Exposure to acute physical and psychological stress alters the response of rat macrophages to corticosterone, neuropeptide Y and beta-endorphin

Abstract

The objective of the present study was to investigate the effect of acute exposure to electric tail shock stress (ES) and a stress witnessing procedure (SW), as models for physical and psychological stress paradigms, respectively on adherence, phagocytosis and hydrogen peroxide (H₂O₂) release from rat peritoneal macrophages. In addition, we studied the in vitro effects of corticosterone (CORT), neuropeptide Y (NPY) and beta-endorphin (BE) on adherence, phagocytosis and H₂O₂ release from macrophages isolated from control rats and from rats that had been exposed to ES or SW procedures 24 h earlier. ES and SW comparably diminished phagocytosis and H₂O₂ release, but did not influence macrophage adherence. In vitro treatment with CORT and NPY notably suppressed phagocytosis and potentiated H₂O₂ release from macrophages. BE suppressed both phagocytosis and H₂O₂ release from macrophages. Previous exposure to ES and SW altered the responsiveness of the isolated macrophages to their in vitro treatment with mediators of stress, making the cells less sensitive to the influence of CORT and NPY and to a lesser extent to BE. It could be concluded that changes in the local macrophage milieu induced by ES and SW 24 h earlier modify macrophage responses to subsequent in vitro exposure to the stress mimics, CORT, NPY and BE.

• Beta-endorphin (BE)
• corticosterone (CORT)
• electric tail shock stress (ES)
• neuropeptide Y (NPY)
• peritoneal macrophage functions
• stress witnessing procedure (SW)

Introduction

Stress is associated with an altered homeostatic state of the organism, involving behavioural, endocrine and immunological changes (McEwen 1998). Various inputs indicative of stress converge on the final common pathways within the brain. This is illustrated by the combined roles in the central construction of the responses to stress of the corticotropin-releasing hormone (CRH) neurons of the paraventricular nucleus (PVN) of the hypothalamus and CRH neurons in the medulla, together with catecholaminergic neurons of the locus coeruleus and the medulla and pons (Whitnall 1993; Stratakis and Chrousos 1995; Chrousos 2000).

The final products of the activation of the hypothalamic–pituitary–adrenal (HPA) axis during stress are glucocorticoids that prepare the organism for changes in energy and metabolism required to deal with the stress (Munck et al. 1984; Whitnall 1993; Stratakis and Chrousos 1995). Human cortisol and rodent corticosterone (CORT) are commonly connected with stress-induced immunosuppression. However, glucocorticoids influence immune functions in a more complex manner by controlling the individual repertoire of immunological responses including the direction and magnitude of immune reactions (Wilckens 1995; Wilckens and De Rijk 1997).

Sympathetic nerve fibres that innervate various organs, including lymphoid organs, also directly contact macrophages, T-lymphocytes and mast cells from non-organised lymphoid compartments (Stevens-Felten and Bellinger 1997). Cells residing within lymphoid organs (Bellinger et al. 2001) and from non-organised lymphoid compartments (Stevens-Felten and Bellinger 1997) are also exposed to neuropeptide Y (NPY) co-localised and co-released with norepinephrine from noradrenergic nerve fibres upon stimulation. It was noted that in some pathological conditions, such as in septic shock, the sensitivity of dilated blood vessels to norepinephrine significantly decreases while NPY retains its vasoconstrictive properties (Hauser et al. 1995). NPY, a 36 amino acid peptide, is one of the most abundant and widely distributed peptides in the central and peripheral nervous system (Baker and Herkenham 1995). In response to acute stress, NPY mRNA expression increases within discrete regions of the brain (Conrad and McEwen 2000) and the adrenal medulla (Hiremagalur et al. 1994). The release of NPY during stress (Grundemar and Hakanson 1993; Puybasset et al. 1993) provides the basis for a role of this peptide in stress-induced immune alterations (Bedoui et al. 2003). Both lymphocytes and macrophages produce NPY after their activation, a process that facillitates the autocrine/paracrine action of NPY in the immune system (Schwarz et al. 1994; Wheway et al. 2005).

Versatile adaptive responses to stress also involve the release of the opioid peptide beta-endorphin (BE),a 31 amino acid peptide hormone that is cleaved from proopiomelanocortin (POMC) along with several other products with variable physiological activity via a proteolytic process (Heijnen et al. 1991). BE is primarily synthesised in the hypothalamus and the anterior and intermediate lobes of the pituitary (Stratakis and Chrousos 1995; Smith and Funder 1988), but also in monocytes, granulocytes, lymphocytes and macrophages (Mousa et al. 2004). Stress significantly increases the BE concentration in plasma and within immune cells (Sacerdote et al. 1994; Tsukada et al. 2001), which together could contribute to stress-induced immunomodulation (Hale et al. 2001).

We have previously shown that exposure to electric tail shock stress (ES) profoundly suppresses the humoral immune response in rats, whereas witnessing electric tail shock administration (SW) potentiates it (Stanojević et al. 2003). It is suggested that alterations in the immune response observed after stress could be specifically attributed to stress-induced changes in macrophage functions as these cells are intimately involved in the development of the humoral and cellular immune responses (Fleshner et al. 1995a; Kizaki et al. 1996). Therefore, we examined if acute exposure to ES and SW, representing physical and psychological stress paradigms, respectively, affected adherence, phagocytosis and H₂O₂ release from peritoneal macrophages.

Several lines of evidence suggest that a previous stressful experience changes the response to a subsequent stressor via a mechanism involving multiple physiological systems (O'Connor et al. 2003). A previous stressful experience may lead to sensitisation (Piazza and Le Moal 1998) or to habituation (De Boer et al. 1990) of a particular response. This depends on whether the subsequent stressor is homotypic or heterotypic. Both phenomena are evident in many aspects of human stress-related disorder (Tilders and Schmidt 1999). The rationale behind this study was that exposure to acute stress could induce functional changes within macrophages thereby altering their response to artificial stressors as mimicked by CORT, NPY and BE in vitro 24 h later. We tested this by studying the in vitro effects of CORT, NPY and BE on adherence, phagocytosis and H₂O₂ release from peritoneal macrophages isolated from rats that had been exposed to ES or SW procedures 24 h earlier.

Materials and methods

Animals

Young adult male Albino Oxford (AO) rats (60 ± 3 days of age) were obtained from our breeding colony in the Immunology Research Centre “Branislav Janković” in Belgrade. Rats were housed individually in perspex-walled cages with free access to food and water. Colony conditions were maintained using 12 h light and 12 h dark cycles (lights on at 08:00). All animal procedures were approved by our Institutional Animal Care and Use Committee and followed guidelines described in the European Community's Council Directive dated 24th November 1986 (86/609/EEC).

Reagents

BE, CORT, phenol red, phorbol myristate acetate (PMA), horseradish peroxidase (EC 1.11.1.7) and zymosan A (isolated from Saccharomyces cerevisiae) were obtained from Sigma (St Louis, MO, USA). Human/rat NPY was purchased from Polypeptide Laboratories (Wolfenbuttel, Germany). Nitro blue tetrazolium chloride (NBT, 2,2′-di-p-nitrophenyl-5,5′-diphenyl-3,3′-[3,3′-dimethoxy-4,4′-diphenylene]ditetrazolium chloride) was obtained from Serva (Heidelberg, Germany). Thioglycolate medium and phenol red-free minimal essential medium (MEM) were both acquired from the “Torlak” Institute in Belgrade.

Stress procedures

Rats were intraperitoneally injected with 15 ml of thioglycolate medium in order to induce recruitment of circulatory monocytes into the peritoneum and proliferation of resident peritoneal macrophages (Eichner and Smeaton 1983; Melnicoff et al. 1989). Stress procedures were performed six days later and took place between 08:00 and 09:30. Each experimental group consisted of eight rats.

ES was delivered in a soundproof room into which the rats had been moved immediately prior to initiating the stress procedure. During the shock procedure the rats were kept in rectangular perspex-walled boxes (18 × 9.5 × 20 cm) containing a semicircular aperture on the lower part of the rear wall that allowed attachment of the electrodes to the rat's tail and prevented movements of rear paws. Electric shocks were delivered via two silver electrodes (separated by 3–4 cm) connected to the tail with the aid of electrode paste and adhesive tape. Four rats were simultaneously shocked with the same intensity (55 V, 1 mA). A shock session consisted of eighty 5 s unsignalled shocks (seven electrical impulses of 0.3 s duration and 0.6 s pauses) with an average inter-shock interval of 60 s (range 5–120 s). In this way, the total duration was 80–90 min. After the shock session, the rats were returned to their colony. As the experimental model of ES involved the exposure of rats to a series of unpredictable, inescapable and uncontrollable electric tail shocks accompanied by early and late opioid-mediated analgesia, it was regarded as both a physical and a psychological challenge that resulted in immunological changes (Maier et al. 1983; Shavit et al. 1984; Sutton et al. 1994). It has been shown that such a procedure induces a several-fold increase in the plasma level of CORT (Johnson et al. 2002).

SW involved rats witnessing the procedure of shock delivery to the rats in the ES group. Rats were moved to a soundproof room immediately prior to the stress session at the same time as the rats belonging to the ES group. SW rats remained there during the ES procedure. Specifically, SW rats were kept in their regular cages and witnessed the whole ES procedure. After the ES session, SW rats were returned to their colony. The model of witnessing the ES procedure involved exposure of caged rats to pheromones (Fanselow 1985) and ultrasonic vocalisation (Knutson et al. 2002) emitted from rats experiencing the ES procedure. Therefore, this experimental design ensures primarily psychological stress for the witnessing rats. Rats witnessing rats experiencing the ES procedure increase CORT secretion (Ottenweller et al. 1989) and experience opioid-mediated analgesia (Moynihan et al. 2000). This stress paradigm also produces immunological changes thereby confirming that physically stressed rats can influence non-physically stressed rats via mechanisms involving vocalisation and pheromones (Fernandes 2000; Moynihan et al. 2000).

Serving as controls were intact control (IC) rats left undisturbed in their regular cages in the colony.

Isolation of macrophages and their cell surface phenotyping

About 24 h after shock termination (on the seventh day after injection of thioglycolate medium) macrophages were obtained by peritoneal lavage with 10 ml of MEM. Seven days after injection of thioglycolate medium the majority of the cells in peritoneal exudates are macrophages (Segura et al. 1996). Peritoneal lavages were centrifuged (250 g, 10 min) and the resulting cell pellets were washed three times using phosphate buffer in order to prepare the fresh macrophage suspensions for the assays. This procedure allowed adequate recovery of cells (greater than 95% viable as determined by trypan blue exclusion).

The cells (1 × 10⁷/ml) were stained with mouse anti-rat CD11b IgG-FITC (clone number ED8, Serotec, Oxford, UK) and mouse anti-rat CD68-biotin (clone number ED1, Serotec, Oxford, UK) followed by streptavidin RPE (STAR4A/B) as a second step reagent (Becton Dickinson, San Jose CA, USA). Analysis on a FACScan flow cytometer (Cell Quest software, Becton Dickinson) by means of FSC and SSC of a total of 10⁴ flow cytometric events confirmed that the majority (75.8 ± 3.6%) of cells in the peritoneal lavages were ED1 + CD11b+ macrophages.

Macrophage adherence

A previously published method (Oez et al. 1990) was followed. Briefly, 96-well flat-bottomed tissue culture plates (Linbro, ICN Biomedicals, Aurora, OH, USA) were filled with 50 μl of MEM, or CORT (2 × 10^{− 9}–2 × 10^{− 5} M in MEM), NPY (2 × 10^{− 14}–2 × 10^{− 6} M in MEM) or BE (2 × 10^{− 14}–2 × 10^{− 6} M in MEM) together with 50 μl of individual cell samples (2.5 × 10⁶ cells/ml, eight individual cell samples per group, in duplicate). The plates were incubated for 1 h at 37°C in 95% air–5% CO₂ before being washed twice with warm (37°C) MEM. The adherent cells were fixed with methanol, stained with 0.1% methylene blue and then washed thoroughly with distilled water. The plates were dried overnight at room temperature. The dye was dissolved in 0.1 M HCl and the optical density (OD) was determined at 620 nm using an Multiskan Ascent platereader (Labsystems, Helsinki, Finland). The results were expressed as OD (mean ± SEM).

Phagocytosis assay

Phagocytosis was determined as previously described (Pick et al. 1981). Briefly, macrophage suspensions adjusted to 2.5 × 10⁶ cells/ml (eight individual cell samples per experimental group, in duplicate) were plated (100 μl/well) in 96-well flat-bottomed tissue culture plates. The plates were incubated for 2 h at 37°C in 95% air–5% CO₂. Non-adherent cells were removed by washing the plates twice with warm (37°C) MEM. Adherent macrophages were stimulated with 50 μl of zymosan (125 μg/ml) in the absence or presence of CORT (2 × 10^{− 8}–2 × 10^{− 5} M), NPY (2 × 10^{− 12}–2 × 10^{− 6} M) or BE (2 × 10^{− 12}–2 × 10^{− 6} M) and in the presence of 50 μl of NBT (0.5 mg/ml in MEM) for 30 min at 37°C in 95% air–5% CO₂. The cells were then fixed with methanol, the plates were air-dried and ODs were determined at 545 nm. The amount of engulfed zymosan particles was proportional to the reduction of yellow NBT to blue formazan. The results were expressed as OD (mean ± SEM).

H₂O₂ release assay

H₂O₂ release was determined according to a previously published method (Pick and Mizel 1981). Briefly, macrophage suspensions adjusted to 2.5 × 10⁶ cells/ml (eight individual cell samples per group, in duplicate) were adhered to tissue culture plates for 2 h at 37°C in 95% air–5% CO₂ (non-adherent cells were removed by washing the plates twice with warm MEM, as previously described). Adherent macrophages were primed for H₂O₂ release with 100 μl of 25 nM PMA in phenol red solution (10 mM potassium phosphate buffer pH 7, 140 mM NaCl, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of horseradish peroxidase) in the absence or presence of CORT (10^{− 9}–10^{− 5} M), NPY (10^{− 14}–10^{− 6} M) or BE (10^{− 14}–10^{− 6} M). PMA was prepared from stock solution (10^{− 2} M in DMSO, stored at − 70°C). The 96-well plates were incubated for 1 h at 37°C in 95% air–5% CO₂. Incubations were terminated with 10 μl of 0.5 M NaOH and ODs were determined at 620 nm. The concentration of H₂O₂ in the samples was calculated using standard concentrations of H₂O₂ (1–40 μM). The results were expressed as nM of H₂O₂ produced per mg of cell protein (mean ± SEM).

Statistical analysis

Statistical analysis of the data was performed using the StatView II computer programme. The main effects of ES and SW were analysed by one-way ANOVA. The effects of CORT, NPY and BE were analysed by one-factor ANOVA for repeated measures (factor: concentration). Fisher's PLSD test was used for post hoc analysis for the evaluation of differences between groups (indicated in the figures with asterisks, *). Differences were regarded as statistically significant if p < 0.05.

Results

The effect of stress on macrophage adherence, phagocytosis and H₂O₂ release

Macrophage adherence was not affected by ES or SW (F_2,21 = 0.6; p = 0.5617; Figure 1(A)). However, both ES and SW diminished zymosan phagocytosis (F_2,21 = 13.5; p < 0.001; Figure 1(B)) and reduced H₂O₂ release (F_2,21 = 4.4; p < 0.05; Figure 1(C)).

Figure 1 The effect of acute exposure to ES and a SW on: (A) adherence, (B) zymosan particle phagocytosis and (C) PMA-primed H₂O₂ release from peritoneal macrophages isolated 24 h after stress. The values represent the mean (n = 8) ± SEM. Statistically significant differences: *, p < 0.05; and **, p < 0.01 vs. the IC group of rats.

The effect of in vitro treatment with CORT, NPY and BE on macrophage adherence

CORT (10^{− 7}–10^{− 5} M) slightly (but significantly) decreased IC rat macrophage adherence (F_5,35 = 5.3; p < 0.001). In contrast, CORT more effectively diminished both ES and SW rat macrophage adherence (F_5,35 = 10.8; p < 0.0001 and F_5,35 = 4.3; p < 0.01, respectively; Figure 2(A)).

Figure 2 The effect of in vitro treatment with: (A) CORT, 10^{− 9}–10^{− 5} M; (B) NPY, 10^{− 14}–10^{− 6} M; and (C) BE, 10^{− 14}–10^{− 6} M on the adherence of macrophages isolated from IC rats, from rats exposed to acute ES and from rats exposed to a SW 24 h earlier. The values represent the mean (n = 8) ± SEM. Statistically significant differences: *, p < 0.05; **, p < 0.01; and ***, p < 0.001 vs. 0.

Only the highest concentration of NPY (10^{− 6} M) increased IC rat macrophage adherence (F_5,35 = 6.4; p < 0.001). However, a wide range of NPY concentrations decreased both ES and SW rat macrophage adherence (10^{− 12}–10^{− 6} M; F_5,35 = 6.9; p < 0.0001 and 10^{− 14}–10^{− 12} M; F_5,35 = 6.1; p < 0.001, respectively, Figure 2(B)).

BE decreased ES rat macrophage adherence (F_5,35 = 2.7; p < 0.05) but failed to have any effect on both IC and SW rat macrophage adherence (F_5,35 = 0.6; p = 0.7036 and F_5,35 = 2; p = 0.0958, respectively; Figure 2(C)).

The effect of in vitro treatment with CORT, NPY and BE on macrophage phagocytosis

CORT (10^{− 8}–10^{− 5} M) strongly suppressed the phagocytic ability of macrophages isolated from both IC (F_4,28 = 48.7; p < 0.0001) and SW rats (F_4,28 = 30.7; p < 0.0001) but failed to influence the phagocytic ability of macrophages isolated from ES rats (F_4,28 = 2.5; p = 0.0673; Figure 3(A)).

Figure 3 The effect of in vitro treatment with: (A) CORT, 10^{− 8}–10^{− 5} M; (B) NPY, 10^{− 12}–10^{− 6} M; and (C) BE, 10^{− 12}–10^{− 6} M on zymosan phagocytosis by macrophages isolated from IC rats, from rats exposed to acute ES and from rats exposed to a SW 24 h earlier. The values represent the mean (n = 8) ± SEM. Statistically significant differences: *, p < 0.01; and **, p < 0.001 vs. 0.

NPY (10^{− 12}–10^{− 6} M) suppressed the phagocytic ability of macrophages isolated from both IC (F_4,28 = 23.6; p < 0.0001) and SW rats (F_4,28 = 21.4; p < 0.0001) but did not affect the phagocytic ability of macrophages isolated from ES rats (F_4,28 = 2.5; p = 0.0785), in comparison with phagocytosis of zymosan in non-treated macrophages from IC group (Figure 3(B)).

BE significantly decreased the phagocytic ability of macrophages isolated from all three experimental groups of rats (IC, F_4,28 = 69.3; p < 0.0001; ES, F_4,28 = 44.1; p < 0.0001 and SW, F_4,28 = 36.5; p < 0.0001; Figure 3(C)).

The effect of in vitro treatment with CORT, NPY and BE on macrophage H₂O₂ release

A low concentration of CORT (10^{− 9} M) significantly increased IC rat macrophage H₂O₂ release whereas a higher concentration (10^{− 5} M) significantly suppressed macrophage H₂O₂ release (F_5,35 = 15.5; p < 0.0001; Figure 4(A)). CORT (only at 10^{− 5} M) decreased ES rat macrophage H₂O₂ release (F_5,25 = 8.2; p < 0.0001). Lower CORT concentrations (ranging from 10^{− 8} to 10^{− 5} M) strongly inhibited SW rat macrophage H₂O₂ release (F_5,35 = 23.5; p < 0.0001).

Figure 4 The effect of in vitro treatment with: (A) CORT, 10^{− 9}–10^{− 5} M; (B) NPY, 10^{− 14}–10^{− 6} M; and (C) BE, 10^{− 14}–10^{− 6} M on PMA-primed H₂O₂ release from macrophages isolated from IC rats, from rats exposed to acute ES and from rats exposed to a SW 24 h earlier. The values represent the mean (n = 6–8) ± SEM. Statistically significant differences: *, p < 0.05; **, p < 0.01; and ***, p < 0.0001 vs. 0.

NPY (at all tested concentrations) strongly increased IC rat macrophage H₂O₂ release (F_5,30 = 14.6; p < 0.0001). The effect of NPY on H₂O₂ release from macrophages isolated from ES rats was less intense (F_5,25 = 11; p < 0.0001). NPY decreased H₂O₂ release from SW rat macrophages (F_5,25 = 4.7; p < 0.01; Figure 4(B)).

BE exerted interesting effects on macrophage H₂O₂ release. BE (10^{− 10}–10^{− 6} M) decreased IC rat macrophage H₂O₂ release (F_5,35 = 40.1; p < 0.0001), both increased (at 10^{− 12} M) and decreased (at 10^{− 8} and 10^{− 6} M) SW rat macrophage H₂O₂ release (F_5,35 = 19.4; p < 0.0001) and at concentrations from 10^{− 14} to 10^{− 8} M significantly increased ES rat macrophage H₂O₂ release (F_5,35 = 14.3; p < 0.0001; Figure 4(C)).

Discussion

Both ES and SW diminished macrophage phagocytotic activity and H₂O₂ release without affecting macrophage adherence. The diminished response to stress mediators in the early phases of macrophage activation and suppressive influence of stress on later activation steps suggest that after ES and SW macrophages retain the ability to leave their local microenvironment and to adhere to smooth surfaces of both tissue membranes and target cells whilst being unable to mount full inflammatory response. The physiological purpose of such a regulatory mechanism induced by stress could be beneficial, since it reduces inflammation. However, since local inflammation restricts spreading of an infection throughout the body, anti-inflammatory effect of stress could also be detrimental.

Our findings that high concentrations of CORT diminished macrophage adherence while a low concentration increased H₂O₂ release together support the general view that the physiological role of endogenous glucocorticoids is to maintain and optimise the immune response (Fleshner et al. 2001), since low concentrations of CORT produce immunoenhancement while higher concentrations are immunosuppressive (Dhabhar and McEwen 1999). However, CORT diminished zymosan engulfment at all concentrations tested. Phagocytotic capability and H₂O₂ release represent two key macrophage parameters, but both indicate oxidative products after the respiratory burst (Pick and Mizel 1981; Pick et al. 1981). The main difference emanates from the divergence in the route of macrophage activation since zymosan stimulates the respiratory burst by phagocytosis via beta-glucan and CR3 complement receptors (Giaimis et al. 1993; Le Cabec et al. 2000) while PMA directly activates protein kinase C (PKC) and stimulates the oxidative burst without phagocytosis (Johnston and Kitagawa 1985; Chanok et al. 1994). In view of this difference, it may be that by decreasing macrophage adherence CORT allows in vivo a greater number of cells to leave the bloodstream. However, by diminishing phagocytosis CORT tends to restrict the likelihood of tissue damage from the subsequent respiratory burst and H₂O₂ spillover.

In contrast, NPY increased macrophage adherence in line with the finding that NPY increased the adhesion of human neutrophils and a monocytic cell line to endothelial cells (Sung et al. 1991). It has been demonstrated that NPY also increases adherence of lipopolysaccharide (LPS)-stimulated macrophages that, besides the cardiovascular actions of NPY (Hauser et al. 1995), could in part contribute to its ability to improve survival in experimental endotoxemia (Nave et al. 2004). NPY diminished zymosan particle phagocytosis, measured using an assay relying on NBT reduction. However, NPY strongly potentiated the respiratory burst and H₂O₂ release from macrophages stimulated with PMA (which activates PKC) and this is in line with the ability of NPY to activate PKC causing its translocation from the cytosol to plasma membrane (De la Fuente et al. 1993). We have previously shown that NPY augments H₂O₂ release from macrophages isolated from inbred Dark Agouti (DA) rats (Dimitrijević et al. 2005).

BE suppressed zymosan phagocytosis and H₂O₂ release without any influence on macrophage adherence. This result mimicked to some extent the effects of ES and SW. The lack of influence of BE on macrophage adherence is in line with the findings of Ortega et al. (1996). Furthermore, we have recently shown that the suppressive effect of BE on inflammatory cells in vitro paralleled its anti-inflammatory effect in vivo (Stanojević et al. 2006).

The second part of our study sought to determine if exposure to ES or SW paradigms (which exerted comparable effects on adherence, phagocytosis and H₂O₂ release from peritoneal macrophages) could differentially alter macrophage responses to subsequent in vitro treatment with CORT, NPY and BE 24 h later. Our results revealed that ES neutralised the diminishing effect of CORT on zymosan phagocytosis. Moreover, ES and SW counteracted the stimulatory effect of low concentration CORT on PMA-primed H₂O₂ release. It has been reported that chronic social stress induces a state of glucocorticoid resistance in stimulated macrophages (Quan et al. 2003). Interestingly, exposure to acute inescapable stress is sufficient to induce resistance of LPS-induced HPA axis activation to the suppressive effect of dexamethasone 24 h after shock termination (O'Connor et al. 2003), implying long term alterations in the response to glucocorticoids after a single stress episode. A long-lasting increase in the level of CORT coupled with a decrease in corticosteroid-binding globulin after acute stress exposure probably contribute to these observed effects (Fleshner et al. 1995b). Although our study could not establish the precise mechanisms implicated in the changes of reactivity of macrophages isolated from stressed rats to low dose CORT, it could be that acute stress exposure accompanied by a second stimulatory signal, such as zymosan or PMA in vitro, rendered macrophages less sensitive to in vitro treatment with CORT due to stress-induced changes at the level of glucocorticoid receptors or post-receptor signalling events.

Both ES and SW negatively regulated the effects of NPY on all three measured macrophage functions. In contrast, ES and SW only affected H₂O₂ release by BE. From our observations one could speculate that stress exposure readily produces a state of NPY “resistance” in macrophages. To the best of our knowledge, there is no previously available literature concerning stress-induced changes in the sensitivity to NPY and BE of cells within the immune system despite both peptides being involved in the modulation of the immune response during stress. It has been previously demonstrated that the basal rat plasma level of NPY (around 195 fM) increases (by approximately 32%) on handling and increases further during electric shock (Castagne et al. 1987). In addition, it has been reported that foot-shock stress induces a significantly greater increase in the level of BE (up to 800 fM in plasma) than a milder form of psychological stress (up to 300 fM) (Tsukada et al. 2001). Both peptides bind to specific receptors that are members of the seven transmembrane domain/G-protein coupled receptor family. However, significant differences in the downstream signal transduction mechanisms of BE (Shahabi et al. 2003) and NPY (Selbie et al. 1995) probably contribute to their differential sensitivity to the impact of the preceding stress.

During stress stimulation, neuroendocrine mediators released from central and peripheral sites make contact with peritoneal macrophages via the circulation and neural projections (Friedman and Irwin 1995). However, macrophages express all the necessary pathway components to regulate the synthesis, the processing and the release of BE (Mousa et al. 2004). Furthermore, stress significantly increases BE concentration in immune system cells (Sacerdote et al. 1994). It has also been suggested that NPY could be produced by activated macrophages, providing an important autocrine signal for the regulation of macrophage function (Wheway et al. 2005). Thus, stress mediators released from multiple sites could converge on macrophages and modify their functions. Therefore, the in vitro addition of peptides/hormones to macrophages that have already been functionally altered due to their previous exposure to increased levels of stress mediators 24 h earlier could interfere with macrophage activity at the level of membrane receptors or at the level of post-receptor signalling pathways.

In summary, our current study has clearly demonstrated that exposure to acute physical and psychological stressors results in similar effects and that in vitro treatment with CORT, NPY and BE exerts differential effects on macrophage function. Prior exposure to ES and SW affected macrophage responses to chemical mediators of stress responses, namely CORT, NPY and BE in vitro, 24 h later. It can be concluded that macrophages may participate in mechanisms supporting the phenomenon of stress-induced alterations of the immune system's response to subsequent stressors.

Acknowledgements

The technical assistance of Mrs Zuzana Tomaš is gratefully acknowledged. This work was supported by the Ministry of Science and Environmental Protection of the Republic of Serbia (project number 145049).