Introduction

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The alveolar macrophage (AM) is the predominant cell type in the alveolus and serves as the first line of host defense against inhaled pathogens and soluble and particulate molecules. AMs play a complex and central role in regulating the pulmonary immune response and it is therefore clear that any alteration in AM activity will have implications for pulmonary homeostasis (1, 2). Recently, we described a close relationship between pulmonary immune functions and acute mild inescapable electrical footshock stress, a stressor that is considered to reflect a state of emotional stress. Exposure of rats to this stressor resulted in enhanced primary and secondary humoral responses upon subsequent intratracheal immunization (3). Furthermore, stress leads to activation of AMs in such a way that they produce different levels of cytokines upon in vitro lipopolysaccharide (LPS) stimulation compared with control cells (4). Increased interleukin (IL)-1 and tumor necrosis factor- (TNF-) production was found, whereas nitric oxide (NO) secretion was decreased. The mechanism for these stress-induced changes in AM secretory activity remains to be elucidated, but it is likely that stress hormones play a role in this phenomenon. During stress, two major neuroendocrine pathways, the hypothalamo-pituitary-adrenal (HPA) axis and the sympathetic-adrenal-medullary (SAM) system, are activated (5, 6). Activation of the HPA axis results in the release of adrenocorticotropic hormone (ACTH) from the pituitary, which induces the secretion of the glucocorticoids (corticosterone [CORT]) from the adrenal cortex. Stimulation of the SAM system results in the local release of noradrenaline from sympathetic nerve terminals and in the hormonal secretion of adrenaline from the adrenal medulla. Thus, elevated plasma levels of ACTH, CORT, and adrenaline are found during stress, and these hormones, through interactions with specific receptors, are known to affect the function of cells of the immune system (7, 8). Recently, we demonstrated in vitro that the synthetic glucocorticoid dexamethasone can indeed activate AMs, leading to increased LPS-induced cytokine secretion (9). Whether there is a role for CORT in vivo in the stress-induced changes of AM activity is questionable, for it was shown that the intensity of the stressor affected the extent of the stress- induced changes in cytokine production by AMs, but not the plasma levels of ACTH and CORT (10). This suggests that catecholamines, released by the activated SAM system, and/or other stress-related hormones are involved. A role for catecholamines can also be inferred from the fact that, although the sympathetic innervation of the respiratory tract is relatively sparse (11), the density of adrenoceptors in the peripheral lung is high. These receptors are predominantly -adrenoceptors, of which the majority are of the 2-subtype that can be stimulated by circulating adrenaline (12). Autoradiographic studies have indicated that > 90% of all pulmonary -adrenoceptors are located on the alveolar wall (12), and it has been reported that AMs also express 2-adrenoceptors on their surface (16, 17). It can therefore be envisaged that the stress-induced changes in AM activity are mediated via -adrenoceptor activation, either directly via stimulation of -adrenoceptors on the surface of the AMs or indirectly via stimulation of receptors on other pulmonary cell types, resulting in changes that subsequently affect the AMs. In the present study we investigated which mediators are responsible for the observed stress-induced changes in AM activity. We studied the role of the autonomic nervous system and of - and -adrenoceptors in the observed stress effect on secretory functions of alveolar macrophages by blocking in vivo the autonomic output and - and -adrenoceptor activation with chlorisondamine and with specific - and -antagonists, respectively. Furthermore, we studied in vitro effects of catecholamines on the LPS-induced IL-1 secretion by AMs.

	Materials and Methods

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Animals

Specific pathogen-free male Wistar rats weighing 160-180 g were purchased from Harlan CPB (Zeist, The Netherlands). Animals were housed and studied under Institutional Animal Care and Use Committee-approved protocols in the animal facility of the Department of Pharmacology, Vrije Universiteit (Amsterdam). Rats were housed two per cage and kept under standard conditions in which the dark period was from 7:00 P.M. to 7:00 A.M., and water and food were provided ad libitum. A 2-wk acclimation period was allowed before experimental manipulations were initiated. To adjust them to experimental procedures, the animals were handled for 3 subsequent days prior to the experiment by being picked up twice a day. All experimental procedures were carried out between 8:00 A.M. and noon to minimize diurnal variations in endocrine levels.

Stress Paradigm

Animals were subjected to inescapable electrical footshock stress as reported previously (3, 4). In short, two rats per session were placed in a Plexiglas shock box (48 cm long, 24 cm wide, and 32 cm high) with a wired grid floor, separated by a nontransparent partition. Animals were subjected to inescapable scrambled electrical footshocks for 20 min (4 shocks/min, 5 s/shock, intensity 0.8 mAmp). Immediately after the stress session, the animals were decapitated, trunk blood was collected, and alveolar macrophages were isolated. Control animals stayed in their homecages until decapitation.

Blocking Agents

Thirty minutes before exposure of rats to the stress paradigm, animals were intraperitoneally injected with various blocking agents to analyze the effects of the autonomic nervous system, the parasympathetic nervous system, and -adrenergic and -adrenergic activation on alveolar macrophage function. Agents used were the nicotine receptor antagonist chlorisondamine (Ecolid; CIBA-Geigy, Arnhem, The Netherlands), 5 mg/kg; the muscarine receptor antagonist methyl-atropine (atropine methyl bromide, Sigma Chemical Co., St. Louis, MO), 4 mg/kg; the nonselective -adrenergic receptor antagonist timolol (BUFA B.V., Pharmaceutical Products, Uitgeest, The Netherlands), 1 mg/kg; and the nonselective -adrenergic receptor antagonist phentolamine (phentolamine hydrochloride; Sigma), 5 mg/kg. Rats received blocking agents intraperitoneally (i.p.), dissolved in 0.5 ml saline. Control rats received saline only. The effectiveness of the chlorisondamine and timolol treatment could be inferred from the apparent ptosis of the eyelids, whereas the effectiveness of methyl-atropine treatment was clear from the lack of bodily excrement during stress. The effectiveness of phentolamine could be inferred indirectly from the rise in body temperature after stress.

Hormone Assays

Blood was collected in ice-cold heparinized tubes and centrifuged (2,000 × g, 10 min, 4°C). Plasma was stored at 20°C until assayed. ACTH and CORT concentrations in plasma were measured as reported previously (18).

Isolation and Culture of AMs

AMs were obtained by bronchoalveolar lavage (4). Briefly, rats were killed by decapitation and the lungs were lavaged by four consecutive washings with 10 ml of magnesium- and calcium-free Hanks' balanced salt solution with 0.6 mM ethylenediamenetetraacetic acid at 37°C. Isolated cells were washed twice with ice-cold RPMI 1640 (Gibco Life Technologies, Breda, The Netherlands), resuspended to a concentration of 1 × 10⁶ cells/ml in culture medium (RPMI 1640 with 10% heat inactivated newborn calf serum [NBCS; Gibco], 2 mM L-glutamine [Gibco], and Pen/Strep [Sigma]), and cultured in 24-well plates (Delta plates; Nuclon, Kamstrup, Denmark) at 500 µl per well with various concentrations of LPS (Escherichia coli 055.B5; Sigma) for 24 h at 37°C, 5% CO₂. The culture supernatants were centrifuged to remove the debris (2,000 × g, 15 min, 4°C) and divided into aliquots which were stored at 20°C until further assessment for the presence of IL-1 and NO.

Analysis of NO

The presence of NO in AM culture supernatants was determined by measuring the amount of nitrite, a metabolic product of NO (19). In short, 50 µl of the culture supernatant was mixed with 50 µl Griess reagent (0.1% naphthylene diamine dihydrochloride [Sigma], 1% sulfanylamide [Sigma], and 2.5% H₃PO₄ in distilled water) in flat-bottomed 96-well microtiter immunoassay plates (Delta plates). After 10 min incubation at room temperature, the extinction of the reaction product was measured at 550 nm using a Titertek Multiskan. The nitrite amount was calculated from a NaNO₂ (Merck, Darmstadt, Germany) standard curve in culture medium.

Radio-immunoassay for the Detection of IL-1

IL-1 in the culture supernatants was measured by radio-immunoassay (RIA) according to DeRijk and colleagues (18). Briefly, iodogen-labeled rat recombinant IL-1 was used as a tracer in combination with a goat antihuman IL-1 polyclonal antiserum (Searl, St. Louis, MO). Culture supernatants (100 µl) were incubated overnight at 4°C with 50 µl antibody solution (diluted 1:16,000 in RIA buffer: 100 mM TrisHCl [pH 7.3], 0.1% wt/vol gelatin, and 0.02% vol/vol Tween-20). Then 50 µl tracer (10,000 cpm) diluted in RIA buffer was added, followed by another overnight incubation at 4°C. The third day, bound and free label were separated using solid-phase second-antibody precipitation (cellulose conjugated donkey antigoat immunoglobulin; Saccel Wellcome Reagents Ltd, Beckenham, UK). The immune complexes were pelleted after addition of 1:40 diluted horse serum and the radioactivity in the pellet was counted. Rat recombinant IL-1 diluted in culture medium was used as a standard. Sensitivity of the assay was 100 pg/ml culture supernatant.

Statistics

The results are presented as means ± SEM. Data were analyzed by a one-, two-, or three-way analysis of variance (ANOVA) followed by the Fisher Least Significant Difference (LSD).

	Results

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Effects of the Autonomic Nervous System on Secretory Activity of AMs during Stress

An enhanced IL-1 production and a decreased NO secretion by AMs after in vitro stimulation with LPS was found after exposure of rats to 20 min of mild inescapable electrical footshock stress, which is in accordance with earlier experiments (4). Here we demonstrate that these stress- induced changes in secretory activity by AMs were completely abrogated when the output of the autonomic nervous system was blocked by chlorisondamine (Figure 1). The chlorisondamine injections had no effect on the stress-induced elevations of plasma levels of ACTH and CORT (Figure 2), indicating that mediators released by the autonomic nervous system are responsible for the stress effects on AMs.

View larger version (14K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   Figure 1.   Effects of chlorisondamine on the LPS-induced IL-1 and NO secretion by AMs from stressed and control rats. Animals were divided into two groups of eight rats per group and were injected i.p. with either chlorisondamine (5 mg/kg) dissolved in 0.5 ml saline (squares) or with 0.5 ml saline only (circles). After 30 min, four rats per group were exposed to inescapable electrical footshock stress (filled figures) for 20 min (4 shocks/min, 5 s/shock, 0.8 mAmp). Immediately after the stress session, rats were decapitated and AMs were isolated by bronchoalveolar lavage. The remaining four chlorisondamine-treated and four saline-treated rats stayed in their homecages until decapitation (open figures). The AMs of four rats per group were pooled and cultured for 24 h with indicated concentrations of LPS. The LPS-induced IL-1 (A) and NO (B) secretion was measured in the culture supernatant. Results presented are for a single experiment out of four performed. Data are expressed as means ± SEM from three wells and were analyzed with a three-way ANOVA followed by Fisher's LSD. *Statistical significance at P < 0.05. Legends: (open circle) saline i.p., nonstressed; (filled circle) saline i.p., stressed; (open square) chlorisondamine i.p., nonstressed; (filled square) chlorisondamine i.p., stressed.

View larger version (17K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Figure 2.   Effects of chlorisondamine on plasma ACTH and CORT in stressed and control rats. Animals were injected i.p. with chlorisondamine (5 mg/kg) dissolved in 0.5 ml saline (dotted bars, n = 8) or received saline only (black bars, n = 20). After 30 min, rats were exposed to inescapable electrical footshock stress for 20 min (4 shocks/min, 5 s/shock, 0.8 mAmp). Immediately after the stress session, rats were decapitated and trunk blood was collected (STRESS). Control animals stayed in their homecages until decapitation (CTRL). Statistical analysis (two-way ANOVA) of the ACTH (A) and CORT (B) data revealed no differences in plasma hormone levels between chlorisondamine or saline-treated rats.

Effects of the Sympathetic and Parasympathetic Nervous System on Secretory Activity of AMs during Stress

Because the nicotine receptor antagonist chlorisondamine blocks both the sympathetic and parasympathetic nervous system, more specific blocking agents were used to discriminate between these branches and their effects on stress-induced changes in AM activity. Sympathetic nervous system blockade was subdivided into -adrenoceptor blockade and -adrenoceptor blockade by injection of phentolamine and timolol, respectively, whereas the parasympathetic output was blocked by the muscarinic receptor antagonist methyl-atropine. It was shown that blockade of -adrenoceptors prevents the enhancement of LPSinduced IL-1 completely (Figure 3A), and also that the reduction in NO secretion by AMs after stress was no longer significant when rats were pretreated with timolol (P < 0.3, Fisher's LSD) (Figure 3B). The results of the blockade of either -adrenoceptors or parasympathetic output were less clear and did not lead to annulment of the stress-induced alterations in AM secretory activity. On the contrary, both blocking agents resulted in an enhanced IL-1 secretion by AMs from both nonstressed and stressed rats compared with the IL-1 secretion by AMs from saline-treated nonstressed rats, whereas stress did not result in an extra increase in IL-1 secretion (Figures 4 and 5).

View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 3.   Effects of timolol on the LPS-induced IL-1 and NO secretion by AMs from stressed and control rats. Animals were divided into two groups of eight rats per group and were injected i.p. with either timolol (1 mg/kg) dissolved in 0.5 ml saline (squares) or with 0.5 ml saline only (circles). After 30 min, four rats per group were exposed to inescapable electrical footshock stress (filled figures) for 20 min (4 shocks/min, 5 s/shock, 0.8 mAmp). Immediately after the stress session, rats were decapitated and AMs were isolated by bronchoalveolar lavage. The remaining four timolol-treated and four saline-treated rats stayed in their homecages until decapitation (open figures). The AMs of four rats per group were pooled and cultured for 24 h with indicated concentrations of LPS. The LPS-induced IL-1 (A) and NO (B) secretion was measured in the culture supernatant. Results presented are for a single experiment out of three performed. Data are expressed as means ± SEM from three wells and were analyzed with a three-way ANOVA followed by Fisher's LSD. *Statistical significance at P < 0.05. Legends: (open circle) saline i.p., nonstressed; (filled circle) saline i.p., stressed; (open square) timolol i.p., nonstressed; (filled square) timolol i.p., stressed.

View larger version (15K): [in this window] [in a new window]   Figure 4.   Effects of methyl-atropine on the LPS-induced IL-1 secretion by AMs from stressed and control rats. Animals were divided into two groups of eight rats per group and were injected i.p. with either methyl-atropine (4 mg/kg) dissolved in 0.5 ml saline (triangles) or with 0.5 ml saline only (circles). After 30 min, four rats per group were exposed to inescapable electrical footshock stress (filled figures) for 20 min (4 shocks/min, 5 s/shock, 0.8 mAmp). Immediately after the stress session, rats were decapitated and AMs were isolated by bronchoalveolar lavage. The remaining four methyl-atropine-treated and four saline-treated rats stayed in their homecages until decapitation (open figures). The AMs of four rats per group were pooled and cultured for 24 h with indicated concentrations of LPS. The LPS-induced IL-1 secretion was measured in the culture supernatant. Results presented are for a single experiment out of three performed. Data are expressed as means ± SEM from three wells and were analyzed with a three-way ANOVA followed by Fisher's LSD. Legends: (open circle) saline i.p., nonstressed; (filled circle) saline i.p., stressed; (open triangle) methyl-atropine i.p., nonstressed; (filled triangle) methyl-atropine i.p., stressed.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Effects of phentolamine on the LPS-induced IL-1 secretion by AMs from stressed and control rats. Animals were divided into two groups of eight rats per group and were injected i.p. with either phentolamine (5 mg/kg) dissolved in 0.5 ml saline (diamonds) or with 0.5 ml saline only (circles). After 30 min, four rats per group were exposed to inescapable electrical footshock stress (filled figures) for 20 min (4 shocks/min, 5 s/shock, 0.8 mAmp). Immediately after the stress session, rats were decapitated and AMs were isolated by bronchoalveolar lavage. The remaining four phentolamine-treated and four saline-treated rats stayed in their homecages until decapitation (open figures). The AMs of four rats per group were pooled and cultured for 24 h with indicated concentrations of LPS. The LPS-induced IL-1 secretion was measured in the culture supernatant. Results presented are for a single experiment out of three performed. Data are expressed as means ± SEM from three wells and were analyzed with a three-way ANOVA followed by Fisher's LSD. Legends: (open circle) saline i.p., nonstressed; (filled circle) saline i.p., stressed; (open diamond) phentolamine i.p., nonstressed; (filled diamond) phentolamine i.p., stressed.

In Vitro Effects of Catecholamines on Cytokine Production by AMs

Next, experiments were performed to determine whether the -adrenoceptor mediated stress effects on AM activity are a result of either direct or indirect influence of catecholamines on AMs. Therefore, the in vivo stress was mimicked in vitro by preincubating control AMs with various concentrations of adrenaline, noradrenaline, -agonist (L-phenylephrine), or -agonist (isoproterenol) for 2 h. After the cells were washed and incubated overnight with LPS, the IL-1 secretion was analyzed (Figure 6A). From these results, a direct effect of catecholamines on the secretory activity of AMs could not be demonstrated. Also, overnight coincubation of AMs with catecholamines and LPS did not result in altered IL-1 secretion compared with control (Figure 6B).

View larger version (31K): [in this window] [in a new window]   View larger version (27K): [in this window] [in a new window]   Figure 6.   Effects of in vitro preincubation with catecholamines on LPS-induced IL-1 secretion by AMs. Alveolar macrophages were isolated by bronchoalveolar lavage, plated in 24-well plates, and cultured for 2 h with various concentrations (105 M [hatched bars], 107 M [crosshatched bars], or 109 M [dotted bars]) of four distinct catecholamines or, as a control, with medium only (black bars). After 2 h, the cells were washed to remove the extracellular catecholamine, the culture medium was replenished, and cells were further cultured with 100 µg/ml LPS in the absence (preincubation, A) or presence (continuous incubation, B) of the same concentrations of catecholamines. Culture supernatants were collected after 20 h and the amount of IL-1 was measured. Statistical analysis (two-way ANOVA) of the IL-1 data revealed no significant differences. Results presented are for a single experiment out of three performed. Data are expressed as the means ± SEM from three wells. A: adrenaline; NA: noradrenaline; -ago: -adrenergic receptor agonist (phenylephrine); -ago: -agonist (isoproterenol).

	Discussion

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We have recently shown that AMs isolated from rats exposed to 20 min of mild inescapable electric footshock stress secreted significantly more IL-1 and TNF- and less NO upon in vitro stimulation than did AMs from control rats (4). Here, we demonstrate that chlorisondamine blockade of the output of the autonomic nervous system during stress completely abrogates these stress-induced changes in AM activity. This role for the autonomic nervous system could be attributed to sympathetic stimulation of -adrenergic receptors. Furthermore, it was shown that the -adrenoceptor-mediated stress effects are not mediated by direct interaction between catecholamines and AMs.

To block either -adrenoceptors or the parasympathetic output, we used the nonselective -antagonist phentolamine and the muscarinic receptor antagonist methyl-atropine, respectively. These agents are widely used in in vivo experiments and do not pass the blood brain barrier (20, 21). Neither blocking agent resulted in reduction of the IL-1 levels to control levels. In contrast, these drugs by themselves induced an increase in IL-1 secretion by AMs, which can be explained by indirect stimulatory effects of these blocking agents on -adrenoceptors. The 2-adrenoceptor blocking agents are known to increase the sympathetic outflow and the release of noradrenaline from sympathetic nerve terminals (22), and it can therefore be envisaged that the nonselective -blocker phentolamine results in excessive release of catecholamines in the peripheral lung which, through stimulation of -adrenoceptors, is then responsible for the enhanced LPS-induced IL-1 secretion. The effects of methyl-atropine can be explained by receptor cross-talk between muscarinic receptors and -adrenoceptors, as recently discussed by Barnes (25). Muscarinic receptors, coupled to inhibitory G-proteins in the plasma membrane, may counteract the effects of -adrenoceptors that are coupled to stimulatory G-proteins. Muscarinic receptor blockade by methyl-atropine may therefore tip the scales toward increased adrenergic responsiveness, resulting in enhanced LPS-induced IL-1 secretion. In this respect it is noteworthy that muscarinic receptors are demonstrated on the alveolar wall in humans (26), and that their presence has been demonstrated on alveolar type II epithelial cells (27, 28).

Although the expression of adrenergic receptors on AMs has been demonstrated (16, 17), we could not demonstrate a direct modulating effect of catecholamines on LPS-induced IL-1 secretion by AMs in our preincubation experiments. But overnight coincubation of AMs with catecholamines and LPS also did not affect the IL-1 secretion. This is in contrast to previous findings that show the involvement of adrenergic receptors in modulation of the LPS-induced TNF- secretion by peritoneal macrophages and monocytic cell lines (29). It is not clear whether the discrepancy is due to differences in cytokines as readout systems or reflects intrinsic differences between AMs and other macrophage populations.

Together, our results show an indirect -adrenoceptor-mediated stress effect on AMs. A possible mechanism for such indirect activation could involve the alveolar type II epithelial cells. These cells are becoming recognized as important immunoregulatory cells in the alveolar space (reviewed in 33 and 34) and are in close proximity to AMs. Furthermore, the presence of adrenergic as well as muscarinic receptors at the surface of alveolar type II epithelial cells has been demonstrated (27, 28). Preliminary experiments in our laboratory have indeed shown that exposure of rats to stress results in an altered IL-6 secretion by isolated type II cells. These stress effects could also be blocked by pretreatment of rats with chlorisondamine, indicative of a role of the sympathetic nervous system (manuscript in preparation). Involvement of the sympathetic nervous system in alveolar type II epithelial cell function has been demonstrated previously. Increased surfactant secretion in response to circulating and exogenous catecholamines has been demonstrated in both in vitro and in vivo models (27, 35). A direct sympathetic nerve influence on alveolar type II epithelial cells has also been reported by Crittenden and coworkers, who showed that 1-min stimulation of the stellate ganglion, which sends sympathetic fibers to the lung parenchyma, results in the immediate release of surfactant phospholipids from the lamellar bodies (36, 37), a process that is accompanied by progressive ultrastructural changes in alveolar type II epithelial cells with clear hyperplasia of the lamellar bodies (38). Both surfactant release and the ultrastructural changes can be prevented by blockade of adrenergic receptors prior to sympathetic nerve stimulation (36, 37). Because the release of surfactant is instantaneous after adrenergic receptor stimulation, it can be envisaged that the stress-induced changes in AM activity result from -adrenoceptor-mediated alveolar type II epithelial cell activation, leading to the release of surfactant or other factors that may then prime the AM to respond differentially upon in vitro activation with LPS. Increased density of surfactant in the alveolar space after stress might lead to enhanced clearance, a process that involves re-uptake into alveolar type II epithelial cells (about 80%) and phagocytosis by AM (15%) (39), indicating that both cell types can be affected. It has been demonstrated that surfactant can influence the cytokine production by AMs (40) and surfactant components can modulate AM functions such as phagocytosis, chemotaxis, and oxidative burst (reviewed in 39). A role for surfactant can further be inferred from the fact that priming of AMs during stress has to occur by a preformed factor, simply because time for de novo synthesis is lacking in our stress model.

In conclusion, the present study shows that stress-induced changes in AM secretory activity are indirectly mediated by -adrenergic receptors. Thus, AM activity can be influenced by the neuroendocrine system, implying that stress may contribute to the onset or severity of pulmonary inflammatory processes.