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The Journal of Immunology, 2002, 168: 6128-6135.
Copyright © 2002 by The American Association of Immunologists

Catecholamines Inhibit the Antigen-Presenting Capability of Epidermal Langerhans Cells1

Kristina Seiffert*, Junichi Hosoi*, Hideshi Torii*, Hiroaki Ozawa*, Wanhong Ding*, Kristina Campton*, John A. Wagner{dagger} and Richard D. Granstein2,*

Departments of * Dermatology and {dagger} Neurology, Joan and Sanford I. Weill Medical College of Cornell University, New York, NY 10021


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The sympathetic nervous system modulates immune function at a number of levels. Within the epidermis, APCs (Langerhans cells (LC)) are frequently anatomically associated with peripheral nerves. Furthermore, some neuropeptides have been shown to regulate LC Ag-presenting function. We explored the expression of adrenergic receptors (AR) in murine LC and assessed their functional role on Ag presentation and modulation of cutaneous immune responses. Both purified LC and the LC-like cell lines XS52-4D and XS106 expressed mRNA for the ARs {alpha}1A and {beta}2. XS106 cells and purified LC also expressed {beta}1-AR mRNA. Treatment of murine epidermal cell preparations with epinephrine (EPI) or norepinephrine inhibited Ag presentation in vitro. Furthermore, pretreatment of epidermal cells with EPI or norepinephrine in vitro suppressed the ability of these cells to present Ag for elicitation of delayed-type hypersensitivity in previously immunized mice. This effect was blocked by use of the {beta}2-adrenergic antagonist ICI 118,551 but not by the {alpha}-antagonist phentolamine. Local intradermal injection of EPI inhibited the induction of contact hypersensitivity to epicutaneously administered haptens. Surprisingly, injection of EPI at a distant site also suppressed induction of contact hypersensitivity. Thus, catecholamines may have both local and systemic effects. We conclude that specific ARs are expressed on LC and that signaling through these receptors can decrease epidermal immune reactions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neural influences on the immune system provide a mechanism by which emotional state might influence immune function (1, 2, 3). Numerous studies have shown that stress can be immunosuppressive in man and animals (4, 5, 6, 7) and that the sympathetic nervous system plays a role in modulation of immune responses (8, 9, 10). Indeed, sympathetic fibers reach primary and secondary lymphoid organs including the spleen, thymus, and lymph nodes (11, 12), where they terminate near or in direct contact with lymphocytes. The presence of adrenergic receptors (AR)3 on lymphocytes suggests that sympathetic innervation may be important in the modulation of immune responses (3, 13). Although neuronal influences on lymphocytes have been explored, their effect on professional APC has only begun to be studied.

The skin is an easily accessible immunocompetent organ in which these regulatory mechanisms can be examined. Within the epidermis, Langerhans cells (LC) are dendritic APCs that play a key role in the cutaneous immune system (14). They reside in the suprabasilar portion of the epidermis and have been shown to be capable of presenting haptens, immunogenic peptides, and tumor Ags for T cell-dependent immune responses (15, 16, 17, 18). The observation that epidermal LC often lie in apposition with epidermal nerves and that calcitonin gene-related peptide (CGRP), a neuropeptide found in epidermal nerves, can regulate LC Ag-presenting function provides evidence for a regulatory interaction between the nervous system and the immune system within the skin (19).

The sympathetic division of the autonomic nervous system within the skin is supplied by postganglionic fibers of the paravertebral chain ganglia. Sympathetic fibers travel together with sensory nerve fibers to innervate blood vessels, sweat glands, and hair follicles (20). They also appear as single nerve fibers in the dermis and epidermis (21, 22, 23). Because the nerve fibers present in the skin release classical neurotransmitters, we examined whether catecholaminergic neurotransmission affects the immunocompetent cells of the skin. We provide evidence for the presence of distinct ARs on murine LCs and show functional effects of adrenergic agents on Ag presentation.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and cell lines

Complete medium (CM) consists of RMPI 1600 (Cellgro, Herndon, VA) containing 10% FCS (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1 mM nonessential amino acids, 0.1 mM essential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10 mM HEPES buffer (Life Technologies). CM was used in cell culturing unless stated otherwise.

The keyhole limpet hemocyanin (KLH)-specific, I-Ad-restricted Th1-clone HDK-1 was kindly provided by A. Takashima, (Department of Dermatology, University of Texas Southwestern Medical Center, Dallas, TX). It was maintained in CM containing 5 x 105 M 2-ME (Sigma-Aldrich, St. Louis, MO) and 200 U/ml recombinant mouse IL-2 (BD PharMingen, San Diego, CA).

S1509a is an immunogenic spindle cell tumor cell line induced by methylcholanthrene in an A/J mouse (I-Ak, H-2a). It was kindly provided by Dr. M. I. Greene (University of Pennsylvania, Philadelphia, PA).

The XS52-4D subclone of the XS52 cell line and the XS106 cell line are LC-like lines derived from neonatal BALB/c epidermis or A/J epidermis, respectively. They were prepared by limiting dilution cloning (24). These cells are dendritic, capable of Ag presentation, and have several phenotypic characteristics of LCs (25, 26). They were a kind gift of A. Takashima.

Animals

Six- to 12-wk-old female BALB/c (H-2d) and CAF1 (BALB/c x A/J)F1 (H-2d/a) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). They were maintained in the Joan and Sanford I. Weill Medical College animal facility at a light/dark circle of 12 h and given food and water ad libitum.

Test reagents

Epinephrine (EPI), norepinephrine (NE), phentolamine hydrochloride, ICI 118,551 and isoproterenol (ISO) were purchased from Sigma-Aldrich.

Preparation of TAA

S1509a cells were maintained at 37°C in humidified air containing 5% CO2 in CM. A freeze-thaw lysate was used as a source of soluble tumor-associated Ags (TAA) as described previously (27). Briefly, 107 S1509a cells/ml of CM were disrupted by three freeze-thaw cycles and centrifuged at 600 x g for 20 min. The supernatant was harvested and re-centrifuged at 13,000 x g for 1 h. This supernatant was used as a source of soluble TAA.

Preparation of EC

Epidermal cells (ECs) were prepared using a modification of a standard protocol (28, 29). BALB/c mice were sacrificed, shaved with electric clippers, and depilated with Neet (Reckitt & Coleman, Wayne, NJ). Truncal skin was then excised, and s.c. fat and panniculus carnosus were removed by blunt dissection. Skin was subsequently floated, dermis down, on 0.5 U/ml dispase (Boehringer Mannheim, Indianapolis, IN) and 0.375% trypsin (Sigma-Aldrich) in Ca2+/Mg2+-free PBS and incubated for 45 min at 37°C. Epidermal sheets were removed by gentle scraping, washed, and pooled in HBSS supplemented with 2% FCS and stirred for 20 min at 150 rpm to dissociate cells. Then, the suspension was decanted through 40-µm cell strainers (BD Biosciences, Franklin Lakes, NJ) to yield a single cell suspension known to contain ~2–3% LC.

Preparation of eEC

To enrich for LC content, ECs were prepared as described above and incubated with anti-Thy 1.2 mAb (Sigma-Aldrich) at a 1/100 dilution at 4°C for 30 min. Low-toxicity rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) was added at a 1/40 dilution for another 30 min at 37°C to delete epidermal T cells and some keratinocytes (in the mouse, keratinocytes weakly express Thy 1). Dead cells were digested by treatment with 0.05% trypsin and 80 µg DNase per milliliter (Sigma-Aldrich) in Ca2+/Mg2+-free PBS for 4 min at room temperature, and the surviving cells were spun down and washed. This procedure yielded an EC population of ~12% LC by FACS analysis (data not shown).

Preparation of highly purified LC populations

To further purify for LC content, enriched EC (eEC) were prepared as described above and incubated with anti-I-Ad Ab (BD PharMingen) at a 1/50 dilution for 30 min at 37°C. They were then incubated with goat anti-mouse IgG conjugated to magnetic microspheres (Dynabeads M-450; Dynal Biotech, Lake Success, NY) for 10 min under continuous gentle agitation. Cells were then separated into two fractions by repeated washing (up to 10 times) under a magnetic field. Those with attached magnetic beads were designated as purified LC (pLC; I-A+). The purity relates to the number of washes in the magnetic field after Dynabead attachment. Depending on the individual experiment, this procedure yields a population of 90–98% I-A+ cells as assessed by FACS analysis (double staining with FITC mouse anti-mouse I-Ad Ab (BD PharMingen) and propidium iodide (Sigma-Aldrich) to exclude dead cells) (data not shown).

PCR analysis

Total RNA was extracted from >98% purified LC as well as the cell lines XS106 and XS52-4D using a total RNA extraction kit as per the manufacturer’s instructions (RNeasy total RNA Mini kit; Qiagen, Santa Clarita, CA) including an additional DNA degradation step (RNase-free DNase set; Qiagen). RT-PCR was performed for catecholamine receptors using a RT-PCR kit (Gene-Amp RNA PCR kit; PerkinElmer, Norwalk, CT). Briefly, ~0.1 µg of RNA was incubated in a reaction mixture containing 5 mM KCl, 10 mM Tris-HCl, 1 mM dNTPs, 2.5 µM oligo(dT), 1 U/µl RNase inhibitor, and 2.5 U Moloney murine leukemia virus reverse transcriptase per microliter in 20 µl for 10 min at room temperature for extension of the primers, for 15 min at 42°C for annealing, and then for 5 min at 99°C to denature the enzyme. Ten microliters of the reverse-transcribed reaction were transferred to a PCR mixture containing primers (Life Technologies) designed using sequences in GenBank. The reaction mixture consisted of 50 mM KCl, 10 mM Tris-HCl, 2 mM MgCl2, 5 µM primers, and 1 U AmpliTaq DNA Polymerase in a total volume of 50 µl.

Primer sequences were as follows: 5'-CTCCGTATCCACCGTAAAAA-3' and 5'-GTCTTGGCGGCTTTCTTCTC-3' for {alpha}1A, 5'-CTACATTGGGGTGCGTTACT-3' and 5'-GGTTTTGGCTGCTTTCTTTT-3' for {alpha}1B, 5'-TCTTCCTAGCCGCCTTCATC-3' and 5'-CCCCTTGCTACTCTGTGTCC-3' for {alpha}1D, 5'-CTACCGCCCCTCATCTACAA-3' and 5'-TCTTCCACCTCCTCTTCATC-3' for {alpha}2, 5'-CTCGTGGCGCTCATCGTTCT-3' and 5'-TCGCAGCACTTGGGGTCGTT-3' for {beta}1, 5'-TTACCTCCTTTTTGCCTATC-3' and 5'-CCGTTCTGCCGTTGCTATTG-3' for {beta}2, and 5'-CCGCACGCCGAGACTACAGA-3' and 5'-AAGGAGACGGAGGAGGAGAG-3' for {beta}3.

Denaturation for 5 min at 95°C was followed by 35 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 55°C, and extension for 30 s at 72°C repeated with a thermal cycler (Gene Amp PCR System 9700; PerkinElmer), followed by completion for 7 min at 72°C. The PCR product was gel-electrophoresed in 1% agarose, stained with ethidium bromide, and visualized with UV radiation.

GADPH control primers were obtained from Invitrogen (Carlsbad, CA). Amplification of PCR product depended upon reverse transcription.

Presentation of KLH to a responsive T cell clone (HDK1)

EC or pLC were prepared from BALB/c (I-Ad) mice as described above, plated out in 96-well plates at a concentration of 1 x 105 cells/well in CM, and exposed to increasing concentrations of EPI or NE (100 pM, 1 nM, 10 nM, or 100 nM) or the inactive (+) or the active (-) isomer of ISO (100 nM) for 3 h at 37°C. After 3 h, KLH (Sigma-Aldrich) was added to a final concentration of 100 µg/ml. After 18 h, cells were gamma irradiated (3000 rad) and washed three times. Subsequently, EC were plated in 96-well plates at a concentration of 1 x 105 cells/well together with HDK1 cells at a concentration of 5 x 104 cells/well in CM. After 72 h of coculture, levels of IFN-{gamma} in culture supernatants were measured by ELISA as described below. Each assay was performed in triplicate wells at least three times.

Cytokine assays

IFN-{gamma} production by HDK1 cells was quantified by sandwich ELISA (BD PharMingen). Purified rat anti-mouse IFN-{gamma} mAb (4 µg/ml) was used as the primary (coating) Ab, with biotinylated rat anti-mouse IFN-{gamma} mAb as the secondary (detection) Ab. Avidin-HRP conjugate was added at a 1/1000 dilution, followed by color development with ABTS substrate solution (as per the manufacturer’s instructions), and the level of colored reaction product was measured spectrophotometrically at 410 nm.

Immunization of mice and elicitation of DTH

Immunity to the S1509a spindle cell carcinoma was induced by s.c. inoculation of 2 x 106 S1509a (killed by four freeze-thaw cycles) at weekly intervals into naive syngeneic mice (CAF1) for a total of three immunizations. Delayed-type hypersensitivity (DTH) was elicited 1 wk after the last immunization by injecting a hind footpad with TAA-pulsed eEC incubated with or without adrenergic antagonists. Specifically, 5 x 105 CAF1 eEC were incubated at 37°C with 10-8 M EPI or NE for 3 h. In some experiments the {beta}2-blocker ICI 118,551 (30, 31) or the {alpha}-blocker phentolamine at 10-8 M was added as well. Cells were then washed and 5 x 106 cells/ml were pulsed with soluble TAA for 3 h. eEC were washed four times in PBS to remove catecholamines, unbound TAA, and medium, before injection of 7.5 x 105 cells in 50 µl PBS into the right hind footpad of each of five CAF1 mice. Control mice received injections of eEC not pulsed with TAA or pulsed with TAA but not treated with EPI or NE. Footpad swelling was assessed at 24 and 48 h with a spring-loaded engineer’s micrometer (Mitutoyo, Tokyo, Japan) and compared with the noninjected foot.

In vivo EPI injection, immunization to haptens, and challenge to elicit CHS

CAF1 mice received an intradermal injection of 100 µl PBS containing 1 µg EPI on the lower back as described before (32). Successful inoculations were characterized by the appearance of a flattened swelling with defined lateral margins immediately beneath the epidermis (33). Control animals were injected with PBS. Fifteen minutes later mice were sensitized by application of 5 µl 1% 2,4-dinitroflourobenzene (DNFB; Sigma-Aldrich) in acetone/olive oil (4:1) to the injected area with care to avoid spread to the adjacent skin. Control animals were painted with acetone/olive oil (4:1) alone. Seven days later baseline ear thickness was measured using a spring-loaded micrometer (Mitutoyo) and all mice were challenged by applying 0.2% DNFB in acetone/olive oil to each side of each ear. Ear thickness was measured after 24 and 48 h. Specific ear swelling was calculated by subtracting the ear thickness before challenge from the 24- and 48-h values for each mouse.

Additional experiments were performed to examine whether the EPI effect was local or systemic. In these experiments 100 µl of PBS containing 1 µg EPI were administered intradermally to the lower right back 15 min before sensitization with DNFB at a distant site (upper left back). Elicitation of contact hypersensitivity (CHS) was performed as described above.

Data generation and statistical analysis

Statistical differences among groups were determined using Excel software (Microsoft, Redmond, WA) and tested by the two-tailed Student t test for independent samples. Mean values were considered to be significantly different when p <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AR mRNA is expressed in pLC and LC-like cell lines

A major impediment to performing receptor studies in LCs lies in the fact that these cells comprise only ~2% of the total EC population. Significant efforts must be taken to obtain highly purified populations. However, such techniques result in isolation of relatively few cells, making it difficult to examine protein expression or to perform Northern analysis. Thus, the more sensitive method, RT-PCR, was used to examine AR expression in these experiments. Epidermal LCs were enriched to >98% by enzymatic digestion and separation with I-Ad-targeted magnetic beads. This purified LC population expressed mRNA for the ARs {beta}1, {beta}2, and {alpha}1A (Fig. 1Go). The PCR products were of the expected size, and sequence specificity of expressed bands was confirmed by restriction enzyme cleavage at the expected sites (data not shown). The mRNAs for {beta}3, {alpha}1B, {alpha}1D, and {alpha}2 were not detected in the purified LCs. Due to the fact that 98% pure LC may still be contaminated by other EC types (predominantly keratinocytes), we also investigated AR expression in the LC-like clones XS52-4D and XS106. The use of these cells avoids the problems of possible contaminating cells. We were able to confirm the expression of {beta}1-, {beta}2-, and {alpha}1A-AR in XS106 cells as well as the expression of {beta}2- and {alpha}1A-AR in XS52-4D cells (Fig. 1GoB). Because {beta}1-AR was expressed by XS106, but not by XS52-4D cells, it is possible that {beta}1-AR is expressed by a subpopulation of LCs. To test whether functional ARs were produced and to test the consequences of engaging these receptors with their ligands, we examined the effect of adrenergic agents on Ag presentation both in vivo and in vitro.



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  		FIGURE 1. Murine LC express mRNA for ARs. Murine LCs were enriched to >98% purity and poly(A)+ RNA was extracted. Poly(A)+ RNA was also extracted from the LC-like cell lines XS106 and XS52-4D and from murine brain tissue, as a positive control. RT-PCR was performed using primers designed from sequences in GenBank (see Materials and Methods). Purified LC expressed mRNA of the expected size for the ARs {beta}1 (441 bp; A), {beta}2 (600 bp; B), and {alpha}1A (119 bp; C). The LC-like cell line XS106 expressed mRNA for {beta}1-, {beta}2-, and {alpha}1A-AR as well. XS52-4D expressed mRNA for {beta}2- and {alpha}1A-AR, but not for {beta}1-AR. Expression of mRNA for {beta}3-AR (B), {alpha}1B-AR (C), {alpha}1D-AR (D), and {alpha}2-AR (D) was not found in LCs or the LC-like cell lines. Receptor expression was verified with three separate RNA isolations.

 
EPI and NE inhibit Ag presentation by murine EC and LC

We examined the ability of both EPI and NE to affect KLH-specific EC Ag presentation to the T cell clone HDK1 in vitro. HDK1 cells respond to presentation of KLH by I-A+ accessory cells by secretion of IFN-{gamma}. EPI significantly reduced the ability of ECs to stimulate IFN-{gamma} production in HDK1 cells in a dose-dependent manner (Fig. 2GoA). In two separate experiments, (+)-ISO, the structurally similar inactive isomer of the {beta}-adrenergic agonist (-)-ISO, had no effect on the magnitude of T cell IFN-{gamma} production in response to presentation of KLH, while the active isomer decreased it significantly, supporting the specificity of the effect (Fig. 2GoB). Additionally, treatment with EPI (100 nM) did not affect the viability of ECs or LCs as determined by trypan blue exclusion or FACS analysis after propidium iodide stain (data not shown).



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  		FIGURE 2. EPI and NE inhibit T cell cytokine production in response to Ag presentation. Murine ECs were preincubated with EPI, NE (A), or ISO (B) at concentrations from 10-9 to 10-7 M and later pulsed with KLH. Upon coculture with HDK1 cells, Th1 cytokine production in response to KLH presentation by epidermal LC was assessed and compared with pulsing with KLH alone. Seventy-two-hour IFN-{gamma} production in conditioned supernatants was measured by ELISA (mean ± SEM; *, p < 0.05 for EC plus KLH vs EPI 10-9 M and iso (active isomes) 10-7 M; **, p < 0.01 for EC plus KLH vs EPI 10-8 M, NE 10-8 M, and NE 10-7 M). A representative experiment from six individual experiments, including two with additional NE treatments, is shown.

 
Ag-dependent T cell activation depends mostly on the function of certain APCs, but other cells may also elaborate factors that either directly affect the lymphocyte or act indirectly on APCs. Indeed, injury- or irritant-activated keratinocytes have the ability to produce a variety of distinct cytokines that either activate or inhibit T cell function and growth (34). Likewise, these cytokines may affect LC function. To further examine whether the effects of EPI were directly on LCs, the experiments were repeated with highly purified LCs (pLC). pLCs were prepared from BALB/c (H-2d) mice using enzymatic digestion and magnetic separation of Ab-labeled I-A+ cells. This resulted routinely in populations >96–98% I-A+. When I-A+ cells were preincubated with EPI, their ability to present KLH to HDK1 cells was significantly reduced as measured by IFN-{gamma} production of the T cells (Fig. 3Go). The I-A- population of cells that resulted from the magnetic bead separation failed to effectively present Ag (data not shown). Thus, LCs express functional ARs and agonist binding to these receptors inhibits Ag presentation.



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  		FIGURE 3. EPI directly inhibits Ag presentation by purified LCs. LCs were enriched to >98% purity by enzymatic digestion and magnetic separation of Ab-labeled I-A+ cells (pLC) (as described in Materials and Methods). They were preincubated with EPI at concentrations from 10-9 to 10-7 M and later pulsed with KLH. pLCs were cocultured with HDK1 cells and IFN-{gamma} production was assessed in conditioned supernatants after 72 h (mean ± SEM; **, p < 0.01 for EC plus KLH vs EPI 10-7). Results are representative of three experiments.

 
EPI and NE suppress eEC presentation of TAA for elicitation of DTH

To define the effects of EPI on Ag presentation in the elicitation phase of an immune response, a mixed in vitro/in vivo system was used. First, eEC, exposed to EPI or NE and pulsed with soluble TAA in vitro, were used to elicit a DTH response in vivo in tumor-immune mice (35). Subsequently, mice were immunized by s.c. injection of S1509a tumor fragments. Mice treated in this fashion have been shown to be immune to subsequent tumor challenge (36). These mice were challenged 7 days after the third immunization by hind footpad injection of EPI-treated, TAA-pulsed eEC, and DTH was assessed at 24 and 48 h. In vitro exposure of eEC to EPI significantly suppressed their ability to elicit DTH against TAA (Fig. 4Go, upper panel). TAA-treated eECs elicited DTH, while EPI pretreatment reduced DTH to the background level seen in the absence of Ag. In the absence of TAA exposure, eEC elicited only background swelling, regardless of EPI treatment. NE significantly suppressed elicitation of DTH to the same extent that EPI did (Fig. 4Go, lower panel). The reduction to background levels seen in DTH may indicate that the conditions for elicitation of DTH are more stringent in this assay and a small perturbation of APC function in vitro may produce large effects in vivo.



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  		FIGURE 4. EPI and NE inhibit elicitation of DTH in a {beta}-receptor-mediated way. Upper panel, EC were enriched for LC content (eEC) and pretreated with EPI before pulsing with TAA (B). eEC were then injected into the left hind footpad of previously immunized mice and footpad swelling was measured 24 and 48 h later. Control eEC were not treated with EPI, but were pulsed with TAA (A), pretreated with EPI but not pulsed with TAA (C), or not exposed to either agent (D). The mean difference (n = 5) in footpad thickness between the injected and uninjected side is shown at both 24 and 48 h (**, p < 0.01 for A vs B). Middle and lower panels, In addition to pretreatment with EPI or NE (F or J, respectively) some groups were also incubated with the {beta}2-receptor antagonist ICI 118,551 (H) or the nonselective {alpha}-receptor antagonist phentolamine (L) as indicated. Cells pulsed with TAA only (E or I) or not pulsed with TAA (G or K) served as controls. *, p < 0.05 for E vs F; not significant for E vs H; **, p < 0.01 for I vs J; *, p < 0.05 for I vs L).

 
Coincubation with the {beta}2-adrenergic antagonist ICI 118,551 completely abrogated the EPI-induced suppression of DTH, while the {alpha}-adrenergic antagonist phentolamine had no effect on the NE-induced suppression (Fig. 4Go, middle and lower panels). Thus, catecholamines have a profound effect on Ag presentation in the elicitation phase of an immune response in a {beta}-AR-mediated fashion.

Intradermal injection of EPI inhibits the induction of CHS to epicutaneously administered haptens

Next, we tested the ability of EPI to modify the sensitization phase of immune reactions in vivo by studying CHS, a prototype of T cell-mediated immunity. This reaction is characterized by inflammation and edema at the site of challenge and there is a positive correlation between the intensity of the immune reaction and the increase in pinna thickness (as reviewed by Dhabhar and McEwen in Ref. 37). CAF1 mice that were injected intradermally with EPI before application of the contact sensitizer DNFB at the injected site showed a reduced response upon DNFB challenge 7 days later (Fig. 5GoA) compared with mice that were injected with vehicle only. Rapid exit from the site of injection or degradation of catecholamines after administration in vivo may be responsible for a lesser magnitude in inhibition, as compared with the inhibition seen in vitro.



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  		FIGURE 5. Intradermal injection of EPI inhibits CHS induction. A, CAF1 mice were injected intradermally with 1 µg EPI or the diluent saline alone 15 min before immunization with DNFB (1%) at the injected site (EPI/DNFB and saline/DNFB). B, In a second set of experiments CAF1 mice were injected intradermally with saline or 1 µg EPI at either the side to be immunized or at a distant site. Control groups were not immunized. Seven days later all mice were challenged on both ears with DNFB (0.2%) and 24-h swelling was assessed. **, p < 0.01 for DNFB/saline vs EPI/saline or saline alone; *, p < 0.05 for DNFB/saline vs DNFB/EPI local or DNFB/EPI distant.

 
In additional experiments, we tested the specificity of the catecholamine effect on CHS by injecting the active or inactive isoform of ISO, (-) or (+), respectively (data not shown). When (-)-ISO was administered at 1 µg per mouse no effect was seen. Three experiments were performed at a dose of 10 µg per mouse. Significant inhibition was induced in one experiment. In a second experiment, inhibition was observed but did not reach statistical significance, while the third experiment failed to show inhibition. No inhibition was seen with the inactive isomer at 1 and 10 mg per mouse. We conclude that (-)-ISO probably has an effect on the induction of CHS but is less potent than EPI.

Because there are functional ARs on LCs, EPI and NE can be expected to act locally, but these agents may also have a systemic effect. To determine whether EPI acts only locally or also systemically, we determined the effect of administering EPI at a distant site. Mice received intradermal injections of EPI on the lower right back. Control animals received injections of PBS at the same site. Fifteen minutes later mice were sensitized with DNFB on the upper left back. Administration of EPI at a distant site inhibited CHS equivalently to local administration (Fig. 5GoB). Thus, catecholamines may act both locally and systemically to affect Ag presentation in the sensitization phase of CHS.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that epidermal LC express ARs and that these receptors are functional in Ag presentation, and, thus, most probably are involved in regulating cutaneous immune functions such as DTH and CHS.

To date AR are subdivided into nine types termed {beta}1, {beta}2, {beta}3, {alpha}1A, {alpha}1B, {alpha}1D, {alpha}2A, {alpha}2B, and {alpha}2C (38). In human and murine skin, {beta}2-AR seems to be the most abundant adrenoceptor (23, 39, 40), although {alpha}-ARs are present as well (41, 42). Among the AR-expressing cell populations within the skin are keratinocytes, melanocytes, fibroblasts, and mast cells (43, 44, 45). Specifically, human keratinocytes as well as the nontumorogenic keratinocyte cell line HaCaT have been shown to express {beta}2-AR and {beta}1-AR by autoradiographic mapping and radioligand binding experiments (46, 47), and {beta}-agonists (predominantly {beta}2) and {alpha}1-agonists stimulate adenylate cyclase activity and cAMP production in vitro (48). We have examined in this work the expression of ARs by LC and we have also investigated the LC-like clones XS52-4D and XS106 to confirm our results in a system without the possibility of contamination by other cells. We found mRNA for the ARs {beta}2-AR and {alpha}1A-AR to be expressed not only on murine LCs but also in LC cell lines. Expression by XS52-4D and XS106 cells verifies the finding with fresh LC and makes it very unlikely that the results with LC are due to contamination with another cell type. Because increasing concentrations of catecholamines directly reduce the Ag-presenting capability of murine ECs in vitro in comparable inhibitory potencies, LCs also express functional ARs. The inhibitory concentrations in our assay correspond to levels catecholamines can reach locally, in the immediate vicinity of a synapse or close to a sympathetic varicosity during stressful events (50–500 nM) (49, 50). Physiologically, however, EPI and NE could be derived from other sources. Epidermal keratinocytes and melanocytes are capable of producing catecholamines (51) and EPI of adrenomedullary origin may reach skin cells as a hormone. Thus, the true physiological roles of ARs on LC and their function in states of sympathetic arousal remain to be elucidated.

Our work indicates that catecholamines affect both phases of the immune response, sensitization, and elicitation, in which APC such as LC play a crucial role (52). Repression of Ag presentation in a {beta}2-AR-mediated way provides a mechanism that may contribute to the effects of stress on cutaneous immune functions such as DTH and CHS. Indeed, it has been shown that immobilization stress in mice inhibits elicitation of DTH when the adrenal axis is intact (53). Likewise, CHS reactions in mice have been reported to be suppressed by psychological and social stress induced by immobilization and overcrowding, respectively (54, 55). Interestingly, in regard to CHS, a recent study by Flint et al. (56) demonstrated that acute restraint before sensitization diminished CHS, whereas restraint administered before challenge enhanced CHS.

While demonstrating that intradermal EPI injections reduce the sensitization phase of CHS, we cannot exclude the possibility that local vasoconstriction, undoubtedly induced by EPI, contributes to the immune reduction, perhaps by interfering with immune cell trafficking. If vasoconstriction alone is sufficient to impair the development of CHS, this effect should be seen with other vasoconstricting substances as well. However, preliminary experiments provide evidence that intradermal injection of PYY does not reduce CHS reactions in the same model (data not shown) despite being an equipotent vasoconstrictor in certain organs (57, 58, 59). Thus, it seems unlikely that vasoconstriction alone explains the observed effect.

In addition to our findings that injection of EPI at the site of hapten sensitization inhibits CHS reactions, it is able to do so even when injected a considerable distance away. This effect may be either systemic or due to diffusion into the surrounding tissues. Indeed, it has been shown that noradrenergic varicosities can release their catecholamines at considerable distances from the effector tissues (12), permitting them to diffuse through the surrounding region more easily than neuropeptides and, thus, to reach even remote targets (20). This might explain why other neuropeptides such as substance P and CGRP promote CHS induction only by a local effect (60, 61), although CGRP, when used in similar assays, has been shown to result in comparable degrees of inhibition (19, 61). Nevertheless, it is also possible that the catecholamines are taken up systemically and affect immune cell trafficking and other related functions. Indeed, in a recent study, Maestroni (62) found NE to be both a chemoattractant and a chemokinetic factor for immature DC and showed that immature DC participate in cell mobilization and migration to regional lymph nodes in an {alpha}1B-AR-mediated fashion. The reasons why ISO was less potent than EPI in inhibiting CHS are speculative. However, ISO is a pure {beta}-agonist, and our findings do not rule out the possibility that additional {alpha}-mediated effects of EPI in vivo may also play a role here. Additional markers of LC maturation in relation to migration, such as their chemokine receptor profile, need to be investigated in future studies. For example, only activated, not resting, LCs express CCR7. Through this receptor, their migration is directed toward stromal cells in T cell zones of lymph nodes and lymphatic endothelial cells in peripheral tissues, such as the dermis, that express the CCR7 ligand secondary lymphoid tissue chemokine (63).

Interactions between the different arms of the immune system must be taken into consideration when examining the in vivo effects of stress on immune function. The varying results of studies examining the effect of stress on immunity in general emphasize the complexity of immune reactions. CHS in particular has been described to be either suppressed (54, 55, 56) or enhanced (56, 64) by psychological and social stress in mice. Differences in these studies regarding how long and in which phase of the immune reaction the animals where stressed emphasize the importance of timing and duration of stress. In evaluating these in vivo observations, one has to consider that stress activates several systems, accompanied by a release of glucocorticoids, catecholamines, endogenous opioids, and other pituitary hormones with effects on all kinds of immune cells. In an elaborate feedback system glucocorticoids regulate catecholamine biosynthesis in the adrenal medulla and catecholamines stimulate adrenocorticotropin release from the anterior pituitary. Additionally, signals from the periphery are sent back to the brain where they coordinate behavioral and physiological responses to infection and inflammation (65). Compensatory changes appear with prolonged stress (66), partly due to a feedback regulation of activated immunologic cells themselves (67, 68). Considering these tightly regulated feedback processes that ultimately determine the direction of an immune response, it seems worthwhile to elucidate isolated effects of neurotransmitters on specific immune cell function, as done in the present study. Although the (patho-) physiological roles of EPI and NE are not completely understood, we can infer that catecholamines are a regulatory element in the cutaneous stress response. We provide a cellular mechanism by showing that EPI and NE isolated directly inhibit LC Ag-presenting function.

As to the exact mechanisms mediating the immune-inhibitory effects, it remains possible that catecholamines affect expression of class II and costimulatory molecules on APC (69), the efficiency of Ag processing and presentation, as well as changes in cytokine production and response to cytokines. For example, in studies by Hosoi et al. (54) and Kawaguchi et al. (55), LCs are found to be the stress-affected cell population, with significant decreases in Ia expression as well as a marked alteration in LC morphology (decreased number of dendrites and cell size). In fact, preliminary data from our group provide evidence that prolonged exposure of LC to EPI in vitro down-regulates I-A expression, at least in combination with down-regulation induced by anti-I-A Abs. Furthermore, catecholamines down-regulate proinflammatory cytokines such as TNF-{alpha} and elevate anti-inflammatory cytokines such as IL-10 via a {beta}-adrenoceptor-dependent pathway in immune cells (50, 70, 71, 72). Interestingly, preliminary data from our group (73) indicate that EPI and NE up-regulate IL-1{beta} and down-regulate TNF-{alpha} expression in the LC-like cell line XS52. Both cytokines are implemented in E-cadherin expression in fetal murine LC-like dendritic cells and dissociation of fetal murine LC-like dendritic cell aggregates (74). There is considerable evidence that immature LCs are retained in their epidermal environment by E-cadherin-mediated adhesion to keratinocytes (75). These findings might indicate an involvement of stress hormones in cell adhesion.

Thus, abnormal adrenergic regulation of the immune response at the level of LCs may contribute to skin disorders such as atopic dermatitis (which is associated with anxiety where circulating catecholamines are elevated) or psoriasis (which is known to be exacerbated upon treatment with {beta}-adrenergic antagonists).


   Acknowledgments
 
We thank Dr. A. A. Sinha for his critical review and helpful suggestions in preparation of the manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AR 42429 (to R.D.G. and J.A.W.), a grant from the Abercrombie Foundation, a grant from the Edith C. Blum Foundation, contributions from the Ann L. and Herbert J. Siegel Philanthropic Fund, and support from Shiseido Co. Ltd. K.S. was supported by a Dermatology Foundation (Cosmetic, Toiletry, and Fragrance Association) Research Fellowship. Back

2 Address correspondence and reprint requests to Dr. Richard D. Granstein, Department of Dermatology, Joan and Sanford I. Weill Medical College of Cornell University, 525 East 68th Street, New York, NY 10021. E-mail address: rdgranst{at}med.cornell.edu Back

3 Abbreviations used in this paper: AR, adrenergic receptor; CHS, contact hypersensitivity; DNFB, 2,4-dinitroflourobenzene; DTH, delayed-type hypersensitivity; EC, epidermal cell; eEC, enriched EC; EPI, epinephrine; NE, norepinephrine; ISO, isoproterenol; LC, Langerhans cell; CGRP, calcitonin gene-related peptide; TAA, tumor-associated Ag; CM, complete medium; KLH, keyhole limpet hemocyanin; pLC, purified LC. Back

Received for publication August 24, 2001. Accepted for publication April 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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