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CUTTING EDGE |
Divisions of
*
General Internal Medicine and
Gastroenterology and Hepatology, Department of Internal Medicine, University of Innsbruck, Innsbruck, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulated immature cell migration
only slightly. Checkerboard analyses revealed a true chemotactic
response induced by neuropeptides. Upon maturation of DC, neuropeptides
inhibited spontaneous, macrophage-inflammatory protein-3- and
6Ckine-induced cell migration. Maturation-dependent changes in
migratory behavior coincided with distinct neuropeptide-induced signal
transduction in DC. Peripheral neuropeptides might guide immature DC to
peripheral nerve fibers where high concentrations of these peptides can
arrest the meanwhile matured cells. It seems that one function of
sensory nerves is to fasten DC at sites of
inflammation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC can be found in many nonlymphoid tissues such as the airway epithelium and unlike other immune cells that rapidly transit into the airways, the DC remain within the epithelium during the acute inflammatory response (5). Using calcitonin gene-related peptide (CGRP) and DC (PGP9.5), immunostaining anatomic connections between DC and nerve fibers have been observed in human (6, 7). In the liver, contacts between nerve fibers staining for substance P (SP), CGRP, and vasoactive intestinal polypeptide (VIP), and DC were observed (8). Pulmonary DC bind 125I-labeled SP and in vitro SP increases motility but has no effect on accessory activity of these cells (9). In contrast, CGRP, released from nonadrenergic-noncholinergic sensory nerve fibers, specifically inhibited the accessory function of cutaneous Langerhans cells (6).
The sensory neuropeptides SP, CGRP, VIP, and secretoneurin (SN) have been identified as potent mediators of inflammatory and immunologic reactions involving leukocytes other than DC (10, 11, 12). CGRP-induced intracellular cAMP increase in pulmonary DC could be blocked by a specific receptor antagonist (13), and more recently the expression of CGRP receptors type 1 on peripheral blood DC could be demonstrated (14). Except VPAC1- and VPAC2-receptor expression and VIP-induced cAMP formation in pulmonary DC (13), no further activities of the latter neuropeptide or of other neuropeptides, including SN have been described in any DC so far.
Therefore, we investigated the effects of nervous system-derived mediators on the migratory behavior of DC and found contrasting responses depending on the maturation stage of the cells. Peripheral neuropeptides can directly attract immature DC, but they may arrest mature DC at sites of neurogenic inflammation. This opposite behavior was accompanied by changes in signal transduction pathways of neuropeptide receptors in immature and mature DC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC were generated from peripheral mononuclear cells as
previously described (15, 16). In brief, peripheral
mononuclear cells were allowed to adhere in cell culture flasks. After
removal of nonadherent cells, adherent cells were cultured in medium
supplemented with 1 x 103 U/ml IL-4
(Schering-Plough, Kenilworth, NJ) and 1 x
103 U/ml GM-CSF (Leucomax; Novartis, Vienna,
Austria). The harvested DC were further purified by magnetic cell
separation using a cocktail of mAb against CD3, CD14, CD19, and CD56
(PharMingen, Hamburg, Germany), followed by addition of sheep
anti-mouse IgG-coated magnetic beads (Dynabeads M-450; Dynal,
Hamburg, Germany). Maturation was induced by incubation of purified DC
in culture medium supplemented with IL-4, GM-CSF, TNF-
(1 x
103 U/ml), and 10 µM PGE2
for 72 h. The resulting DC population yielded a purity of >97%,
as determined by cytofluorometry analysis after CD1a, CD3, CD14, CD19,
CD56, CD40, and CD83 (HB15A; Immunotech, Vienna, Austria) staining.
Cytofluorometric analysis of DC surface phenotype
A total of 5 x 105 DC were washed in PBS/2% FCS, resuspended in 250 µg/ml human IgG/PBS/2% FCS. After pelleting, DC were incubated alternatively with 10 µg/ml anti-CD1a, anti-CD14, anti-CD83, or anti-HLA-DR mAbs and the respective isotype-matched control Igs. After washing in PBS/2% FCS, a 1:40 dilution of FITC-anti-mouse IgG in PBS/2% FCS was incubated for 30 min at 4°C. Cells were immediately analyzed on a FACScan after the addition of 1 µg/ml propidium iodide (PI). Analysis was performed on PI- cells (e.g., viable cell population) with CellQuest software (BD Biosciences, Mountain View, CA) (15, 16).
Chemotaxis assay
Migration of DC into cellulose nitrate was measured as described
recently (15, 16, 17). In brief, using a 48-well
microchemotaxis chamber (Neuroprobe, Bethesda, MD) in which a 8-µm
pore sized filter (Sartorius, Göttingen, Germany) separates the
upper and lower chamber, cells migrate toward gradients of soluble
attractants. The migration medium was RPMI 1640/0.5% BSA. Aliquots (30
µl) of chemoattractant solution or control medium (RPMI 1640/0.5%
BSA) were put in the lower wells of the chamber. A total of 50 µl of
cell suspension (1 x 106 cells/ml) were
seeded in the upper chamber. For checkerboard analyses cells were
resuspended in RPMI 1640/0.5% BSA containing various concentrations of
chemoattractants just before transferring them to the upper chamber.
Migration time was 4 h. After this period, the filters were
dehydrated, fixed, and stained with hematoxylin-eosin. Migration depth
of DC into nitrocellulose was quantified by microscopy, measuring the
distance (micrometers) from the surface of the filter to the leading
front of cells, before any cell had reached the lower surface (leading
front assay). All neuropeptides (CGRP, VIP, SN, secretin, helodermin,
SP, -endorphin) and the selective CGRP-receptor type 2 agonist
[Cys(Et)2,7]CGRP were obtained from Neosystem (Strasbourg, France).
RANTES, macrophage-inflammatory protein-3 (MIP-3), human
exodus-2/secondary lymphoid tissue chemokine (6Ckine), and stromal
cell-derived factor-1 (SDF-1) were obtained from PeproTech
(London, U.K.).
Signal transduction experiments
To rule out distinct signal transduction pathways immature and
mature DC were incubated for 30 min at optimal concentrations of 500 nM
bisindolylmaleimide I (GFX; protein kinase C (PKC) blocker; inhibits
PKC pathway], 10 ng/ml tyrphostin-23 (tyrosine kinase inhibitor;
inhibits receptor associated/cytosolic tyrosine kinases), 10 nM
wortmannin (WTN; PI3 kinase-inhibitor; PI-3,4,5-phosphate
(PtdIns(3, 4, 5)P3)-related signaling), 10 µM rolipram
(phosphodiesterase blocker; inhibits the cAMP-related pathways), or
medium control. After washing twice, immature DC were attracted by 0.1
nM CGRP, 0.1 nM VIP, and 10 nM SN. Because neuropeptides are not
directly chemotactic on mature DC, untreated and pretreated cells
migrated toward 10 nM MIP-3, 1 µg/ml 6Ckine, or 1 µg/ml SDF-1
and the neuropeptides concomitantly in the lower wells. Thereafter, the
assay was proceeded as described above.
Statistical analyses
Data are expressed as mean and SEM of the "chemotaxis index." Means were compared by Kruskal-Wallis ANOVA and by Mann-Whitney U test. A difference with p < 0.05 was considered significant. Statistical analyses were calculated using the StatView software package (Abacus Concepts, Berkley, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Highly purified monocyte-derived DC were obtained by 6-day culture
of monocytes in IL-4 and GM-CSF and by further depletion of
contaminating CD3+, CD14+,
CD19+, and CD56+ cells. The
phenotype of the resulting day-6 DC (immature) population was
determined cytofluorometrically after staining for CD1a and CD83 and
revealed a purity of >97% (Fig. 1
, a–d). Full maturation of DC can be induced by
further incubation of day-6 cells for 72 h with TNF- and
PGE2. These mature phenotypes hardly express CD1a
Ag, while CD83 expression is markedly up-regulated (Fig. 1, e–h).
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The neuropeptides CGRP, VIP, secretin, and SN (each 0.01 pM-1
µM) induced DC chemotaxis with bell-shaped dose-response curves,
indicating a receptor-mediated effect. Maximal effects were seen at 0.1
nM of VIP or secretin and at 1 nM or 10 nM of CGRP or SN, respectively
(Fig. 2). Only the highest concentration
of SP (1 µM) slightly stimulated cells. The selective CGRP receptor
type 2 agonist [Cys(Et)2,7]CGRP and -endorphin were inactive and
helodermin, which prefers VPAC2, showed a flat linearly increasing
dose-response while the highest concentration (1 µM) produced
approximatly 60% of the secretin-induced effect at a concentration
four logarithmic decades below. RANTES (0.001 pM-0.1 µM), which is
inactive in mature DC, served as positive control for immature DC
chemotaxis, whereas MIP-3 (0.01 pM-1 µM), known to be most active
in mature DC (4), failed to induce immature DC migration
(Fig. 2).
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Cells were resuspended in medium alone or medium containing
various concentrations of attractants immediately before transferring
them to the upper wells of the chemotaxis chamber. Therefore, positive
concentration gradients between the upper and the lower wells can be
formed. Data are presented, not as numbers within a matrix, but in line
charts. Analyses revealed a true chemotactic response of immature DC
toward RANTES (1 pM-10 nM), CGRP, VIP (each 0.01 pM-0.1 nM), and SN (1
pM-10 nM); increasing concentrations in the upper wells diminish DC
migration depth toward any concentration in the lower wells. Because
equal concentrations of neuropeptides in the upper and the lower wells
still slightly stimulated cell migration, chemokinetic activities of
these neuropeptides on immature DC cannot be excluded; lines of the
intermediate and in part of the highest concentrations (0.1 nM, 1 pM,
and 0.01 pM) parallel the x-axis at a higher level (not
strictly increasing), indicating partial chemokinesis (Fig. 3).
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Untreated cells migrated toward various concentrations of
neuropeptides (0.01 pM-1 µM) in the lower wells of the chemotaxis
chamber. In contrast to immature DC, CGRP, VIP, and SN inhibited mature
DC migration. The migration distance was 
70% of control medium;
therefore, the rarely seen effect of "negative" chemotaxis can be
excluded. When mature DC were attracted by 10 nM MIP-3 the highest
concentrations of VIP or SN diminished DC chemotaxis nearly to baseline
levels (neuropeptides remained concomitantly with MIP-3 in the lower
wells); CGRP reduced migration >50% compared with control. Only a
high dose of SP slightly stimulated mature DC migration and inhibited
MIP-3-induced chemotaxis (Fig. 4, left). Also strong inhibition of mature DC chemotaxis toward
1 µg/ml 6Ckine was observed with CGRP or VIP, whereas SN and SP
failed to show such an effect (Fig. 4, right). In contrast,
neuropeptides did not affect SDF-1-induced mature DC chemotaxis
(Fig. 4, inset).
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The enzyme blockers GFX, tyrphostin-23, WTN, and rolipram had no
effect on either cell phenotype migration. The VIP- and CGRP-induced
immature DC chemotaxis was blocked by tyrphostin-23 and WTN.
Additionally, CGRP receptor type 1 signal transduction was rolipram
sensitive. Contrasting in mature cells, where CGRP- or VIP-inhibited
MIP-3- or 6Ckine-stimulated migration was restored by blockade of
PKC with GFX. Additionally, CGRP-affected migration toward 6Ckine was
WTN and rolipram sensitive. Effects of VIP on MIP-3- or
6Ckine-induced mature DC migration were also abolished by tyrphostin-23
or rolipram, respectively. SN involves PtdIns(3, 4, 5)P3 and
phosphodiesterases in its signal transduction in immature DC but
SN-induced inhibition of mature DC chemotaxis was reversed by tyrosine
kinase blockade. The solely MIP-3-attracted mature DC used products
of the polyphosphoinositide cycle and phosphodiesterases for their
chemotactic response, whereas 6Ckine- and SDF-1-stimulated migration
can be blocked by pretreatment of mature DC with tyrphostin-23 or WTN
(Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SP has been reported to have no effect on accessory activities of
pulmonary DC, and it failed to increase intracellular cAMP levels in
Langerhans cells (immature DC) (9, 21). This is consistent
with our recent study where SP activates immature and mature DC
migration only at high concentrations. Although the maturation stage
determines the migratory properties of these cells (22),
SP was shown to induce pulmonary DC migration (9); high
concentrations of SP can act via fMLP receptors (23),
which may also be the case in DC (18). CGRP, VIP, and SN
were chemotactic on immature DC in our experiments and this response
was confirmed as true chemotaxis in checkerboard analyses (Fig. 3
),
although lower concentrations of neuropeptides yielded in part
chemokinetic effects. The fact, that [Cys(Et)2,7]CGRP, a selective
CGRP receptor type 2 agonist, failed to affect DC migration and that
CGRP-induced chemotaxis was diminished by tyrphostin-23 and WTN, as it
was shown for adrenomedulin (a CGRP receptor type 1 agonist)-mediated
signaling in smooth muscle cell proliferation (24),
coincides with the recently demonstrated CGRP receptor type 1 mRNA
expression in immature and mature peripheral blood-derived DC
(14). VIP is a monocyte and lymphocyte chemoattractant and
receptors mediating this effect are VPAC1 and VPAC2, which are
preferentially activated by secretin and helodermin, respectively.
Signaling of the first receptor is known to be tyrosine
phosphorylation dependent (25) and WTN
sensitive. In contrast to VIP and secretin, which induced signaling
characteristics of VPAC1, helodermin did not stimulate immature cell
migration in our experiments. These facts give strong evidence of
functional VPAC1 expression in DC. Recently, a receptor for the novel
sensory neuropeptide SN has been identified (26) and in
previous investigations we uncovered its signaling in
eosinophils (27). In the present DC chemotaxis
assays, the bell-shaped dose-response curve and signaling
characteristics of the SN receptor suggest its presence also on
immature DC.
Differences in receptor expression between immature and mature DC can
be seen for several chemokines/CCR (2), as was seen in our
study. MIP-3 and 6Ckine were potent chemoattractants for mature DC
but failed to influence immature cells, which lack expression of CCR7
(2). Interestingly, in our in vitro experiments,
neuropeptides lost their ability to attract mature DC and on the
contrary, the cells were immobilized by high neuropeptide
concentrations. In this context it is important to note that some
neuropeptides inhibited MIP-3- or 6Ckine-induced mature DC migration
(both CCR7 ligands) (28) but all neuropeptides lacked such
an effect when cells were attracted by SDF-1, which also acts
chemotactic on immature DC (CXCR4 ligand). This indicates a specific
inhibitory effect of neuropeptides on mature DC. Because receptors for
neuropeptides are described to be expressed on both immature and mature
DC (14) changes in responsiveness to neuropeptides during
DC development occurred not due to altered neuropeptide receptor
distribution between immature and mature cells but more likely are
caused by a switch to another signal transduction pathway of the same
receptor. Depending on maturation stage of DC, results ruled out two
different signaling pathways, for CGRP and VIP on the one hand and for
SN on the other hand (Table I).
Cell polarization is imperative for directed migration, and
chemokinesis describes enhanced random migration. Data from
checkerboard analyses suggest the ability of neuropeptides to affect
both cell functions in immature DC. Lower concentration gradients can
improve the motility of DC and, once stimulated, they can be guided by
strong concentration gradients to the source of the neuropeptides,
primarily sensory nerve fibers. When cells reach these fibers they may
undergo functional and phenotypical maturation, which will keep them
arrested at this site of high neuropeptide concentration. This concept
is supported by the finding that VIP synergizes with TNF- in
inducing DC maturation (29). For example in the lung,
which is a rich source of different neuropeptides that dramatically
increase upon inflammation (9), DC are located in
immediate proximity to unmyelinated nerve fibers. Because neonatal
capsaicin treatment of rats (leads to loss of neuropeptide
production/release) inhibits the accumulation of DC around small
pulmonary vessels during a pulmonary response to inhaled Ag
(9), our in vitro finding may be of pathophysiological
relevance.
We conclude that some sensory neuropeptides can guide immature DC migration and can arrest mature DC at sensory nerve fibers. These effects are mediated via specific receptors, which switch signal transduction pathways in DC of distinct maturation stages. Our concept based on in vitro findings may provide evidence of a novel link between adaptive immunity and the nervous system and shows a new aspect in neuroimmunology.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Christian Wiedermann, Department of Internal Medicine, University Hospital Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria.
3 Abbreviations used in this paper: DC, dendritic cells; CGRP, calcitonin gene-related peptide; GFX, bisindolylmaleimide I; MIP-3, macrophage-inflammatory protein-3; SDF-1, stromal cell-derived factor-1; SN, secretoneurin; SP, substance P; VIP, vasoactive intestinal polypeptide; WTN, wortmannin; 6Ckine, human exodus-2/secondary lymphoid tissue chemokine; PI, propidium iodide; PKC, protein kinase C; PtdIns(3,4,5)P3, phosphatidylinositol-3,4,5-phosphate.
Received for publication August 2, 2000. Accepted for publication December 11, 2000.
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References |
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-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 286:525. in inducing human dendritic cell maturation. J. Immunol. 163:3071.This article has been cited by other articles:
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