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Laboratories of Cellular Physiology and Immunology and
Molecular Biology and Biochemistry, Rockefeller University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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50%),
especially at low DC:T cell ratios (1:360). This effect was not
observed with CGRP-treated, immature DCs. In contrast, CGRP-treated
mature or immature DCs were no less efficient than untreated DCs in
driving syngeneic T cell-proliferative responses to staphylococcal
enterotoxin B. We conclude that mature and immature DCs express type 1
CGRP receptors and that signaling through these receptors may dampen
mature DC-driven T cell proliferation most likely via down-regulation
of CD86 and HLA-DR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although neuropeptides can influence many cell types in the immune system including LCs, their effects on human blood-derived DCs have not been extensively characterized. These professional APCs express the two signals (8) required by T cells: one signal consisting of processed Ag presented on MHC class I or II; and second signals for T cell binding and activation via such molecules as ICAM-1 (CD54), LFAs (CD2, CD11a, CD58), and B7s (CD80, CD86). DCs derived from monocytes exposed to IL-4 and GM-CSF express MHC class II and CD86 but revert to a macrophage phenotype if GM-CSF or IL-4 is withdrawn (9). Further culture with monocyte conditioned medium (MCM) yields fully mature DCs. We show that immature and mature DCs possess type 1 CGRP receptors and that treatment with CGRP during the maturation phase can dampen DC stimulation of T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used RPMI 1640 (Cellgro, Fisher Scientific, Springfield, NJ), supplemented with 2 mM L-glutamine (Life Technologies, Grand Island, NY), 50 µM 2-ME (Sigma, St. Louis, MO), 10 mM HEPES (Life Technologies), penicillin (100 U/ml)-streptomycin (100 µg/ml) (Gemini Bio Products, Calabrasas, CA), and 1% human plasma (heparinized).
Cytokines
Recombinant human GM-CSF was purchased from Immunex (Seattle, WA), and recombinant human IL-4 was purchased from R&D (Minneapolis, MN).
Monocyte-conditioned medium (MCM)
IgG-coated bacteriologic plates (100 mm, Falcon, Lincoln Park,
NY) were prepared immediately before use by addition of 4 ml human
-globulin (10 mg/ml, Cappel Laboratories, Organon Teknika,
Westchester, PA) for 3 min. Plates were washed three times with PBS
before use. Peripheral blood was obtained from buffy coats purchased
from the New York Blood Center (New York, NY). PBMCs were isolated by
sedimentation in Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden). T
cell-depleted (erythrocyte rosette (ER)-) fractions were prepared by
rosetting with neuraminidase-treated sheep RBC as described previously
(10). T cell-depleted (ER-) cells (5 x
107) or 9 x 107 PBMCs
were cultured on IgG-coated plates for 1 h at 37°C. Nonadherent
cells were washed off with gentle aspiration. The IgG-adherent cells
were cultured with 10 ml fresh medium at 37°C for 24 h. The
medium was collected and frozen at -20°C before use.
Monoclonal Abs
mAbs to the following Ags were used: HLA-DR; CD3; CD14; CD20; CD25 (PE conjugate, Becton Dickinson, Mountain View, CA); CD83 (IgG1, unlabeled or PE conjugate, Coulter, Miami, FL); and CD86 (PE conjugate, PharMingen, San Diego, CA). Cell populations were phenotyped with these mAbs using a FACScan (Becton Dickson Immunocytometry Systems, San Jose, CA).
Generation of DCs from human blood
Peripheral blood was obtained from normal human donors in heparinized syringes or from buffy coats purchased from the New York Blood Center. T cell-enriched (ER+) or T cell-depleted (ER-) fractions from PBMCs were prepared by rosetting with neuraminidase-treated sheep RBC as described (10). ER- cells (2.5 x 106/well) were plated in each well of a six-well plate (Falcon). Alternatively, adherent monocytes were derived by plating 8 x 106 PBMCs/well in 3 ml culture medium for 1 h at 37°C. Nonadherent cells were removed, and adherent cells washed with warm RPMI. GM-CSF (1000 U/ml) and recombinant human IL-4 (100 U/ml) were added to the ER- or adherent monocytes on day 0 of culture and replenished on days 2, 4, and 6 by removal of 0.3 ml medium followed by addition of 0.5 ml medium with cytokines. To generate mature DCs, cultures were supplemented with MCM (50% v/v) on day 7. To generate immature DCs, cultures were supplemented with cytokines on day 8 but were not supplemented with MCM.
Purification of DCs
In all experiments, CD14-negative, immature, and mature DCs were purified by negative selection by incubation with anti-CD2 and anti-CD19 Dynabeads (Dynal, Oslo, Norway) per manufacturer’s instructions. After two cycles of separation as described (11), purity was >99% and was confirmed by FACS analysis.
Generation of T cells
PBMCs from heparinized blood from normal human donors or from buffy coats were isolated by sedimentation in Ficoll-Hypaque as described for generation of DCs. T cell-enriched (ER+) fractions were prepared by rosetting with neuraminidase-treated sheep RBC (10). Alternatively, T cells were purified from nonadherent fractions by negative selection by incubation with Dynabeads coated with anti-HLA-DR according to the manufacturer’s instructions. Purity was confirmed by FACS analysis.
Neuropeptides and neuropeptide treatment
CGRP and CGRP type 1 receptor antagonist, CGR8–37, were purchased from Phoenix Pharmaceuticals (Belmont, CA), and CGRP type 2 receptor agonist, diacetoamidomethylcysteine CGRP (CYS (ACM 2, 7) CGRP) was purchased from Sigma. The neuropeptide treatment protocol was a modification of the one described by Fox et al. (12). Immature DCs, after 7 days of culture with IL-4 (100 U/ml) and GM-CSF (1000 U/ml), were cultured with CGRP (10-7 M) for 2 h at 37°C. Then cells were cultured for 2 additional days in medium supplemented with MCM and CGRP. As mentioned earlier, addition of MCM to the culture media for 2 days promotes DC maturation. We refer to this cell population as CGRP-treated, mature DCs. In parallel plates, DCs were cultured for additional 2 days with CGRP, but without addition of MCM in the culture medium. Under these conditions, DCs do not mature and remain as immature DCs. We refer to this cell population as CGRP-treated, immature DCs. At day 9, CGRP-treated, mature DCs and CGRP-treated, immature DCs were cultured again with CGRP (10-7 M) for 2 h at 37°C and washed twice before phenotypic analysis and functional assays.
Allogeneic mixed leukocyte reactions
To test for allogeneic T cell-stimulatory function, 50–5000 purified CGRP-treated or untreated, mature or immature DCs were added as stimulators for 2 x 105 allogeneic T cells in 96-well flat-bottom plates (Costar, Cambridge, MA) for 5 days at 37°C. Each combination was set up in triplicate, and proliferation was determined on day 5 by measuring the uptake of [3H]TdR (4 µCi/ml) during the final 8 h of coculture. Results are expressed as the mean cpm incorporated from triplicate wells (mean cpm). In some experiments, CGRP (10-7 M in each well) was added to cocultures on days 0, 2, and 4.
Staphylococcal enterotoxin B (SEB) assay
As another assay for DC presenting function, 300–10,000 CGRP-treated or untreated, mature or immature, purified DCs were added to 1 x 105 T cells with 5 ng/ml SEB (Toxin Technology, Sarasota, FL) in 96-well flat-bottom plates and cultured for 3 days at 37°C. Each combination was set up in triplicate, and proliferation was determined on day 3 using [3H]TdR (4 µCi/ml) uptake during the final 8 h (mean cpm). In some experiments, CGRP (10-7 M in each well) was added to cocultures on days 0 and 2.
Assay for intracellular calcium
Mature or immature, purified DCs (2–8 x 106) were collected in 17- x 20-mm conical tubes (Falcon), pelleted at 2000 rpm for 2 min, washed twice with EBSS-H (125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM NaH2PO4, 26 mM HEPES, 5.6 mM glucose, 0.1% w/v BSA, 2 mM CaCl2), and resuspended in 1 ml EBSS-H at 1–4 x 106 cells/ml. The fluorophore fluo-3-acetoxymethyl ester (fluo-3-AM) (Molecular Probes, Eugene, OR) was added to 2 µM, and the DCs were incubated for 60–75 min at room temperature on a nutator. The fluo-3-AM-loaded DCs were pelleted and resuspended in EBSS-H, washed again, and incubated in EBSS-H for 30–45 min. The DCs were pelleted and transferred to cuvettes at 2–4 x 106 cells/ml, and the fluorescence due to intracellular calcium was measured at room temperature in a Hitachi F-2000 fluorescence spectrophotometer using excitation and emission wavelengths of 505 and 525 nm, respectively.
RT-PCR for detection of CGRP-R1 transcripts
Total RNA from 1–2 x 106 purified mature or immature DCs was extracted in 2 ml RNAzol (Tel-Test, Friendswood, TX) mixed with 0.2 ml chloroform and centrifuged at 12,000 x g for 15 min. The aqueous phase was transferred to a fresh tube, isopropanol was added to precipitate the RNA, and the pellet was dissolved in 20 µl double-distilled H2O and stored at -20°C. First-strand cDNA was reverse transcribed from 5 µg total RNA in a 20-µl reaction volume in 20 mM Tris-HCl; 50 mM KCl; 2.5 mM MgCl2; 10 mM DTT; 500 nM oligo(dT); 500 µM each dATP, dCTP, dGTP, dTTP; and 200 U Super Script II RT (Life Technologies, Gaithersburg, MD) for 50 min at 42°C. Two µl of the 20 µl of RT reaction mixture were PCR amplified. Primers for human CGRP-R1 were synthesized based on a published sequence (13). The sense primer 5'-TCT GGT TCT CTT GCC TTT TTT TAT G-3' corresponded to nucleotides 581–605, and the antisense primer 5'-GTC CAT GTT CTG TTG CTT GCT G-3' was complementary to nucleotides 898–919. Thirty cycles of PCR were run at 94°C for 1 min, 56°C for 1 min, and 72°C for 1 min with 2.5 U Taq polymerase (Perkin-Elmer, Norwalk, CT).
Automated DNA sequencing
This was done by Taq FS dye terminator cycle sequencing using a Model 377 stretch sequencer (Perkin-Elmer/Applied Biosystems, Foster City, CA) (14).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because immunomodulatory effects of CGRP on LCs had been
described, we set out to identify CGRP receptors on DCs derived from
human PBMCs and then study their immunoregulatory potential. Total RNA
was isolated from purified blood-derived, mature, and immature DCs.
Results of RT-PCR experiments indicated that mRNA specific for CGRP
type 1 receptor (CGRP-R1) (705 bp) was expressed in mature and immature
DCs (Fig. 1
). Sequence analysis confirmed
98 and 99% identity with the human CGRP-R1, as recorded in GenBank,
for mature and immature DCs, respectively.
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To determine the functional potential of CGRP-R1 on DCs, we
measured the ability of CGRP to trigger calcium fluxes in mature and
immature cells. The DCs were loaded with fluo-3 AM, a dye that exhibits
increased fluorescence in the presence of calcium. When either mature
or immature, fluo-3-AM-loaded DCs were treated with CGRP
(10-7 M), intracellular calcium mobilization was
noted as determined by fluorometry (Fig. 2). Mobilization of intracellular calcium
was abrogated by the presence of equimolar concentrations of
CGRP8–37, a CGRP-R1 antagonist, whereas calcium flux was
not triggered by diacetoamidomethylcysteine CGRP (CYS (ACM 2, 7) CGRP),
an agonist for the type 2 CGRP receptor (CGRP-R2) (data not shown).
Mobilization of intracellular calcium by CGRP confirmed the
functionality of the CGRP-R1 receptor on both mature and immature
DCs.
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As shown previously, CD25 and CD83 are expressed on mature DCs,
whereas the levels of the MHC class II molecule, HLA-DR, and the
costimulatory molecule, CD86, are increased (9). Previous
studies indicated that CGRP suppressed CD86 in the murine, LC-like,
XS52 cells (6), so we conducted similar studies on human
DCs, as a model for the in vivo situation in which DCs are exposed to
neurogenic stimuli before or concomitant with the exposure to Ag.
Treatment of purified DCs with CGRP (10-7 M) for
2 h before and during culture with MCM, and for an additional
2 h after the MCM-induced maturation phase, yielded CGRP-treated,
mature DCs and resulted in decreased expression of CD86 and HLA-DR
(Fig. 3). Expression of CD25, the 
subunit of the IL-2 receptor, and CD83, a maturation marker, were
unchanged by CGRP treatment. Treatment of immature DCs with CGRP
(10-7 M) subsequently cultured with GM-CSF and
IL-4 but without MCM, yielded CGRP-treated, immature DCs with decreased
expression of CD86 but unchanged expression of HLA-DR (not shown). CD25
and CD83 expression on immature DCs remained negative after CGRP
treatment. Decreased expression of CD86 and HLA-DR was observed with
DCs from 60% of the buffy coats and blood donors we studied. In all
cases in which regulation was observed, the changes in expression of
HLA-DR and CD86 occurred conjointly. In the case of CGRP-treated,
mature DCs, these phenotypic changes correlated with diminished
induction of allogeneic T cell proliferation.
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The down-regulation of CD86 on CGRP-treated, mature, and immature
DCs suggested that CGRP treatment might modulate their Ag-presenting
capability. This was investigated using the allogeneic MLR assay.
Mature DCs vigorously stimulate allogeneic T cells and at low DC:T cell
ratios (9), whereas previous studies revealed a decrease
in allogeneic T cell stimulation by CGRP-treated LCs (6).
Therefore, purified, CGRP-treated or untreated, mature or immature DCs
were used to stimulate allogeneic T cells in the MLR assay. The
proliferative responses were lower than those induced by untreated,
mature DCs (25–50%), especially at low DC:T cell ratios (1:360 in
Fig. 4). Addition of CGRP
(10-7 M) to DC:T cell cocultures on days 0, 2,
and 4 of the MLR assay had no additional effect on T cell-proliferative
responses. In contrast, allogeneic T cell-proliferative responses
driven by CGRP-treated, immature DCs were not different from those
driven by untreated, immature DCs.
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Finally, we examined presentation in a superantigen (SEB) system
that is less sensitive to subtle changes in surface expression of
HLA-DR. Varying numbers of purified, CGRP-treated or untreated, mature
or immature DCs were cultured with syngeneic T cells and SEB, for 3
days, to determine whether CGRP treatment might decrease this DC-driven
response. However, T cell-proliferative responses were unchanged (Fig. 5). Further addition of CGRP
(10-7 M) to the cocultures on days 0 and 2 did
not affect the response to SEB (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our results extend the findings that some neuropeptides may regulate immune responses (15). Neuropeptides, including CGRP, pituitary adenylate cyclase-activating peptide, vasoactive intestinal peptide, and substance P, can regulate the function of B cells, T cells, and macrophages (1). The interaction of LCs and neuropeptides, in particular CGRP, has been described (3). Hosoi et al. (16) demonstrated that LCs often lie in apposition with epidermal nerves containing CGRP. Also CGRP is required for UVB-mediated suppression of contact hypersensitivity (CH) (17, 18). Other studies support the potential suppression of delayed-type hypersensitivity as well as CH by CGRP (19). Finally, Fox et al. (12) reported that treatment of human monocytes with CGRP for 2 h decreased their presentation of tetanus toxoid.
The expression of CGRP-R1-specific mRNA (Fig. 1
) by DCs alludes to the
potential for functional regulation of DCs by neuropeptides. At least
two subtypes of CGRP receptors have been identified, termed CGRP-R1 and
CGRP-R2 (20). Sequence identity with human CGRP-R1
supports the presence of the type 1 receptor on human blood-derived
DCs, consistent with other data of CGRP-R1 on LCs
(21).
CGRP-R1 mobilizes intracellular calcium in human DCs (Fig. 2). The CGRP receptor is a member of a superfamily of transmembrane
receptor proteins. On ligand binding, the receptor couples to a
membrane-associated heterotrimeric G protein that initiates signaling
pathways, ultimately leading to increased intracellular calcium
(22). The inhibition of calcium fluxes by the CGRP-R1
antagonist and the lack of a calcium flux to CYS (ACM 2, 7)
CGRP, a CGRP R-2 agonist, further supports the presence of a functional
CGRP R1 on DCs. The identity of specific G protein subunits involved
with CGRP-mediated signaling in DCs remains to be determined.
Dampening of DC-driven immune responses by CGRP may involve regulation
of costimulatory molecules, alterations in cytokine induction, or
direct effects on Ag uptake. We observed a decreased expression of CD86
and HLA-DR by CGRP-treated, mature DCs (Fig. 3) but only a decrease in
CD86 in CGRP-treated, immature DCs. Because the stimulatory activity
for allogeneic T cells was decreased only for mature DCs (Fig. 4), the
down-regulation of HLA-DR may be more significant. CGRP also did not
reduce DC-driven T cell proliferation to SEB, a phenomenon that is less
susceptible to subtle down-regulation of HLA-DR (23),
supporting a role for HLA-DR down-regulation in the dampening of
allogeneic T cell proliferation to CGRP-treated, mature DCs.
As stated earlier, the phenomenon of phenotypic regulation was observed in only 60% of blood donors. The reasons for this are not clear and may involve differences in the level of expression of CGRP-R1 on DCs or the presence of defective receptors in some individuals. However, in all cases in which regulation was observed, the changes in expression of HLA-DR and CD86 occurred in concert, and, in CGRP-treated, mature DCs, phenotypic changes correlated with reduced allogeneic T cell stimulation.
Suppression of CD86 on human monocytes by CGRP has been described and has been reported to involve IL-10 (12). Other studies have demonstrated CGRP-mediated induction of IL-10 and suppression of IL-12 and CD86 in the epidermal LC-like cell line XS52 (7). The dampened DC function that we observed still needs to be addressed at the levels of these cytokines and Ag uptake. Dampened immune responses in another system has been suggested by Khachatryan et al., who showed that CGRP expression in pancreatic ß cells suppresses development of diabetes in nonobese diabetic mice (24).
We considered the idea that CGRP could have a direct effect on T cells.
CGRP potentiates T cell proliferation in the presence of
IFN--treated, class II-bearing, intestinal smooth muscle cells
(25) but suppresses mitogen-stimulated murine T cells
(26). In our studies, addition of CGRP
(10-7 M) to DC:T cell cocultures had no effect
on DC-driven allogeneic T cell proliferation or responses to SEB (Figs. 4 and 5). The expression of CGRP-R1 mRNA by immature and mature DCs,
coupled with effects of CGRP on intracellular calcium, HLA-DR, and
CD86, suggests that CGRP acts on DCs directly.
On the basis of previous and current findings, we speculate that the release of neuropeptides in the face of inflammatory stimuli, including certain inflammatory skin disorders, can suppress APCs including DCs. The cellular mechanisms involved in regulation of CH induction by UV radiation, especially the role of LCs, have been studied (17, 18, 19, 27). The potential therapeutic effects of UV radiation in inflammatory disorders, including atopic dermatitis and psoriasis, are also described (28). Because CGRP is required for the suppression of CH by UVB (18, 19), CGRP may influence responses to phototherapy. Accordingly, selective activation of CGRP-R1 on DCs might prove useful in the treatment of inflammatory skin disorders.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Dermatology, Yale University School of Medicine, 4th Floor, Yale Physicians Building, 800 Howard Avenue, New Haven, CT 06519.
3 Current address: Institute for Infectious Diseases, Department of Medical Microbiology and Infectious Diseases Immunology, Free University of Berlin-University Hospital Benjamin Franklin, Hindenburgdamm 27, 12203 Berlin, Germany.
4 Address correspondence and reprint requests to Dr. Svetlana Mojsov, 204 Bronk Building, The Rockefeller University, 1230 York Avenue, New York, NY 10021. E-mail address:
5 Abbreviations used in this paper: CGRP, calcitonin gene-related peptide; DCs, dendritic cells; LCs, Langerhans cells; ER, erythrocyte rosette; MCM, monocyte-conditioned medium; SEB, staphylococcal enterotoxin B; fluo-3-AM, fluo-3-acetoxymethyl ester; CGRP-R1, type 1 CGRP receptor; CGRP-R2, type 2 CGRP receptor; CYS (ACM 2, 7) CGRP, diacetoamidomethylcysteine CGRP; CH, contact hypersensitivity.
Received for publication June 7, 1999. Accepted for publication January 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, Control DCs; 
, CGRP-treated DCs;
•, control DCs with CGRP added during coculture; 
, CGRP-treated
DCs with CGRP added during coculture.
, CGRP-treated
DCs.