ToxSci Advance Access originally published online on May 28, 2003
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Toxicological Sciences 74, 325-334 (2003)
Copyright © 2003 by the Society of Toxicology
IMMUNOTOXICOLOGY |
Immune Changes during Acute Cold/Restraint Stress-Induced Inhibition of Host Resistance to Listeria
Laboratory of Clinical and Environmental Endocrinology and Immunology, Wadsworth Center, New York State Department of Health, Albany, New York 12201
Received January 22, 2003; accepted May 3, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiments were conducted to delineate the cellular changes modulated by acute cold/restraint stress (ACRS), a physical and psychological stressor, in response to a Listeria monocytogenes(LM) infection. In addition to wild type (WT) BALB/c mice, CD4-deficient (CD4–/–) BALB/c mice, which have no effective adaptive immunity, were used to determine the involvement of adaptive versus innate immunity. ACRS-induced suppression of host resistance to LM was not observed in CD4–/– mice, suggesting the involvement of CD4+T cells in the acute cold/restraint stress (ACRS)-induced inhibition. The in vivo splenic leukocyte phenotypes and activities of WT BALB/c mice after infection and in vitro lymphocyte responses to heat-killed LM (HKLM) also were examined. There were no significant differences in the numbers of splenic T and B lymphocytes, natural killer cells, macrophages, or neutrophils between nonstressed and ACRS-treated WT mice. However, higher levels of activated T cells and non-T lymphocytes were observed in the ACRS-treated mice; ß-adrenergic receptor (ß-ADR) antagonists (propranolol and atenolol) eliminated these elevated levels of activation, as well as the ACRS-induced suppression of host resistance. ACRS and control mice also had equivalent activation of macrophages. With in vitro HKLM stimulation, splenocytes from ACRS-treated mice produced significantly higher levels of IFN
and slightly higher levels of IL-6 in comparison with the nonstressed mice, although equivalent levels of lymphocyte proliferation were obtained. Additionally, ACRS-treated mice showed comparable elevation of serum nitric oxide after infection, indicating macrophage bactericidal activity similar to nonstressed mice. Thus, it appears that ACRS inhibits host resistance through regulatory CD4+ T cells and/or effector cell functions downstream of CD4+ T cell activation, as well as through ß-ADR signaling, in that blockage of these receptors appears to aid host defenses by means other than elevation of helper T cell activity. Because CD4 T cell deficiency and ß-ADR blockage produced equivalent effects, ß-ADR+ CD4+ T cells may have a negative role on host defenses after ACRS. Key Words: acute cold/restraint stress; host resistance; Listeria monocytogenes; CD4/mice; nitric oxide.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Physical and psychological stressors have been reported to modify multiple aspects of immune responses, which includes decreasing natural killer (NK) cell activity (Iwakabe et al., 1998
; Saperstein et al., 1992; Wu et al., 2000), suppressing macrophage phagocytosis (Lu et al., 1998) and release of nitric oxide (NO) and TNF
(Aarstad et al., 1991; Brown and Zwilling, 1994; Kizaki et al., 1996), altering responsiveness to IFN (Zwilling et al., 1992), inhibiting T cell proliferation, producing IL-2 and IFN (Iwakabe et al., 1998; Li et al., 1997), modulating MHC class II expression (Zwilling et al., 1990), affecting the type-1/type-2 T cell (Th1/Th2) balance (Iwakabe et al., 1998), and modulating antibody production (Fukui et al., 1997; Komori et al., 1996; Okimura et al., 1986). It is also well accepted that, as a consequence of the modulatory effects of stress on immune responses, physical or psychological stress can modulate host defenses against invading pathogens and can alter susceptibility to infectious diseases (Biondi et al., 1997; Yang and Glaser, 2000). Chemical stressors are often studied, but physical and psychological stressors also can substantially influence immune responses and need to be considered as critical environmental parameters.
Studies using chronic physical and psychological stress models have attempted to explore the underlying cellular and molecular mechanisms associated with stress-induced changes in host resistance. For example, daily 12-h restraint stress repeated over 7 days caused increased susceptibility to Listeria monocytogenes (LM) infection accompanied by multiple immunosuppressive effects, including inhibition of leukocyte migration into infected foci, suppression of LM-induced MHC class II expression, and down-regulation of IFN and IL-12, with concomitant up-regulation of IL-4 and IL-6 gene expression (Zhang et al., 1998). Therefore, decreased leukocyte infiltration, reduced antigen presentation, and dysregulated Th1/Th2 balance all appear to be involved in stress-impaired host resistance. Studies with repeated restraint stress and herpes simplex virus-1 (HSV-1) infection (Bonneau et al., 1991) demonstrated that suppression of NK cells and antigen-specific cytotoxic T cell function contributed to the chronic stress-induced susceptibility to HSV-1.
In contrast to the situation for chronic stress, there have been few reports on the effects of acute physical and psychological stressors on host resistance. Previously, others reported that inescapable tail shocks cause reduced inflammatory responses after injection of killed Escherichia coli (Deak et al., 1999). Acute restraint stress-induced leukocyte redistribution led to a significantly enhanced delayed-type hypersensitivity response due to locally accumulated lymphocytes (Dhabhar and McEwen, 1998). In our previous studies, acute cold/restraint stress (ACRS) for 1 h significantly suppressed host resistance to LM in BALB/c mice accompanied with greater inflammatory responses (Cao and Lawrence, 2002; Cao et al., 2002). This inhibition was mediated through the peripheral sympathetic nervous system (SNS) via ß1-adrenergic receptors (ADR) (Cao et al., 2003). Despite these studies, it is still uncertain how acute stress affects host resistance against infectious diseases at the cellular/molecular level. This is especially important to investigate because environmental physical and psychological stressors can have profound effects on health, and these stressors generally are greater in populations of lower socioeconomic status, in which there are increased health concerns.
Listeria monocytogenes is a gram-positive, facultative intracellular bacillus, widespread in nature (Mielke et al., 1997). Mouse LM infection is a well-established model for studying host resistance to infection (Finelli et al., 1999; North and Conlan, 1998). Primary immunity to LM involves both innate and adaptive immunity (mainly cell-mediated immunity) (Busch and Pamer, 1999; Finelli et al., 1999; North and Conlan, 1998; Unanue, 1997b). Innate immunity is critical for the early control of infection. Neutrophils accumulate quickly (increased within 24 h after infection) at infectious foci and phagocytose extracellular bacteria, along with tissue-fixed macrophages (e.g., Kupffer cells) and infiltrating monocytes/macrophages. Upon phagocytosis of bacteria, the macrophages become activated and produce a variety of cytokines (including pro-inflammatory cytokines IL-6, IL-1, and TNF), chemokines, and NO; activate NK cells (innate responses) via IL-12; and begin to activate CD4+ T cells (adaptive responses) through antigen presentation and IL-12. NK cells and differentiated Th1 cells are major sources of IFN, which can fully activate macrophages to become effective killers. In addition, Th1 cells can activate CD8+ T cells to become cytotoxic T cells and kill infected cells. Thus, CD4+ T cell activation is critical in the initiation of adaptive immune response; and macrophages, Th1, and cytotoxic T cells (mainly CD8+ T cells) are the major effector cells during host defense against LM.
Here, following our previous studies, we further investigated ACRS-induced inhibition of host resistance to LM in BALB/c mice at the cellular level and the involvement of ß-ADR. To evaluate the involvement of different cell components, we utilized CD4–/– mice, examined in vivo kinetic changes in splenic leukocyte phenotypes and serum NO levels, and assessed splenic cell proliferation and cytokine production in vitro.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mouse strains and ACRS.
Male BALB/cByJ mice were housed three to four mice per cage in a specified pathogen-free environment with food and water ad libitum. All mice were maintained on a 12-h light/dark cycle with lights on from 7 A.M. to 7 P.M. Animals were allowed at least 1 week of habituation in the animal room before use in experiments. CD4–/– BALB/c mice were bred in the Wadsworth Center animal facility using breeding pairs obtained from Dr. William T. Lee (Wadsworth Center) and were maintained as described above. Tail DNA PCR was performed to verify the targeted mutation by using primers specific to the neomycin cassette used to disrupt IL-4 gene, following protocols from Jackson Laboratory (Bar Harbor, ME). Mice also were randomly tested for CD4+ T cells by flow cytometry, as described later in this section.
For ACRS, as described previously (Cao and Lawrence, 2002; Cao et al., 2002), 10-week-old mice were individually restrained in well-ventilated plastic 60-ml syringes (Sherwood Medical Company, St. Louis, MO) at 4°C for 1 h. Mice can move forward and backward in the syringe but cannot turn head to tail. The ACRS was always performed between 10 A.M. and noon. ACRS is considered a physical and psychological stressor. Control mice were left in their original cages undisturbed during the same time period. The day at which mice were given ACRS was considered day 0 for all experiments. All animal procedures were approved by the IACUC of the Wadsworth Center (protocol 00-278).
Drug administrations.
Both ß-ADR antagonists, atenolol and propranolol, were purchased from Sigma (St. Louis, MO). Drugs were dissolved in sterile saline and injected ip according to individual body weight (BW). The nonselective ß-ADR antagonist propranolol was given at 20 mg/kg BW, and the selective ß1-ADR antagonist atenolol was given at 20 mg/kg BW. Either drug or vehicle control were injected into the experimental mice immediately before ACRS. The doses were chosen based on our previous studies (Cao et al., 2003), in which both drugs significantly eliminated the inhibition of host resistance by ACRS. In addition, both propranolol (cross-blood–brain barrier; BBB) and nadolol (not cross-BBB) were used in the previous study. No difference in host resistance (i.e., organ LM levels at day 3 after infection) was seen between these two drugs, indicating that the ACRS-induced inhibition of host resistance is mediated through peripheral but not central ß-ADRs. Thus, either drug can be used in this study. We chose propranolol here. No toxic effects were evident after any drug treatments.
LM infection and determination of viable LM.
Listeria monocytogenes was originally isolated from an LM meningitis patient and was passaged in mice for multiple (>9) generations before LM stocks were prepared, as described previously (Cao et al., 2002). Mice were intravenously injected with 5–6 x 103 colony-forming units (CFU) LM/mouse on day 0 immediately after ACRS. Mice were sacrificed at the indicated times postinfection. Upon sacrifice, blood was collected after CO2 anesthesia by cardiac puncture, and sera were obtained by centrifugation and kept at –20°C for later cytokine detection by enzyme-linked immunosorbent assay (ELISA) or NO determination. Spleens and livers were removed aseptically and homogenized for enumeration of viable LM, as described before (Cao and Lawrence, 2002; Cao et al., 2002). LM burdens in the organs are presented as viable LM per organ. Spleen homogenates were further centrifuged after addition of 1% NP-40 (Sigma), and the supernatants were kept at –20°C until cytokine analysis.
Analysis of splenic leukocyte phenotype by flow cytometry.
Following ACRS and/or infection as described above, at the indicated time, mice were anesthetized with CO2, and spleens were removed aseptically and weighed. Single-cell suspensions were prepared by grinding the tissue between two frosted microscope slides (Erie Scientific Company, Portsmouth, NH). Red blood cells were lysed with RBC-lysing buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.2–7.4). Cells were washed twice with PBS, then total cell numbers were determined with a Coulter Counter ZM (Coulter Corp., Miami, FL). Cells (2 x 106 per tube) were used for further staining. All monoclonal antibodies were purchased from PharMingen (San Diego, CA): purified antimouse CD16/CD32 Fc receptor, PerCP-antimouse CD3, PE-antimouse CD69, PE-antimouse CD19, PE-antimouse CD11b, FITC-antimouse CD8, FITC-antimouse panNK, and FITC-antimouse I-Ad. Briefly, cells were first treated with antimouse CD16/CD32 Fc receptor (1 µg/tube, in 100 µl staining buffer: 2% FBS and 0.09% NaN3 in PBS) for 15 min on ice to block Fc receptors, then stained with a combination of two or three antimouse monoclonal antibodies (1 µg/tube for each) for 30 min on ice. After washing twice with PBS, cells were analyzed by a FACScan flow cytometer with Cell Quest software (Becton Dickinson, San Jose, CA). The following combinations of antibodies were used: (tube 1) CD3, CD69, and CD8, with CD3+/CD8– representing CD4+ T cells, CD3+/CD8+ representing CD8+ T cells, and CD69+ (expressed on activated T, B, NK cells and macrophages) used to identify activation of the T cell subsets and CD3– cells; (tube 2) CD3, CD19, and panNK, with CD3-/CD19+ representing B cells and CD3–/panNK+ representing NK cells; and (tube 3) CD11b and I-Ad, with CD11b+/I-Ad+ cells representing macrophages, CD11b+/I-Ad– cells representing neutrophils, and the intensity of I-Ad on macrophages representing macrophage activation. Cell numbers of each cell population were calculated based on the total cell numbers and percentage of each cell population. Activation of T cells and of CD3– cells was expressed as the percentage of CD69+ cells within an indicated cell population.
In Vitro spleen cell culture and proliferation assay.
Spleen single-cell suspensions were prepared from mice immediately after nonstressed control or ACRS time period but without LM infection, as described in the preceding section. Heat-killed LM (HKLM) was prepared by heating living LM at 70°C for 1 h, and the number of colonies was determined by culturing of a small portion of living LM before heat inactivation as described. Unfractionated single-cell splenic leukocyte preparations (2 x 105/well/200 µl) were stimulated with HKLM at various concentrations (0, 105, 106, and 107 CFU/well) in complete RPMI1640 media for 72 h in a low-O2 incubator (37°C, 7% CO2, and 5% O2). During the last 6 h of incubation, cells were incubated with 0.5 µCi [3H]-thymidine (25 µl/well; Perkin Elmer, Boston, MA). After incubation, cells were harvested with a semiautomatic cell harvester (Skatron, AS, Norway), and radioactive signals were then analyzed with a FUJIX BAS2000 phosphorimager (Fuji, Minamiashigara City, Japan). The average of the photostimulating luminescence (PSL, manufacturer’s units for the intensity of radioactivity) minus background PSL (PSL-Bkg) of triplicate wells was calculated. The proliferation was expressed as the percentage of the average PSL-Bkg of treated wells, relative to the average PSL-Bkg of nonstimulated wells, which was set at 100%. In addition, similar spleen cell cultures were set up at the same time for cytokine analysis. Following incubation (72 h), the supernatants were collected after centrifugation and were stored at –20°C until quantification of cytokines by ELISA.
Cytokine ELISA.
IL-6 was determined by DuoSet ELISA development kit (R&D, Minneapolis, MN) according to manufacturer’s protocols. The sensitivity of the kit is 
3 pg/ml. IFN and IL-4 were detected with antibody pairs (Caltag, Burlingame, CA) following a modified protocol (Heo et al., 1996). Briefly, 96-well easy-wash EIA/RIA plates (Costar, Corning, NY) were coated with 2 µg/ml capture antibody (50 µl/well) in coating buffer (0.1 M NaHCO3, pH 8.2) and incubated at 4°C overnight. The next day, plates were washed with PBS/0.05% Tween 20, and nonspecific binding was blocked with 10% FBS/PBS (200 µl/well) for 2 h at room temperature. Then after washing, diluted samples and standards were loaded, and plates were incubated at 4° C overnight. On the third day, detection antibody was added at 1.5 µg/µl in 10% FBS/PBS (50 µl/well) after washing of the plates. Following 45 min incubation at room temperature, avidin-HRP (Sigma) was added (1:400, 50 µl/well), followed by 30 min incubation at room temperature. Next, plates were washed again, and TMB substrate (PharMingen) was added (50 µl/well). After 10 min development in the dark, 2 N H2SO4 stop solution was added (25 µl/well), and plates were read at 450 nm with an EL310 ELISA reader (Bio-Tek, Burlington, VT). IFN or IL-4 concentrations were calculated, based on the standard curve. The sensitivity of these assays is 75 pg/ml for IFN and 15 pg/ml for IL-4. Culture supernatant and serum cytokine levels were expressed in pg/ml, and splenic cytokines were normalized based on individual protein concentration (i.e., pg/mg protein), which is determined by BCA protein assay reagents (Pierce, Rockford, IL) following manufacturer’s protocol.
Serum NO determination.
Serum collected from nonstressed control and ACRS mice at various time points after LM infection were analyzed for NO content with an NOA280 nitric oxide analyzer (Sievers Instruments, Inc., Boulder, CO) following manufacturer’s operation manual. Serum samples were deproteinized by precipitating with cold absolute ethanol (sample/ethanol = 1:3 in volume). The supernatants were used for NO analysis. Nitrate standards were prepared in a range from 0 to 10 mM. The detection limit is 1 pM. The results were calculated from the standards and presented as µM of NO for each sample.
Statistical analysis.
For comparison of multiple groups, appropriate ANOVA was used initially. If a significant main effect or interaction was identified by the ANOVA analysis (p < 0.05), respective group means were compared using the Student-Newman-Keuls post hoc test; p < 0.05 was considered a significant difference.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Host Resistance to LM in CD4–/– Mice
To evaluate the involvement of innate versus adaptive immunity with regard to ACRS-induced suppression, we used CD4–/– BALB/c mice, which have about 90% of their peripheral T cells as CD8+ T cells and markedly decreased adaptive responses (Rahemtulla et al., 1991
). Based on our analysis of CD4–/– mouse spleens, over 80% of the T cells were CD8+, and there were normal amounts of B cells, NK cells, macrophages, and neutrophils (data not shown). As reported previously, in the wild type (WT) mice, the ACRS treatment resulted in significantly (p < 0.01) higher numbers of viable LM in both spleen and liver, which was prevented by propranolol (Fig. 1). However, similar to SCID mice (Cao et al., 2003), no ACRS-induced increase of LM burden was observed in CD4–/– mice. Because blockage of ß-ADRs has been reported to lessen ACRS-induced suppression of host resistance to LM, propranolol was used to determine whether it would be able to lessen the suppression further in CD4–/– mice. There was no difference in host resistance among vehicle-treated and propranolol-pretreated CD4–/– mice, with or without ACRS. Sickness behavior parameters (BW and food/water intake) were monitored after infection as an index of the differential kinetic changes between WT and CD4–/– mice. Unlike the WT mice, the CD4–/– mice, with or without propranolol, didn’t show any significant weight loss or decrease in food/water intake (data not shown). Thus, the day 3 LM burden correlates well with the measures of sickness behavior, as previously reported (Cao and Lawrence, 2002). Interestingly, all CD4–/– mouse groups had significantly lower LM burdens than did the WT BALB/c mice.
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Splenic Cellular Changes after ACRS/LM Infection
To assess the effects of ACRS on immune cell components during LM infection in vivo, we analyzed the splenic leukocyte populations and their activation. One nonstressed control group and one ACRS group of mice were sacrificed immediately after the ACRS period without infection and served for baseline values (day 0). All other groups of mice were infected immediately after the ACRS period and were assayed on days 1–3 after infection. Although splenic weights and total splenic leukocytes increased with time after infection, no significant differences in these measurements were observed between the nonstressed and ACRS-treated mice (Fig. 2
). Also, all subpopulations of leukocytes, including total T cells, CD4+ and CD8+ T cells, B cells, NK cells, macrophages, and neutrophils substantially increased after infection (Fig. 3). However, there were no significant differences in the numbers of any of these subpopulations between nonstressed and ACRS-treated mice (Fig. 3). The activation of leukocytes also was evaluated based on the expression of the early activation marker CD69 or the intensity of I-Ad staining (activated macrophages) (Fig. 4). In both nonstressed and ACRS-treated mice, CD69 expression and I-Ad intensity were significantly increased after infection. CD69 expression peaked at day 2 postinfection and slightly decreased by day 3, whereas I-Ad intensity showed a continuous elevation after the infection. The levels of CD69 expression, which were most evident at days 1 and 2 postinfection, were significantly higher on CD4+and CD8+ T cells and on non-T cells (mainly B cells) from ACRS-treated mice, compared with nonstressed mice (Figs. 4A–4C). Similar I-Ad intensities on macrophages were observed with or without ACRS (Fig. 4D).
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Because ß-ADR blockage had been shown to prevent ACRS-induced inhibition of host resistance to LM in vivo (Cao et al., 2003
), mice were pretreated with saline, propranolol, or atenolol to assess their effects on the cellular responses to infection after ACRS. Neither propranolol nor atenolol affected splenic weights, total leukocyte numbers, or numbers of the leukocyte subpopulations, compared with saline-treated nonstressed or ACRS-treated mice (data not shown). However, although the percentages of the activated lymphocyte and macrophage populations increased similarly over time (Fig. 4
), both propranolol and atenolol significantly prevented the higher level of lymphocyte activation in ACRS-treated WT mice at day 1 after infection (Fig. 5).
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In Vitro Splenic Cellular Responses to HKLM
Because activation (indicated by expression of CD69) does not necessarily represent T cell proliferation or differentiation into effectors, in order to evaluate whether ACRS can affect T cell function in response to LM, we assessed in vitro splenic cellular responses to HKLM. Naive spleen cells from nonstressed and ACRS-treated mice were stimulated with various concentrations of HKLM, and cell proliferation and cytokine production were determined after 72 h. There was no significant difference in spleen cell proliferation between nonstressed and ACRS-treated mice (Fig. 6A
). IL-6 levels were greater in the culture supernatants of cells from ACRS-treated mice, although the difference was not statistically significant (Fig. 6B). IFN production by cells from ACRS-treated mice was significantly greater (Fig. 6C p, < 0.05). The increased secretion of cytokines in vitro is consistent with the elevated activation of lymphocytes in vivo, and these results confirm that there was no ACRS-induced deficiency of T cell activation, proliferation, or IFN production. Supernatant IL-4 levels were undetectable.
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Serum NO after ACRS and/or LM Infection
To evaluate the bactericidal activity of macrophages during the LM infection after ACRS, we determined the NO levels in serum at various time points (7 h or 1, 2, 3, 5, 7, or 9 days) after LM infection in both the nonstressed and ACRS-treated mice (Fig. 7
). In both groups, the NO levels were elevated after infection and peaked at day 3 postinfection. The NO levels of the ACRS-treated mice were slightly but not significantly higher than those of the nonstressed controls, indicating a comparable bactericidal activity of macrophages with regard to NO production, which is consistent with the equivalent activation of macrophages (Fig. 4D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study was conducted to delineate the cellular components involved in ACRS-induced inhibition of host resistance to LM in BALB/c mice and those modified by ß-ADR blockage, which enhances host resistance (Cao et al., 2003
). The general host defense responses are outlined in Figure 8, as described in the introduction. Because CD4+ T cell activation is crucial for effective host defense, we used CD4-/– mice, which have severely impaired adaptive immunity (Rahemtulla et al., 1991), to evaluate initially the involvement of innate versus adaptive immunity, particularly CD4+ T cells, in the ACRS-induced inhibition. ACRS-induced suppression of host resistance was not observed with the CD4–/– mice, which suggests at least two possibilities.
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First, the ACRS-induced inhibition may be primarily caused by ACRS-induced changes of innate immunity. However, the excessive numbers of CD8+ T cells in CD4–/– mice (Rahemtulla et al., 1991
) may provide extra protective effects strong enough to overcome the suppressive effects of ACRS on innate immunity. It is known that substantial bystander activation of naive CD8+ T cells can be induced by a primary LM infection (Busch et al., 2000), and adoptive transfer experiments have shown that even naive CD8+ T cells can provide significant protection from LM infection, although with delayed time kinetics (Bhardwaj et al., 1998). Within innate defenses against LM, NK cells mainly function as a source of IFN to activate macrophages, whereas neutrophils and macrophages are the major bactericidal cells (Unanue, 1997a) (Fig. 8, left side). Similar responses with regard to cell numbers (total neutrophil, NK, and macrophage numbers) and cell activation (macrophage MHC class II expression and serum NO levels) were detected with nonstressed and ACRS-treated mice after infection, indicating equivalent leukocyte infiltration, macrophage activation, and macrophage production of the bactericidal agent NO. Thus, these results do not support the proposal of suppressed innate immunity by ACRS. Nevertheless, the excessive numbers of CD8+ T cells may contribute to the observed significantly enhanced host resistance in the CD4–/– mice, compared with the WT mice. On the other hand, the absence of ACRS-induced suppression in CD4–/– mice may suggest that CD4+ T cells themselves or other immune responses affected by activated CD4+ T cells are inhibitory under ACRS conditions. T cell, especially helper T cell, functions were then further investigated. No propranolol-induced changes were observed with CD4–/– mice, suggesting that this ß-ADR blocker may act either directly or indirectly on CD4+ T cells or on a downstream immune component following CD4+ T cell activation. These accumulative findings suggest a negative or at least nonpositive role for CD4+ T cells. CD4+ T cells appear to be required for inhibition of host resistance by ACRS, possibly via inhibition of effector CD8+ T cells. Alternatively, the ACRS-induced suppression may be dependent on the CD4/CD8 ratio or the absolute number of CD8+ T cells throughout the host, in that host resistance to LM is suppressed when the numbers of CD8+ T cells are low (WT mice) but not when they are elevated (CD4–/– mice).
To evaluate helper T cell functions further (Fig. 8, right side), we examined the CD4+ T cell phenotype after infection in vivo and the in vitro HKLM-stimulated splenocyte proliferation and cytokine production by cells from nonstressed and ACRS mice. Equal expansion and enhanced activation (represented by CD69 expression) in vivo were observed in ACRS mice. In agreement, splenocyte proliferation and elevated IFN production were observed in vitro with cells from ACRS mice. Altogether, these indicate that CD4+ T cells from ACRS mice can be activated, can proliferate, and can together with NK cells release reasonable amounts of IFN to activate macrophages further. Therefore, lack of activation of helper T cells and/or lack of IFN do not appear to contribute to the ACRS-inhibited host resistance. In addition, the ß-ADR antagonists propranolol and atenolol blocked enhancement of CD4+ T cell activation in ACRS mice, consistent with the idea that the SNS is responsible for the ACRS-impaired host resistance via ß1-ADRs (Cao et al., 2002, 2003). The association between inhibited host resistance and increased CD4+ T cell activation possibly could be due to: (1) ACRS-induced preferential development of Th2 cells, leading to predominant humoral responses and inhibition of the cell-mediated responses needed to defend against LM infection or (2) regulatory/suppressor T cell (CD4+CD25+ T cells) activity (Thornton and Shevach, 1998) enhanced by ACRS. However, either of these possibilities requires further direct investigation.
In terms of the major effector cells, macrophages, and cytotoxic T cells (effectors downstream of CD4+ [helper] T cell activation, Fig. 8), there was similar CD8+ T cell expansion, higher activation of CD8+ T cells, no reduction of MHC class II expression on splenic macrophages, and no suppression of NO production in ACRS mice, in comparison with the nonstressed mice. These outcomes suggest normal activation of macrophages and cytotoxic CD8+ T cells, and comparable NO-induced bactericidal activity by macrophages. In fact, both CD8+ T cell activation and serum NO levels were higher in ACRS mice, correlating with the enhanced CD4+ T cell activation discussed earlier. Thus, these studies have detected no significant dysfunction in either of these effector cells under ACRS conditions. ACRS-induced inhibition of host resistance does not seem to be due to qualitative depression of cell-mediated defenses.
Use of CD8–/– mice or cytotoxic T cell activity assays will help to evaluate further the cytotoxic T cell function in ACRS-induced changes of host resistance, but other possible defects in bacteria killing also need to be considered. Studies using MHC class I-deficient mice demonstrated that MHC class I peptide recognition is critical for cytotoxic T cell-specific killing of infected cells (Ladel et al., 1994; Seaman et al., 1999). Interestingly, MHC class I has also been associated with nonimmune functions, such as central nervous system development (Corriveau et al., 1998). Moreover, MHC class I exhibited molecular associations with several hormone receptors, including insulin receptors, epidermal growth factor receptors, ß-ADRs, and muscarinic cholinergic receptors. Antibodies directed against MHC class I could stimulate ß-ADRs and could induce the downstream intracellular secondary messengers, resulting in physiological changes in the targeted cells (Gremaschi and Sterin-Borda, 1994). Therefore, we speculate that ACRS-induced activation of ß-ADRs could affect MHC class I expression on LM infected cells, and lack of effective targeting on infected cells could lead to an inefficient elimination of infected bacteria by cytotoxic T cells and, thus, a higher LM burden in ACRS mice. However, with this scenario it is not obvious why CD4+ T cells would be needed to observe the ACRS-induced inhibition of host resistance.
An alternative scenario could be that there is increased growth of LM in response to the stress hormone NE, leading to the higher LM burdens in ACRS mice. Certain bacteria, such as E. coli, have been reported to respond to NE, showing an enhanced growth (Freestone et al., 2000; Lyte and Ernst, 1992). However, our previous study showed similar NE levels in spleen and liver in nonstressed and ACRS mice (Cao et al., 2002), and other studies have shown that NE has no effects on LM growth rate in vitro (Rice et al., 2001). Furthermore, it is not obvious why such a direct effect would occur only between days 2 and 3 after infection (Cao and Lawrence, 2002).
In summary, ACRS inhibition of host resistance does not seem to be due to suppressive effects on innate immunity, inhibition of the bactericidal activity of macrophages, lack of T cell activation, or inhibited IFN production. However, the suppression of host resistance by ACRS is strongly associated with the presence of CD4+ T cells. The major defense pathway following CD4+ T cell activation needs to be fully investigated in order to uncover the mechanisms by which ACRS inhibits host resistance. It is most interesting that ß-ADR antagonists (in WT mice) and absence of CD4+ T cells eliminated the suppressive effects of ACRS on host resistance to LM. Furthermore, blockage of ß-ADR signaling lowered the number of LM-activated (CD69+) lymphocytes in nonstressed and ACRS-treated mice while improving host resistance. Clearly, activated lymphocytes are needed to defend against infection, suggesting that the mechanisms responsible for the ACRS-induced suppression must involve regulatory mechanisms still unresolved; however, CD4+ T cells and ß-ADRs are implicated.
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ACKNOWLEDGMENTS |
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We are grateful to Dr. William Lee for kindly providing the CD4–/– mice. We also thank the Molecular Genetics Core facility of the Wadsworth Center for genotyping of the CD4–/– mice and the Immunology Core of the Wadsworth Center, particularly Joan Pederson-Lane, for helping with the flow cytometric analysis of spleen cells and for use of the phosphorimager.
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NOTES |
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1 Present address: Department of Psychiatry, University of Rochester, P. O. Box PSYCH, 300 Crittenden Blvd., Rochester, NY 14642.
2 Present address: Department of Medicine, SUNY Upstate Medical School, Syracuse, NY 13210.
3 To whom correspondence should be addressed at Wadsworth Center, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, NY 12201-0509. Fax: (518) 474-1412. E-mail: david.lawrence{at}wadsworth.org.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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