1 Department of Biology, College of Dentistry, 2 Department of Molecular Virology, Immunology, and Medical Genetics, College of Medicine, 3 Institute of Behavioural Medicine Research, Ohio State University, 4179 Postle Hall, 305 West 12th Avenue, Columbus, OH 43210, USA
Correspondence to: F. S. Dhabhar; E-mail: dhabhar.1{at}osu.edu
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Abstract |
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Keywords: evolution, hormone, Langerhans cell, leukocyte trafficking, memory lymphocyte, psychophysiological stress, skin diseases, surveillance, survival
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Introduction |
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Although the word stress generally has negative connotations, stress is a familiar aspect of life, being a stimulant for some, but a burden for others. Stress is defined as a constellation of events, comprised of a stimulus (stressor), that precipitates a reaction in the brain (stress perception), that activates physiologic fight-or-flight systems in the body (stress response) (1). It is often overlooked that a stress response has salubrious adaptive effects in the short run (1, 35), although stress can be harmful when it is long lasting (1, 68). An important distinguishing characteristic of stress is its duration. We define acute stress as stress that lasts for a period of minutes to hours, and chronic stress as stress that persists for several hours a day for weeks, months or years (1). We have previously studied the immunosuppressive effects of chronic stress, as have numerous other investigators (1). Here we examine the immunoenhancing effects of acute stress on the sensitization or immunization phase of cell-mediated immunity (CMI).
2,4-Dinitro-1-fluorobenzene (DNFB)-induced CMI, also known as contact hypersensitivity, is a well-characterized immune response (911). Following first time exposure to DNFB (immunization/sensitization phase), epidermal dendritic cells (DCs), chiefly Langerhans cells, help generate an antigen-specific repertoire of CD4+ Th cells and CD8+ CTLs in draining lymph nodes (LNs). T cells and macrophages are critical for antigen elimination: CD4 cells produce cytokines necessary for accessory and effector cell recruitment and activation (12) and CD8 cells induce keratinoyte apoptosis (13), using either the perforin or the Fas/FasL pathway or both for their lytic activity (14). Macrophages are crucial for removing hapten-modified cells and debris and for setting the stage for restoration of tissue integrity. T lymphocytes also generate memory cells that respond during future antigen exposures. Thus, the induction phase is critical because of its inherent capacity to generate immediate effector and subsequent memory cohorts of antigen-specific lymphocytes.
The role of DCs, keratinocytes and macrophages in the initiation of an immune response to DNFB is well documented (11, 15). DNFB-carrier conjugates are internalized by antigen-presenting cells (APCs), chiefly DCs and macrophages, that migrate from the epidermis to the dermis and on to draining LNs where they present the antigen to naive T lymphocytes. Effective antigen presentation in the LNs leads to clonal expansion of affinity-matured lymphocytes. In addition, DCs, keratinocytes and endothelial cells further promote the immune response by producing chemokines and cytokines that recruit and activate leukocytes at the site of antigen exposure (15, 16).
We have previously shown that an acute stress-induced enhancement of skin CMI is eliminated following adrenalectomy and restored by acute administration of physiological stress levels of corticosterone and epinephrine, indicating that the immunoenhancing effects of stress are mediated by these principal stress hormones (13, 17, 18). In contrast, we showed that synthetic glucocorticoid hormones like dexamethasone are potently immunosuppressive as would be expected given their clinically well-known anti-inflammatory effects (2).
Although the mechanisms mediating the immune response to haptens like DNFB are well established and the endocrine mechanisms mediating a stress-induced immunoenhancement are known, little is known about the immunologic mechanisms by which acute stress enhances skin CMI. Here we elucidate the effects of stress on five important aspects of the immunological cascades that mediate the immunization or sensitization phase of CMI: (i) changes in chemokine and cytokine gene expression, (ii) DC maturation and trafficking in skin and LNs, (iii) macrophage infiltration and activation, (iv) T cell activation in LNs and (v) T cell recruitment to skin. Acute stress was administered just once at the time of antigen exposure. The effects of stress were examined 6 and 24 h after antigen exposure as these time points represent important early and late events during sensitization (10). Results suggest that acute stress increases the efficacy of critical processes that occur during a primary immune response. This may in turn increase immunologic memory formation resulting in subsequent immunoenhancement at the time of secondary antigen exposure.
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Methods |
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Experimental design
Three groups (n = 10 per group) of mice were used in each experiment for each time point. The first group was acutely stressed (STR) for 2.5 h while the second group remained in home cages as the non-stressed (NS) control. The NS and STR groups were both exposed to antigen. The third group served as the antigen-naive control (NAIVE).
Restraint stress paradigm
Acute stress was administered by placing animals (without squeezing or compression) in well-ventilated wire mesh restrainers for a single session of 2.5 h beginning at 9:00 a.m. (lights in the animal room were turned on at 6:00 a.m.). This procedure mimics stress that is largely psychological in nature because of the perception of confinement on the part of the animals (19, 20). The psychological component of restraint stress is thought to arise from it mimicking a collapsed tunnel that is intrinsically stressful for these burrow-dwelling animals (21). It has also been suggested that restraint stress may have a neurogenic component due to forced positioning (22). This stressor activates the sympathetic nervous system (23) and the hypothalamicpituitaryadrenal (HPA) axis (24, 25) and results in the activation of adrenal steroid receptors in tissues throughout the body (24, 25). Antigen was administered at the end of restraint (11:30 a.m.).
Induction of CMI
Animals were sensitized with DNFB (Sigma Aldrich Co., St Louis, MO, USA). Prior to sensitization, baseline pinna thickness was recorded using a constant-loading dial micrometer (Mitutoyo, Tokyo, Japan). On the day of sensitization, 15 µl of DNFB [0.5% (w/v) in 4 : 1, acetone : olive oil] was applied on the dorsal aspect of the pinnae. Pinna thickness was measured at 6 and 24 h after DNFB exposure. Naive animals were exposed to vehicle alone (4 : 1, acetone : olive oil). Following each measurement, groups of animals were euthanized (via CO2 inhalation), and pinnae and draining cervical LNs were collected. Each animal's right and left pinnae were pooled for immediate preparation of epidermal and dermal cell suspensions. Similarly, the right and left LNs were pooled for immediate preparation of LN cell suspension. In another identical experiment, pinnae were halved and frozen for real-time PCR and histological analyses.
RNA isolation and cDNA synthesis
Chemokine and cytokine mRNA analysis was performed using semi-quantitative PCR. Briefly, total RNA from frozen pinnae was extracted by homogenization in Trizol (Invitrogen, Carlsbad, CA, USA). mRNA was then selected from 20 µg of total RNA from each sample using Dynabeads® Oligo(dT)25 (Dynal Biotech, Lake Success, NY, USA). Reverse transcription of mRNA was carried out at 42°C for 60 min, 90°C for 5 min in a total volume of 25 µl using AMV Reverse Transcriptase Reaction Buffer (Promega, Madison, WI, USA), 1 mM of each deoxynucleoside triphosphate (Invitrogen), 25 U RNase Inhibitor (Roche Applied Science, Indianapolis, IN, USA), 0.6 µg Oligo(dT)12-18 primer (Invitrogen) and 15 U reverse transcriptase (Promega).
Primer and probe design and real-time PCR
Sequences from GenBank were used in the Primer Express 1.5a software (Applied Biosystems, Foster City, CA, USA) to design primers and probes. Probes were labeled at the 5' end with FAM and at the 3' end with TAMRA (Biosearch Technologies, Novato, CA, USA). Primers were synthesized by Invitrogen. TaqMan® Rodent GAPDH Control Reagents were obtained from Applied Biosystems. The ABI Prism 7500 Sequence Detection System (Applied Biosystems) was used to quantify gene expression. Threshold cycles (Ct value) were determined using the 7500 Sequence Detection software.
Preparation of epidermal and dermal cell suspensions
Epidermal and dermal suspensions were prepared as previously described by Glade et al. (26). Mice pinnae were collected in HBSS (Invitrogen) on ice. The dorsal and ventral halves were pulled apart using fine forceps. The dorsal (DNFB-exposed) halves were incubated (overnight, 4°C) in PBS containing 0.5 mg ml1 thermolysin and 10 mM HEPES (Sigma Aldrich Co.). Following incubation, epidermis and dermis were carefully peeled away from one another. To disperse the cells further, epidermis and dermis were incubated (20 min, 37°C) separately in PBS containing 0.25 mg ml1 trypsin + 3.0 mg ml1 dithioerythritol (Sigma Aldrich Co.). Cells were then washed with HBSS containing 20% FBS and were passed through a 70-µm sieve to obtain a single-cell suspension. Total cell counts were determined using a hematology analyzer (Hemavet, Oxford, CT, USA) and viable cell counts were confirmed via trypan blue exclusion (Sigma Aldrich Co.).
Preparation of draining LN cell suspensions
Cervical LNs were harvested, weighed and stored immediately in HBSS (Invitrogen) on ice. A single-cell suspension was prepared by squeezing the LNs between frosted ends of microscope glass slides (Fisher Scientific, Pittsburgh, PA, USA). Cells were then filtered through a 70-µm nylon sieve into HBSS. The cellular suspension thus obtained was washed twice and re-suspended in HBSS. Cell numbers were then counted on a hematology analyzer (Hemavet) and stained for flow cytometric analysis.
Flow cytometry
Specific leukocyte subtypes were measured by immunofluorescent antibody staining and analyzed using multicolor flow cytometry (FACSCalibur; Becton Dickinson, San Jose, CA, USA). Epidermal, dermal and LN cell suspensions were incubated with BD Fc BlockTM (clone: 2.4G2; Becton DickinsonPharMingen, San Diego, CA, USA) for 15 min on ice to inhibit non-specific binding. Cells were then incubated with specific mAbs for 30 min at room temperature, washed with PBS and analyzed on the FACSCalibur. Each panel consisted of either single or multiple antibodies. Directly conjugated rat anti-mouse CD3 (clone: 145-2C11), CD4 (RM4-5), CD8 (53-6.7), Ly6G (1A8), CD11a (2D7), CD11b (M1/70), CD11c (HL3), CD28 (37.51), CD44 (IM7), CD54 (3E2), CD62L (MEL-14), CD71 (C2), CD162 (2PH1) and IAb (AF6-120.1) (Becton DickinsonPharMingen) and F4/80 (clone: CI:A3-1; Caltag Laboratories, Burlingame, CA, USA) were used. Approximately 3000 events were counted from the epidermal and dermal cellular preparation, and 10 000 from the LN cellular preparation. Control samples matched for each fluorochrome and each antibody isotype were used to set negative staining criteria. Data were analyzed using CellQuest software (Becton Dickinson). Keratinocytes were identified as described by Tani et al. (27) and were subtracted from the neutrophil, monocyte and lymphocyte gates. DCs were identified as CD11c+ cells, neutrophils were identified as CD11b+Ly6G+ cells and macrophages/monocytes were identified as CD11b+F4/80+ cells and by their respective forward versus side scatter patterns.
Hematoxylin and eosin staining
Hematoxylin (Sigma Aldrich Co.) and eosin (Fisher Diagnostics, Middletown, VA, USA) staining was performed on 10-µm tissue sections from frozen pinnae. Photomicrographs of representative sections were obtained at a constant magnification (x40).
Data analysis and statistics
Each experiment was performed twice. Figures and tables represent data from one representative experiment. Data are expressed as means ± SEM. Since data were normally distributed, differences between groups and time points were analyzed using Student's t-test. Means that differed significantly are indicated by symbols that are defined in the figure legends. A computer statistics package was used for statistical analyses (Statview; SAS Institute Inc., Cary, NC, USA).
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Results |
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Acute stress increases the accumulation of macrophages in antigen-exposed skin and draining LNs
We examined total and activated (IAbhi) macrophages in skin and draining LN. Compared with NS animals, STR animals showed higher numbers of total macrophages in the epidermis at 6 h and in the dermis and LNs at 24 h (Table 2). With respect to activated macrophages, STR animals showed significantly higher numbers in the epidermis and dermis at 6 h and in the LNs at 24 h. These results show that acute stress enhances macrophage migration and activation in the skin and increases macrophage infiltration into draining LNs following primary antigen exposure.
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Acute stress enhances T cell migration into inflamed skin
Table 3 also shows the effects of acute stress on CD162+ T cells at the site of cutaneous antigen exposure. There was no difference in CD162+ T cell numbers between NS and STR animals at 6 h. However, STR animals showed significantly higher numbers of CD162+ Th cells and CTLs in the dermis at 24 h. These results suggest that acute stress increases the number of skin-homing T cells infiltrating the site of primary antigen exposure.
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Discussion |
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Contrary to the popular notion that stress is necessarily immunosuppressive, we initially suggested and showed that acute or naturalistic stressors have novel adjuvant-like immunoenhancing effects while chronic stress is immunosuppressive (2, 3, 18, 40). While numerous studies have examined the immunosuppressive effects of stress, few studies have elucidated the mechanisms mediating stress-induced immunoenhancement. Elucidation of such mechanisms is critical because stress-induced immunoenhancement has important clinical consequences. Such enhancement is likely to be beneficial if the augmented immune response is directed towards an infectious agent, vaccinating antigens or towards healing a wound. However, stress-induced immunoenhancement is likely to be harmful if directed against innocuous or self-antigens (e.g. hypersensitivity or autoimmune diseases) or against inflamed tissue (e.g. endothelial plaques in cardiovascular disease or gum tissue in gingivitis).
We have previously elucidated the endocrine mechanisms mediating a stress-induced enhancement of skin immune function (2). The studies described here identify the immunological mechanisms mediating these novel adjuvant effects of the psychophysiological stress response. Stress increases the efficacy with which professional APCs mature and migrate from the cutaneous site of antigen exposure to draining LNs. This results in greater activation of T cells within the LNs. As a result, acute stress may also increase the generation of antigen-specific memory T cells to induce long-term immunoenhancement. Understanding and further elucidating these mechanisms is important and likely to be clinically beneficial in two ways. First, physiological factors like stress hormones could be used to therapeutically manipulate specific immunological mediators and increase immunoprotection during infection, wound healing, cancer or vaccination. Second, these mediators may also be manipulated to dampen or eliminate stress-induced exacerbation of inflammatory and autoimmune diseases.
A stress-induced enhancement of skin immune function makes sense when viewed from an evolutionary perspective. An acute stress response is an evolutionarily adaptive psychophysiological survival mechanism (18, 41, 42). One primary function of the brain is to perceive stressors, warn of danger and promote survival. Stress-responsive neurotransmitters and hormones are the brain's signals to the body. Since stressful natural encounters often result in wounding and infection, it is unlikely that eons of evolution would select for a system exquisitely designed to escape the jaws and claws of a lion to only have it eaten inside out by bacteria. It has also been hypothesized that the adaptive value of a stress-induced immunosuppression is that it may decrease the development of autoimmunity following tissue damage. It is well established that an inflammatory stress-driven activation of the HPA axis, which results in systemic glucocorticoid secretion, is responsible for an endogenous glucocorticoid-induced suppression of autoimmune reactions (43, 44). In contrast, the results described here suggest that stress experienced at the time of antigen exposure promotes an antigen-specific immune response. It is possible that these differential effects may be due to differences in glucocorticoid sensitivity or receptivity of the immune response that may depend on the phase (early versus late) of the response. At the beginning of an immune response, factors such as leukocyte trafficking, antigen presentation, helper T cell function, leukocyte proliferation, cytokine and chemokine function and effector cell function may be receptive to stress hormone-mediated immunoenhancement. In contrast, at a later stage, these components may be more receptive to immunosuppression. Studies examining the effects of corticosterone on T lymphocyte proliferation in vitro support this hypothesis (45). These studies have shown that during the early stages of T cell activation, low levels of corticosterone potently enhance anti-TCR-induced lymphocyte proliferation. However, during later stages of culture, the same levels of corticosterone suppress T lymphocyte proliferation (45). Further in vivo studies are needed to test this hypothesis.
Rapid recruitment of leukocytes to a site of immunization is critical for immunoprotection. Acute stress induced a significant increase in pinna thickness at 6 and 24 h following primary DNFB exposure. Histological analyses of cellular infiltration revealed the presence of neutrophils, macrophages and lymphocytes. Antigen-exposed skin of STR animals showed dense leukocyte infiltration, which was absent in NS animals. Similarly, the draining LNs of STR animals showed a significant increase in weight and cellularity indicating a heightened immune response. Tissue myeloperoxidase levels (data not shown) were not different between NS and STR animals suggesting that neutrophils, though present, were unaffected by stress in this model. We believe that neutrophils may play a role in stress-induced immunoenhancement in other situations, especially those involving wound healing and phagocytic function.
Acute stress also significantly up-regulated MCP-1, IL-6 and IFN- gene expression at 6 h and MIP-3
, MCP-1, IL-6, TNF-
, IL-1
and IL-1ß at 24 h in antigen-exposed skin. The induction phase of a CMI response is propelled by these chemokines and cytokines produced by resident keratinocytes, Langerhans cells and tissue macrophages and infiltrating neutrophils, macrophages, DCs and T lymphocytes. These chemokines and cytokines are critical mediators of the immunization phase of CMI. They up-regulate MHC class II on APCs and thus increase the efficiency of antigen presentation (46). They also trigger adhesion molecule function and expression on endothelial cells and increase leukocyte extravasation while also activating newly recruited leukocytes (47). Thus, greater numbers of leukocytes may have migrated in the STR animals in response to increases in the expression of MIP-3
, MCP-1, TNF-
, IL-1
and IL-1ß which would result in greater DC recruitment and activation and increases in MCP-1, IL-6, TNF-
and IFN-
which would result in greater macrophage and T cell recruitment and activation (4850). Taken together, these results suggest that acute stress-induced enhancement of chemokine and cytokine gene expression following immunization may be an important mechanism contributing to the enhancement of skin CMI.
Interestingly, in contrast to the enhancing effects of stress on pro-inflammatory cytokines and the Type 1-cytokine, IFN-, we did not observe an effect of stress on the Type 2-cytokine, IL-4. The lack of differences in IL-4 gene expression in the context of a significant stress-induced increase in IFN-
gene expression suggests that in the context of skin CMI reactions, acute stress may shift the Type 1Type 2 cytokine balance in favor of Type 1 cytokines that contribute to the enhancement of CMI. We previously identified IFN-
, which is a potent stimulator of macrophages, as a critical local mediator of stress-induced enhancement of the challenge or recall phase of CMI (18). Here, we demonstrate that IFN-
gene expression is enhanced by acute stress as early as 6 h after primary antigen exposure. Given this early time point during a primary immune response, it is likely that DCs or NK cells rather than activated T cells may be the source of IFN-
. These observations regarding changes in gene expression need to be further confirmed by examining corresponding changes in protein levels, which we hope to analyze in future studies.
In contrast to these results, numerous studies have shown that stress and glucocorticoid hormones induce a shift from Type 1 to Type 2 immune responses (5153). One explanation for the differences between findings may be that most studies showing a stress-induced Type 2 cytokine bias involve chronic stressors or in vitro manipulations using glucocorticoid hormones, whereas the studies described here are conducted in vivo and specifically examine the effects of acute stress (which results in endogenous effects of catecholamine and glucocorticoid hormones) experienced at the time of primary antigen exposure. It is important to bear in mind that most in vivo immune responses are a combination of innate and adaptive, cellular and humoral, Type 1 and Type 2 cytokine-driven mechanisms. We hypothesize that acute stress experienced at the time of antigen exposure can promote either a Type 1- or a Type 2-biased immune response, with the response-initiating APCs and/or antigen being critical for determining the bias of the response.
These studies also elucidated the effects of acute stress on APCs by examining the kinetics of DC maturation and migration and macrophage recruitment and activation. Cutaneous DCs are a heterogenous population whose recruitment, maturation and emigration involve dynamic processes (54, 55). At 24 h, STR animals showed higher numbers of mature DCs in the dermis and draining LNs. Our data are in agreement with a recent finding showing enhanced accumulation of hapten-carrying DCs in draining LNs following acute stress (56). Macrophages are pivotal cells for CMI (57) because they function as important APCs as well as potent effector cells (58). At 6 h, we observed a stress-induced increase in numbers of IAbhi macrophages in DNFB-exposed skin. At 24 h, this increase was also observed in LNs where macrophages are known to engage in antigen presentation (59). Thus, our results indicate that stress enhances macrophage infiltration, activation and potential antigen-presenting capability, all of which may contribute to a more robust primary immune response.
A local reaction to antigens launches the process of developing immunity (60). However, the timing of antigen localization to draining LNs by activated APCs is critical for activation of naive T cells (61). The observed stress-induced increase in activated DC and macrophage numbers in draining LNs corresponded with a significant increase in activated T cell numbers. Expression of CD62L, CD44, CD11a, CD28 and CD162 on T cells differentiates antigen-primed effector T cells from resting naive cells (49, 6265). At 24 h after antigen exposure, we observed a stress-induced increase in the numbers of CD62L, CD44+, CD11a+, CD28+ and CD162+ Th cells and CD44+, CD11a+ and CD162+ CTLs. This shows that acute stress enhanced T cell activation in draining LNs, which may be the result of stress-induced increases in DC and macrophage migration as discussed above.
An important finding revealed in this study is that acute psychological stress increases T cell surveillance in skin. Acute stress increased the numbers of skin infiltrating CD162+ CTLs and Th cells 24 h following antigen exposure. Skin surveillance T cells express surface CD162, which enables skin homing (36, 37, 39). These findings suggest that acute stress may enhance immune surveillance during cutaneous immune activation. They also present a potential mechanism by which stress exacerbates skin inflammatory and autoimmune diseases like psoriasis (66, 67).
The studies described here support our initial novel finding that short-term or acute stressors have potent immunoenhancing effects. These studies are important because they are specifically designed to mimic the temporal relationship between stressor and antigen exposure that is often observed in nature and during many clinical situations. For example, when a gazelle is wounded during an escape from a lion, the gazelle is acutely stressed during the chase after which it is exposed to antigens and pathogens that enter its wounds. When a pediatrician or nurse approaches a toddler with a vaccine-filled syringe, the toddler remembers its prior painful needle stick and mounts a stress response before and during the process of being immunized. Adults may also mount a similar response. The stressors experienced during many such conditions are acute and proximal to the time of antigen exposure. Our results suggest that the physiological changes accompanying acute stress have adaptive immunoenhancing effects that are established early during an immune response and that it may be clinically beneficial to understand and harness these psychophysiological adjuvant mechanisms.
The effects of stress on immune function may be especially relevant during in vivo immune responses because stress is a ubiquitous fact of life and stress hormones are important components of an individual's psychophysiological milieu. Immune cells express receptors for, and respond to, these hormones (2, 42, 68). In vivo immune responses necessarily involve intricate interactions between both the innate and adaptive arms of the immune system. Interestingly, our experiments show that acute stress enhances recruitment and activation of critical molecular and cellular components of both innate (DCs and macrophages) and adaptive (T cells) immunity at the cutaneous site of antigen exposure and in draining LNs. These studies are in agreement with others that have shown acute stress-induced enhancement of immune responses (3, 18, 56, 6972).
The studies described here elucidate the immunological mechanisms through which acute stress enhances the immunization/sensitization phase of skin CMI. Taken together with our findings showing stress-induced enhancement of the recall phase of CMI (3, 18), these findings may pave the way for developing biomedical treatments designed to harness endogenous physiological mediators to selectively augment immune responses during vaccination, wound healing, infections or cancer. These findings may also increase our understanding of mechanisms mediating stress-induced exacerbation of pro-inflammatory and autoimmune diseases like psoriasis.
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Acknowledgements |
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Abbreviations |
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APC | antigen-presenting cell |
CMI | cell-mediated immunity |
DC | dendritic cell |
DNFB | 2,4-dinitro-1-fluorobenzene |
HPA | hypothalamicpituitaryadrenal |
LN | lymph node |
MCP-1 | macrophage chemoattractant protein-1 |
MIP-3![]() | macrophage inflammatory protein-3![]() |
NS | non stressed |
STR | acutely stressed |
TNF | tumor necrosis factor |
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Notes |
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Received 20 November 2004, accepted 23 May 2005.
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References |
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