| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
in Inducing Human Dendritic Cell Maturation
Centre d’Immunologie Pierre Fabre, Saint-Julien en Genevois, France
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
to induce DC
maturation. In the presence of a suboptimal concentration of TNF-,
which has no detectable effect on DC by itself, VIP induces the
production of high levels of bioactive IL-12, the neoexpression of CD83
on almost all the DC population (with an effect significant at 10 and
0.1 nM, respectively), and the up-regulation of various adhesion and
costimulatory molecule expression. Moreover, DC exposed to VIP plus a
suboptimal concentration of TNF- are as potent as mature DC obtained
by treatment with an optimal concentration of TNF- in stimulating
allogenic T cell proliferation. Our data suggest that, in inflammatory
sites, VIP may cooperate with proinflammatory mediators, such as
TNF-, to induce DC maturation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production
by LPS-stimulated monocytes and macrophages (8, 9, 10), and
protects mice from endotoxemia through the inhibition of IL-6 and
TNF- production (11). VIP also modulates Ig production
by B cells (12, 13), induces mast cell degranulation
(14), and is mitogenic for connective tissue, epithelial
cells, and keratinocytes (15, 16).
Dendritic cells (DC) are crucial for the initiation of a primary immune
response. They are distinguishable from other APC by their potent
Ag-presenting capacity. In peripheral nonlymphoid sites, resident DC
are immature and highly effective at processing foreign Ags
(17, 18, 19). After Ag challenge in vivo or stimulation with
inflammatory stimuli, resident DC capture Ag and migrate to the
secondary lymphoid organs (17, 18, 19, 20). During their
migration, DC are thought to undergo modulations of phenotype and
function, referred to as DC maturation. They increase in size, express
increased levels of surface Ags important in T cell activation (such as
CD40, CD54, CD58, CD86, and MHC class I/II), produce cytokines (such as
TNF- and IL-12), neoexpress some molecules (such as CD83 on human
cells), and lose their capacity to process Ag (i.e., decrease of uptake
and proteolytic digestion of Ags and disappearance of the class II
compartment) (17, 18, 19, 21). In the T cell-dependent areas
of the lymphoid organs, myeloid migratory DC have potent
immunostimulatory properties and sensitize recirculating naive
Ag-specific T cells (17, 18, 19).
In the present work, we have investigated the effect of different
neuropeptides, SOM, SP, and VIP on human immature DC function. We show
that VIP induces DC maturation. Although the effect of VIP by its own
remains moderate, VIP synergizes with TNF- in inducing DC
maturation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PBMC were isolated from healthy volunteers by standard density
gradient centrifugation on Ficoll-Paque (Pharmacia Biotech, Uppsalla,
Sweden). Monocytes were purified from PBMC by positive selection using
a magnetic cell separator (MACS; Miltenyi Biotex, Bergisch
Gladbach, Germany) according to the manufacturer’s instructions.
Purity assessed by FACS analysis using a FITC-labeled anti-CD13 mAb
(Cymbys, Hants, U.K.) was >95%. Monocytes were cultured in culture
medium (CM) consisting in RPMI 1640 medium supplemented with 10% FCS,
2 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml
streptomycin, 10 mM HEPES, and 0.1 mM nonessential amino acids (all
from Life Technologies, Cergy Pontoise, France) at 5 x
106 cells/5 ml/well in 6-well tissue culture
plates (Costar, Cambridge, MA) with 20 ng/ml IL-4 and 20 ng/ml GM-CSF
(R&D Systems, Abingdon, U.K.). On day 6, cells were analyzed by FACS,
as described above; only the homogeneous immature DC population
characterized by high levels of CD1a (mean fluorescence intensity (MFI)
from 100 to 800) and no CD83 expression was used. DC were then
recultured at 105 cells/200 µl/well in 96-well
flat-bottom tissue culture plates (Costar) in cytokine-containing CM
with or without different concentrations of the following
neuropeptides: VIP (Calbiochem, La Jolla, CA), SP, or SOM (both from
Sigma, St. Louis, MO). In some experiments, DC were also stimulated
with TNF- (R&D Systems) or LPS (from Escherichia coli
isotype 0111:B4) (Sigma). In other experiments, immature DC have been
exposed to VIP in the presence of 5 µg/ml polymyxin B sulfate or of
10-6 M of a VIP receptor antagonist
(VIP6–28, reference V4508) (both from Sigma)
(22).
Flow cytometric measurement of surface Ag expression
FACS analysis was performed using a FACSvantage cytofluorometer (Becton Dickinson, Erembodegem, Belgium) with the following mAbs: FITC-labeled anti-CD1a (Immunoquality Products, Groningen, The Netherlands), anti-CD80 and anti-CD86 (both from PharMingen, San Diego, CA), anti-CD54 and anti-HLA-DR (both from Becton Dickinson), and anti-CD40 (Serotec, Oxford, U.K.) mAbs. The binding of the anti-CD83 mAb (Immunotech, Marseille, France) was revealed by FITC-labeled anti-mouse IgG Ab (Silenus, Hauworth, Australia). Control isotype mAbs were from Becton Dickinson. Results are expressed in MFI values after subtraction of the MFI obtained with the control mAb or as a percentage of positive cells.
Quantification of bioactive IL-12
Day 6 DC were incubated for 48 h with various stimuli, as described above, and the biologically active IL-12 p40/p35 heterodimer (IL-12 p75) was measured in cell-free culture supernatants by ELISA using a commercial kit (R&D Systems) according to the manufacturer’s recommendations (sensitivity of 0.5 pg/ml). Results are expressed in pg/ml.
Primary allogenic mixed lymphocyte reaction
Day 6 DC were washed, recultured at 2.5 x
105 cells/5 ml/well in 6-well culture plates in
cytokine-containing CM and were either unstimulated or stimulated with
0.2 ng/ml TNF-, 10-6 M VIP, 0.2 ng/ml TNF-
plus 10-6 M VIP, or 20 ng/ml TNF-. After 4
days, DC were washed two times in CM, irradiated (3000 rad), and
cultured in quintuplicate at 104 cells/200
µl/well in 96-well flat-bottom culture plates with 5 x
104 allogenic T cells. T cells were purified from
PBMC from healthy volunteers by rosetting with sheep RBCs; the purity
assessed by FACS analysis using a FITC-labeled anti-CD3 mAb
(Immunotech) was >95%. After 5 days, cells were pulsed during the
last 16 h with [3H]thymidine (0.25
µCi/well) (Amersham, Amersham, U.K.). Results are expressed in cpm
(mean ± SD of quintuplicate values) or in proliferation index
(PI) defined as follows: A/B, where A and
B are the cpm values obtained in the presence or absence of
stimulus, respectively.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have evaluated whether some neuropeptides may affect DC
maturation. Day 6 DC were exposed to
10-10–10-6 M SOM, SP, or
VIP. The neoexpression of the maturation marker CD83 (23)
was evaluated 4 days later. As expected, unstimulated DC retain an
immature phenotype, whereas TNF--treated and LPS-treated DC acquire
CD83 expression (Table I
and Fig. 1A, respectively). Among the
neuropeptides tested, VIP is the only one that induces CD83 expression
on a fraction of DC: its effect is dose-dependent, significant at
10-8 M, and maximal at
10-6 M, the highest concentration tested
(16 ± 5% and 45 ± 8% of CD83+
cells, respectively, mean ± SD, n = 5) (Fig. 1B). PCR analysis confirmed the constitutive expression of
VIP-R1 and, at a lower level, of VIP-R2 on day 6 DC (data not
shown).
|
|
on DC maturation (17, 18, 19, 23). Day 6 DC were
exposed to 10-10–10-6 M
VIP in the presence of increasing concentrations of LPS (from 0.5 to
100 pg/ml) or TNF- (from 0.2 to 20 ng/ml), and the neoexpression of
CD83 was evaluated 4 days later. VIP synergizes with TNF- in
inducing CD83 expression. This synergistic effect is maximal when VIP
is used together with 0.2 ng/ml TNF-: VIP induced-CD83 expression is
significant at 10-10 M (16 ± 4% of
CD83+ cells, mean ± SD, n =
5) and maximal at 10-6 M (95 ± 10% of
CD83+ cells) (Fig. 1
B). In contrast,
0.2 ng/ml TNF- alone fails to induce CD83 expression (Fig. 1).
Whatever the concentration of LPS used, we report no synergistic or
antagonistic effect of LPS on VIP-induced CD83 expression (data not
shown).
To exclude an effect of contaminating endotoxin, cells were treated
with 5 µg/ml polymixin B. Polymixin B down-regulates LPS-induced CD83
expression (decrease of 72 ± 8%, n = 3) but not
VIP- or VIP plus TNF--induced CD83 expression (Fig. 1A).
To verify the specificity of VIP-induced CD83 expression, VIP and/or
0.2 ng/ml TNF- were added to immature DC together with a VIP
antagonist. This inhibitor does not induce CD83 expression by itself
and partly prevents VIP-induced and VIP plus TNF--induced CD83
expression (decrease of 59 ± 11% and 66 ± 8%,
respectively) (Fig. 1A). These data obtained using VIP
provided by Calbiochem have been reproduced using VIP purchased from
another commercialized source (Sigma) (data not shown).
VIP synergizes with TNF- in up-regulating adhesion and
costimulatory molecules expression on DC
In addition to CD83 neoexpression, DC maturation is associated
with a marked up-regulation of adhesion and costimulatory molecules,
such as CD40, CD54, CD80, CD86, and HLA-DR (17, 18, 19). Table I shows that day 6 DC exposed to 0.2 ng/ml TNF- still retain an
immature phenotype. However, exposure of immature DC to VIP plus 0.2
ng/ml TNF- for 4 days results in a dramatic increase in CD40, CD54,
CD80, and HLA-DR expression, and in an induction of CD86 expression on
virtually all the DC of the population (98 ± 8% of
CD86+ cells, mean ± SD, n =
3) (Table I). Phase contrast microscopy also reveals a pronounced
morphology of mature DC with large veils (data not shown). Therefore,
these cells exhibit a mature phenotype similar to that of cells treated
with an optimal dose of TNF- (Table I) (17, 18, 19, 23). In
contrast, 10-6 M VIP used alone induces CD86
expression on a limited number of cells (58 ± 12%) and
moderately enhances CD40, CD54, CD80, and HLA-DR expression (Table I).
These results indicate that VIP synergizes with suboptimal
concentrations of TNF- to confer a mature phenotype to DC.
VIP synergizes with TNF- in inducing IL-12 production by DC
The process of maturation is associated with the production of
cytokines including IL-12 (17, 18, 19, 24, 25). We have thus
analyzed the production of p75 bioactive IL-12 by VIP-treated DC. In
the absence of stimulus, immature DC produce undetectable levels of
IL-12. The amount of IL-12 induced by VIP remains low: 3 ± 1 and
9 ± 1.8 pg/ml (mean ± SD, n = 4) at
10-7 M and 10-6 M,
respectively. Although 0.2 ng/ml TNF- alone does not induce
detectable levels of IL-12, in the presence of 0.2 ng/ml TNF-, VIP
dose dependently stimulates IL-12 production with a significant effect
at 10-8 M and maximal at
10-6 M (8 ± 1.3 and 22 ± 2.5 pg/ml,
respectively, mean ± SD, n = 4). Polymixin B does
not affect VIP-induced and VIP plus TNF--induced IL-12 production
(data not shown) but inhibits LPS-induced IL-12 production (decrease of
92 ± 15%, mean ± SD, n = 3). In contrast,
a VIP receptor antagonist partly prevents VIP-induced and VIP plus
TNF--induced IL-12 production (decrease of 85 ± 21% and
52 ± 13%, respectively). In additional experiments, as it has
been suggested that autocrine IL-12 may affect murine bone
marrow-derived DC maturation (26), we have tested the
effect of a neutralizing anti-IL-12 Ab on VIP-induced and VIP plus
TNF--induced CD86 and CD83 expression on DC. Our results show that
this Ab does not modulate the effect of VIP (data not shown), thereby
suggesting that endogenous IL-12 has no or a limited role in
VIP-induced human monocyte-derived DC maturation. Taken together, these
data indicate that VIP synergizes with TNF- in inducing bioactive
IL-12 production by DC.
VIP and TNF- cooperate to enhance DC accessory capacity
Due to higher levels of costimulatory molecule expression and
cytokine production, mature DC are more efficient than immature ones in
stimulating T cells (17, 21). Based on the observation
that VIP-treated DC have a mature phenotype, we have analyzed their
accessory cell capacities. In allogenic mixed lymphocyte reaction
assays, allogenic T cells from two subjects were cultured with DC that
were previously exposed to VIP and/or TNF- for 4 days. Results show
that DC treated with 10-6 M VIP plus 0.2 ng/ml
TNF- or with 20 ng/ml TNF- are the most potent in stimulating T
cell proliferation (PI = 24 and 15, and 25 and 20, respectively)
(Fig. 3). DC treated with VIP alone also enhance T cell proliferation,
but to a lesser extent (PI = 8 and 3), whereas DC treated with 0.2
ng/ml TNF- have no significant effect compared with untreated DC
(Fig. 3). Thus, immature DC stimulated with VIP plus a suboptimal
concentration of TNF- present potent costimulatory properties.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in inducing full DC maturation. The treatment of immature DC
with VIP plus TNF- results in the induction of CD83 expression and
IL-12 production, in the up-regulation of different adhesion and
costimulatory molecule expression, and in the acquisition of potent T
cell stimulatory capacity.
DC residing in the peripheral tissues are immature. The presence of
inflammatory mediators prompts DC to migrate to the secondary lymphoid
organs. During their migration, DC are thought to undergo a maturation
process controlled by different factors. Inflammatory mediators such as
TNF-, IL-1ß, PGE2, and LPS induce DC
maturation; the ligation of CD40 expressed on DC also results in the
generation of hyperactivated DC (17, 18, 19, 24, 25). In
contrast, IL-10 converts immature DC into tolerogenic APC (19, 27). Based on our results, VIP appears as a novel DC maturation
factor.
Data from the literature suggest that VIP has a dual role on T cell
stimulation. Although VIP decreases proliferation and IL-2 but not
IFN-
production by murine and human T cells stimulated with
anti-CD3 mAb (with or without phorbol ester) or with mitogenic
lectins (5, 6, 7), it enhances IL-2 and IFN- production by
T cells specifically stimulated by Ag-pulsed APC (28, 29).
In agreement with our results, this last observation suggests that VIP
acts on APC to favor Ag-dependent T cell stimulation (29).
In addition, as VIP is found in the skin, its effect on murine
Langerhans cells (LC), the immature DC of the epidermis, has been
investigated. In agreement with our observation, LC express VIP-RI and
-RII mRNA (30). Moreover, the VIP receptors are coupled to
adenylate cyclase, and it has been observed that VIP increases cAMP in
murine LC (31). In agreement with the absence of effect of
SP on DC reported here, others have also observed that SP does not
affect the APC capacity of LC (32). Finally, all these
data suggesting that VIP may act on APC are reinforced by the
observation that VIP induces human DC maturation.
The binding of VIP to different immune cells results in an increase in intracellular cAMP (1, 2, 3). Agents that increase intracellular cAMP (forskolin, dibutyryl cAMP, and PGE2) have been recently shown to induce CD83 expression and IL-12 production by immature DC, suggesting that the cAMP signaling pathway is involved in the DC maturation process (25). Therefore, it is tempting to speculate that this pathway is involved in VIP-induced DC maturation.
We show that the effect of VIP on DC maturation is potentiated by
TNF-. VIP is released locally in functionally relevant
concentrations during immune and inflammatory responses (1, 2). TNF- is produced early by different cell types in
response to stimulation with LPS or inflammatory mediators
(33). Thus, in inflammatory sites, VIP and TNF- might
be present concomitantly. The mechanism by which these molecules
cooperate to induce the maturation of resident DC remains undetermined.
As the 5'-flanking regions of the VIP-R genes contain cytokine-related
elements (34), TNF- may up-regulate VIP-R expression.
Nevertheless, previous data showing that TNF- synergizes with cAMP
in up-regulating IL-1ß synthesis by human monocytes (35)
and with PGE2 in inducing human DC maturation
(25), also suggest a possible cooperation between cAMP and
TNF- transduction pathways. Although PGE2 and
other cAMP-inducing agents synergize with TNF- in inducing IL-12
production by DC (25), they inhibit LPS-induced IL-12
production by monocytes and DC (9, 25). Thus,
cAMP-inducing agents may have a dual effect on IL-12 production when
used in combination with TNF- or LPS. In agreement with this
hypothesis, we report that VIP synergizes with TNF- in inducing
IL-12 production by DC, whereas others have shown that VIP
down-regulates LPS-induced IL-12 production by macrophages (9, 10).
VIP prevents mice from endotoxemia (11). Moreover, VIP
inhibits TNF- and increases IL-10 production by LPS-stimulated
macrophages (10, 11, 36). As these molecules enhance and
prevent DC maturation, respectively (27, 37), VIP may
limit the potent effect of LPS on DC maturation in vivo. In addition,
the recent observation that cAMP-inducing agents decrease LPS-induced
IL-12 production by DC (25) also suggests that VIP may
have a direct inhibitory effect on LPS-stimulated DC.
In response to inflammatory mediators, DC migrate from the peripheral
tissues to the T cell areas of the secondary lymphoid organs where they
present Ag to recirculating T cells. The observation that VIP
potentiates TNF--induced DC maturation and IL-12 production suggests
that VIP, in combination with proinflammatory cytokines, may amplify
the initiation of specific T cell responses and the generation of
IFN--producing T cells. This point is reinforced by the recent
observation that VIP strikingly enhances IFN- production by
Ag-stimulated T cells (29). In addition, VIP has been
shown to favor T cell migration through basement membranes and
connective tissues by increasing expression of adhesive proteins,
stimulating chemotaxis, and inducing secretion of matrix
metalloproteinases (1). Based on these data, it is
tempting to speculate that VIP may favor the development of specific T
cell responses by enhancing DC activation and T cell trafficking.
Nevertheless, in vitro experiments performed with murine T cells, in
the absence of APC, using mitogenic lectin or phorbol ester as stimulus
(4, 5, 6, 7), also suggest a potential down-regulatory effect of
VIP on previously activated T cells.
In conclusion, these data show that VIP synergizes with TNF- in
inducing human DC maturation and thereby suggest that VIP may
participate in the control of specific T cell responses. As a
consequence, by acting on the DC maturation process, VIP-targeted
pharmacological agents may have interesting therapeutic
applications.
|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Pascale Jeannin, Centre d’Immunologie Pierre Fabre, 5, Avenue Napoléon III, F-74164 Saint-Julien en Genevois, Cedex, France. E-mail address:
3 Abbreviations used in this paper: VIP, vasoactive intestinal peptide; DC, dendritic cells; SP, substance P; SOM, somatostatin; MFI, mean fluorescence intensity; PI, proliferation index; LC, Langerhans cells; CM, culture medium.
Received for publication April 8, 1999. Accepted for publication June 30, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
from stimulated murine T lymphocytes. J. Neurosci. 47:147.
and IL-6. J. Immunol. 162:1200.-interferon production by antigen-stimulated type 1 helper T cells. FASEB J. 13:347. in human monocytes. J. Immunol. 155:836.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  D. Fabricius, M. S. O'Dorisio, S. Blackwell, and B. Jahrsdorfer Human Plasmacytoid Dendritic Cell Function: Inhibition of IFN-{alpha} Secretion and Modulation of Immune Phenotype by Vasoactive Intestinal Peptide J. Immunol., November 1, 2006; 177(9): 5920 - 5927. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Ashwood, S. Wills, and J. Van de Water The immune response in autism: a new frontier for autism research J. Leukoc. Biol., July 1, 2006; 80(1): 1 - 15. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Delgado, D. Pozo, and D. Ganea The Significance of Vasoactive Intestinal Peptide in Immunomodulation Pharmacol. Rev., June 1, 2004; 56(2): 249 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Delgado, A. Reduta, V. Sharma, and D. Ganea VIP/PACAP oppositely affects immature and mature dendritic cell expression of CD80/CD86 and the stimulatory activity for CD4+ T cells J. Leukoc. Biol., June 1, 2004; 75(6): 1122 - 1130. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. STRAUB and H. O. BESEDOVSKY Integrated evolutionary, immunological, and neuroendocrine framework for the pathogenesis of chronic disabling inflammatory diseases FASEB J, December 1, 2003; 17(15): 2176 - 2183. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Burzyn, C. C. Jancic, S. Zittermann, M. I. Keller Sarmiento, L. Fainboim, R. E. Rosenstein, and H. E. Chuluyan Decrease in cAMP levels modulates adhesion to fibronectin and immunostimulatory ability of human dendritic cells J. Leukoc. Biol., July 1, 2002; 72(1): 93 - 100. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Ganea and M. Delgado VASOACTIVE INTESTINAL PEPTIDE (VIP) AND PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) AS MODULATORS OF BOTH INNATE AND ADAPTIVE IMMUNITY Crit. Rev. Oral. Biol. Med., May 1, 2002; 13(3): 229 - 237. [Abstract] [Full Text]
|
|
|
|
 |
|
 H. Ni, J. Capodici, G. Cannon, D. Communi, J.-M. Boeynaems, K. Kariko, and D. Weissman Extracellular mRNA Induces Dendritic Cell Activation by Stimulating Tumor Necrosis Factor-alpha Secretion and Signaling through a Nucleotide Receptor J. Biol. Chem., April 5, 2002; 277(15): 12689 - 12696. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Lipscomb and B. J. Masten Dendritic Cells: Immune Regulators in Health and Disease Physiol Rev, January 1, 2002; 82(1): 97 - 130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Stober, R. Schirmbeck, and J. Reimann IL-12/IL-18-Dependent IFN-{{gamma}} Release by Murine Dendritic Cells J. Immunol., July 15, 2001; 167(2): 957 - 965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Wilkin, X. Duhant, C. Bruyns, N. Suarez-Huerta, J.-M. Boeynaems, and B. Robaye The P2Y11 Receptor Mediates the ATP-Induced Maturation of Human Monocyte-Derived Dendritic Cells J. Immunol., June 15, 2001; 166(12): 7172 - 7177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Dunzendorfer, A. Kaser, C. Meierhofer, H. Tilg, and C. J. Wiedermann Cutting Edge: Peripheral Neuropeptides Attract Immature and Arrest Mature Blood-Derived Dendritic Cells J. Immunol., February 15, 2001; 166(4): 2167 - 2172. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or
presence of 5 µg/ml polymixin B (
) or of 10-6 M
VIP-receptor antagonist (
). The expression of CD83 was analyzed by
FACS after 4 days. Results are expressed in percentage of positive
cells, mean ± SD of three separate experiments. B,
VIP synergizes with TNF-
