines is thought to change over time, which provides a mechanism for SNS modulation throughout the development of host immunity. For example, the differentiation state of Th1 cells appears to determine the influence of βAR signaling on IFN-γ production, in that naïve T-cells produce more IFN-γ following NE exposure and antigenic stimulation, while mature Th1 clones produce less IFN-γ (113,125). Studies that used in vitro stimulated cell lines suggest that activated Mφ can be less sensitive and mature T-cells more sensitive to βAR-mediated sympathetic stimuli (103). It has long been proposed that cAMP promotes differentiation of immature lymphocytes but inhibits the function of mature lymphocytes (25). Thus, the classical methodologies that have defined βAR expression patterns of immune cells based upon pharmacological analysis may be inconclusive. Determination of β1AR and β2AR expression on immune cells has mainly relied on ligand-binding variations and on the extent of functional responsiveness to these ligands (i.e., cAMP production or AC activity), but these parameters may not be directly interrelated. For example, ligand-binding studies of β2AR distribution in respiratory-tract smooth-muscle tissue have shown the presence of β2ARs to be segment-specific, and AC coupling activity to be inversely correlated with receptor density (134). Additionally, ligand binding likely depends on cell surface topography, as well as on the expression of other surface molecules that influence receptor activity. In any case, a comprehensive analysis of βAR subsets on naïve, resting, and activated immune cells, including quantification of mRNA and protein expression, is warranted.

C. Immunologic Consequences of b-adrenergic Receptor Signaling Catecholamine signaling through βARs is known to modulate a wide range of immune functions, including altered peripheral cytokine levels (126), activation of virus-specific effectors (47), increased DTH responses (31), decreased Mφ IL-1 production (57), increased lymphocyte trafficking (especially of NK cells [30] and monocytes [35]), and decreased splenic and thymic lymphocyte chemotaxis (42). It is well documented

48. Cold-Restraint-induced Immune and Biochemical Changes Inhibit Host Resistance to Listeria

that βAR stimulation on immune cells inhibits production of pro-inflammatory cytokines (TNF-α, IL-12, and IFN-γ) and enhances the production of IL-6 and IL-10. For example, Epi and NE have both been shown to inhibit IL-12 by T-cells and to enhance IL-10 production by Mφ (32). Catecholamine signaling through β2ARs has been found to be associated with functional changes in Bcells, CD4+ lymphocytes, Mφs, NKs, and DCs. β2ARs promote T-cell-dependent antibody responses (115) and mediate antigen-specific follicular cell expansion and germinal center formation in the spleen (58). Overexpression of β2AR in a macrophage cell line significantly reduced IL-12 mRNA and protein expression following lipopolysaccharide (LPS) stimulation (50); interestingly, the β2AR inhibition of IL-12 expression by peritoneal macrophages was eliminated by exercise training of mice, which suggests that exercise downregulates β2AR activity. Other studies that utilize β2AR-specific agonists or antagonists have shown β2AR-associated inhibition of antigen-presentation by Langerhans cells (117) and β2AR-specific inhibition of IL-10 production early in LPS activation of DCs, whereas inhibition of IL-12 appeared to be mediated by both α2AR and β2ARs (79). In studies of human immune cells, the β2AR-specific agonist salbutamol blocked IL-12, but not IL-1α, IL-1β, IL-6, or IL-10, production by LPS-stimulated monocytes in vitro (95). The effects of β2AR signaling on Th1 cell function are reported to be both suppressive and enhancing, depending upon the mode of activation (i.e., LPS [95] or antigen-bound MHC class II [125], respectively), or on the developmental stage of the Th cell studied (i.e., naïve [125] or mature [114]). While these studies provide strong evidence for a role of β2ARs in many immune cell subsets, analyses of β1AR regulation of immune responses have been less intensively investigated. Furthermore, the mutual effects of β1AR and β2AR on immunity have been largely unexplored.

D. Proposed Mechanisms of b-adrenergic Receptor-Mediated Immunosuppression and Future Research Directions Both tissue-specific and cell type-specific mechanisms can account for cold-restraint–induced immunosuppression, and both β1AR- and β2AR-signaling pathways are likely to be involved. Based upon LM colonization results, cold-restraint treatment induces a more dramatic influence on host immunosuppression in liver than in spleen (Figure 3). It is possible that the expression of βARs differs between these tissues, or that the blood supply (supplying Epi) and sympathetic innervation (supplying NE) differs between

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these tissues, thereby altering the impact of catecholamine actions following cold-restraint stress. In fact, it was shown recently that β2AR mRNAs are more abundant in bovine hepatic tissue than are mRNAs of either β1AR or β3AR (with β2AR > β1AR > β3AR) (21). Furthermore, immunologic regulation in the liver is unique (131) and neuroimmune interactions in hepatic tissue require further study in a cold-restraint stress model. Since it is well documented that NK cells are required for efficient LM clearance, a simple explanation for the enhanced immunity observed in β1AR−/− mice with or without cold-restraint treatment may be that blockade or elimination of β1ARs has no impact on NK function, if NK cells do not normally express this receptor subtype (52). Our studies show that β2AR−/− mice succumb to cold-restraint treatment to an extent equal to or worse than that for wild-type mice. Perhaps, in mice lacking β2AR signaling, the beneficial stress-induced activation of NKs (via β2AR) is eliminated; inadequate activation of NK cells would be detrimental to defensive mechanisms against LM. Alternatively, β1AR-dependent mechanisms may directly influence immune cell subsets that likely express β1ARs, namely the monocyte (33,56,134) and DC populations (79). It is reported that βAR stimulation by an effective agonist requires the reduction of a disulfide bond in the active binding site, in order to initiate optimal coupling to AC (62,98,135). This activation is controlled by thiol-disulfide redox reactions, which can be disrupted by oxidative stress. In fact, oxidative stress of heart tissue has been shown to reduce βAR functioning by decreasing levels of cAMP production (45). We hypothesize that βAR activity is altered by and may contribute to stress-induced oxidation, the consequences of which are detrimental to host innate immune defenses. An association between oxygen radicals and β2AR expression by human T lymphocytes has been demonstrated; combined isoproterenol and H2O2 treatment caused a 70% reduction in sequestration of β2ARs (72). In these studies, H2O2 treatment reduced G-protein–coupled receptor kinase 2 (GRK2) protein levels and activity, but not mRNA. Expression of GRK2 is known to regulate G-protein–coupled receptor desensitization and re-sensitization. Thus, an oxidative environment may promote more β2AR expression on lymphocyte surfaces in times of stress, thereby subjecting cells to prolonged β2AR activation. In a homeostatic environment, oxidation is required to activate resting lymphocytes (37); the source of the immunoenhancing oxygen radicals is typically activated Mφ and neutrophils. Too much of a good thing

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can turn bad, in that excessive oxidation and a critical loss of surface thiols can alter CD8+ cytotoxic activity (106) and prevent T-cell activation (90). Intracellular redox regulation is also critical for lymphocyte trafficking or scavenging. For example, selective oxidation of Rho GTPase (but not Rac), by the arsenical compound PAO, causes the inhibition of stress-fiber formation and membrane ruffling, two essential processes for phagocytosis and cell migration (43). Finally, it has been proposed that the redox state of the Mφ is decisive in promoting either a cell-mediated or a humoral immune response (89,99). Resting Mφs are known to contain high levels of oxidant scavengers such as GSH and thioredoxin so as to maintain a reduced microenvironment for optimal activation of nearby lymphocytes since, as stated previously, an oxidized lymphocyte surface is associated with inhibited activation (90). A Mφ with greater reducing potential promotes Th1 responses through regulated production of NO and the cytokines IFN-γ and IL-12, whereas a Mφ with lowered reducing potential has diminished GSH and promotes a Th2-mediated host response, due to the elevated synthesis of IL-10 and IL-4 (89). We believe that the critical effect of cold restraint with our LM model is to alter the redox state of the resting macrophage, promoting a Th2-mediating phenotype, which weakens host defenses against LM. The initial cause of the altered redox state and whether βARs play a direct role are uncertain. A possible scenario is that βAR-induced inhibition of protein synthesis alters available GSH for the intracellular and extracellular maintenance of the optimal redox status. Whether the βAR-specific immunosuppressive effects of cold restraint are a consequence of βAR activity in immune or non-immune cells is poorly understood. Nonetheless, cold-restraint activation of stress mediators followed by the initiation of a systemic inflammatory response (LM infection) impairs host innate and/or adaptive immune responses. An increased understanding of several factors is crucial, if we are to attain a more comprehensive knowledge of βAR expression and function on immune cell subsets. First, molecular analysis of mRNA transcription and surface expression of all βAR subsets on immune cells would shed light on βAR-induced immunologic changes. While many years of study have elucidated the functional consequences of β2AR signaling on immune cells, little knowledge exists for β1AR and almost none for β3ARs. A single study has reported immune alterations induced by β3AR agonists in obese rats (63). Oral administration of the β3AR agonist trecadrine, which is an effective anti-obesity agent, caused an increase of CD4+ and CD8+ T-cells, yet it caused a decrease in lympho-proliferative activity.

Additional studies are needed to clarify the association between βAR subsets and redox changes of immune cells under various states of activation. Finally, a more detailed conception of an individual’s ability to manage allostatic load must be considered in stressresponse studies, for it is well documented that individuals perceive stress differently, depending upon genetic and/or behavioral characteristics (6,61,119). Generally, it appears that dominant or subordinate behavioral patterns can influence the sensitivity of an individual to a stressor, possibly through different inactivation mechanisms of stress factors (61). The mediation of allostatic load, the way an individual manages stress, is also a process relevant to aging and the associated immunosuppression that can occur in some individuals. In fact, diminished immunocompetence that is associated with aging has been linked to altered redox regulation (59,97,111); the relationships among these three parameters require further exploration.

E. The Implications of Short-term Stress for Sickness and Health A thorough understanding of the direct action of βAR signaling on immune cells will have broad therapeutic application in human health and disease. For example, a β2AR-induced increase in cAMP was shown to be coincident with decreases in TNF-α in rheumatoid arthritis patients, but not in controls (73). Therapeutic strategies for the inhibition or stimulation of specific βARs may therefore prove useful in treatment of inflammatory disorders such as arthritis, sepsis (93), allergic asthma (69), and multiple sclerosis (132), as well as in prevention of tumor metastasis (since βAR stimulation has been shown to alter NK activity and resistance to tumor metastasis in rats [118]). Prophylactic strategies like psychological counseling, which are procedures that can induce SNS activity, can improve the outcome of patients undergoing invasive surgeries. Pre-surgical psychosocial intervention has been shown to positively affect breast cancer patients’ post-operative psychological status, as well as their immune responsiveness (66). In terms of host susceptibility to infectious pathogens, stressful conditions that occur at or near the time of antigen exposure have been shown to significantly lower host immunity and to increase susceptibility to infectious disease (10,137). Furthermore, an individual’s self-perceived stress level may influence the ability of that person to mount an optimal response to vaccination (16,138). Since βARs are polymorphic, there may be clinical consequences of altered βAR signaling and susceptibility to tumor growth, pathogenic

48. Cold-Restraint-induced Immune and Biochemical Changes Inhibit Host Resistance to Listeria

infection, or chronic inflammatory disease (136). Our studies and those of many others indicate that βAR signaling influences innate and cellular immune function and survival during systemic inflammation. Delineation of the multi-faceted nature of βAR signaling, through analysis of the effects of cold-restraint stress during an LM infection, should continue to provide insights into the complex interactions between the nervous system and the immune system.

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