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*
Department of Biological Sciences, Rutgers University, Newark, NJ 07102; and
Departamento Biologia Celular, Universidad Complutense, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2. In vivo administration of VIP or PACAP in
Ag-immunized mice reduce the numbers of IFN--secreting cells and
enhance the numbers of IL-4-secreting cells. One of the consequences of
the VIP-/PACAP-induced shift in cytokine profile is a change in the
Ag-specific Ig isotype, increasing IgG1 and decreasing IgG2a levels.
Finally, the preferential differentiation into Th2 effector cells after
Ag stimulation induced by VIP-/PACAP-treated macrophages is mediated
through the up-regulation of B7.2 expression. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
TNF-ß) critical for the generation of a cellular immune response,
thereby activating macrophages, inducing delayed-type hypersensitivity
responses, and stimulating IgG2a Ab production in mice. Th2 cells
produce IL-4, IL-5, and IL-10, which are critical for IgG1 and IgE Ab
production and immunity against helminthic parasites and inhibit
macrophage activation and Ag presentation, thereby down-regulating the
cellular immune responses. Because their respective cytokines act
antagonistically, these two cell subpopulations regulate each other’s
function (1). The molecular basis for the differentiation
of Th cells is just beginning to be delineated. Activation of precursor
Th cells (Th0) results in the production of IL-2 with relatively little
IFN- or IL-4. Subsequent events appear to bias the cells toward
differentiation into the Th1 or Th2 phenotype. Determining factors
include the nature of the APC, the nature and amount of Ag, and the
genetic background of the host, with the cytokine microenvironment as
the dominant factor. Increasing evidence demonstrates that IL-12 and,
to a lesser extent IFN-, direct CD4+ T cells
to differentiate into Th1. In contrast, IL-4 is necessary to induce the
development of Th2. When both IL-4 and IL-12 are added to in vitro
cultures, IL-4 dominates over IL-12, driving naive
CD4+ T cells toward the Th2 phenotype
(1). The activation of naive CD4+ T lymphocytes requires two signals delivered by APCs, leading to enhanced cytokine production and proliferation. The first signal, which confers specificity, is provided by the interaction of the antigenic peptide/MHC complex with the T cell receptor. The second signal is provided by costimulatory molecules expressed on APCs. Among the accessory molecules, the B7 family appears to be the most potent. The B7 costimulatory pathway involves at least two molecules, B7.1 (CD80) and B7.2 (CD86), both of which can interact with their counterreceptors, CD28 and CTLA-4, on T cells (2). B7.1 and B7.2 are expressed on a variety of APCs, including B cells, dendritic cells, Langerhans’ cells, and monocytes (reviewed in Ref. 2) and are regulated separately as demonstrated by the kinetics of their expression on APCs upon activation (3), as well as their regulation by cytokines and inflammatory mediators (4). The biologic significance of the differential expression of B7.1 and B7.2 is not clear. Although signals provided through B7/CD28 interactions appear to be essential for the development of Th2-type cytokine responses (5, 6, 7, 8), the question whether B7.1 and B7.2 play different roles in the differentiation of Th cells is still under debate. Signals or agents that trigger the B7 accessory molecules are likely to be essential for the generation of an immune response, and their dysregulation may be responsible for certain autoimmune diseases.
Vasoactive intestinal peptide (VIP)3 and the structurally related peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), are two neuropeptides present in the immune microenvironment (9, 10), with a broad spectrum of biologic functions, including actions on natural and acquired immunity (reviewed in Refs. 10, 11, 12). VIP and PACAP bind to specific receptors expressed in immune cells (13, 14, 15) and regulate T cell cytokine production and proliferation, and several macrophage functions, expressing predominantly an anti-inflammatory activity (reviewed in Refs. 11 and 16). In a recent study, we reported that VIP and PACAP differentially regulate B7.1 and B7.2 expression in resting murine macrophages (36). Moreover, both neuropeptides enhance the costimulatory activity of macrophages for anti-CD3 and allogeneic stimulated naive T cells, by up-regulating B7.2 expression. In the present study, we investigate the role of VIP and PACAP on macrophage B7 expression and costimulatory function for Ag-primed CD4+ T cells, and on the macrophage-induced regulation of Th1/Th2 differentiation in vitro and in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b) and DBA/2 (H-2d) mice obtained from The Jackson Laboratory (Bar Harbor, ME) were kept in pathogen-free conditions. All mice used were between 7 and 12 wk of age.
Reagents and Abs
Synthetic VIP and PACAP38 were purchased from Novabiochem
(Laufelfingen, Switzerland). Keyhole limpet hemocyanin (KLH) was
purchased from Calbiochem (San Diego, CA). Monoclonal Abs to Mac-1,
CD24, Thy-1, CD8, IgM µ-chain (6B2), B7.1 (IG10, rat IgG2a) and B7.2
(GL1, rat IgG2a), and capture and biotinylated monoclonal
anti-murine IL-4, IL-2, IL-5, and IFN- Abs were purchased from
PharMingen (San Diego, CA). OVA, 5-bromo-4-chloro-3-indolyl-phosphate,
nitroblue tetrazolium, and avidin-peroxidase were purchased from Sigma
(St. Louis, MO).
Cell cultures
All cells were cultured in DMEM (HyClone Laboratories, Logan, UT) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 10 µg/ml streptomycin, and 2% FCS (Life Technologies, Grand Island, NY).
In vivo followed by in vitro Ag-stimulation of T cells
To generate KLH-specific CD4+ T cells, DBA/2 mice were immunized with 150 µg KLH in CFA in the footpads as previously described by DeKruyff et al. (17). Two weeks later, the draining lymph nodes were used as a source for the KLH-primed CD4+ T cells. Single-cell suspensions were depleted of B cells by adherence to goat anti-mouse Ig-coated dishes (5 µg/ml; 100 µl per well) for 1 h at 4°C. The remaining cells were depleted of CD8+ T cells, residual B cells, and other accessory cells by treating the cells with a mixture of anti-CD8 (clone 53-6.7), anti-class II (clone 34-2-12), and anti-CD24 (clone J11d) mAbs on ice for 10 min, followed by complement-mediated lysis (rabbit complement from Pel Freeze, Rogers, AR) at 37°C for 45 min. The purified T cells were >98% CD4+ by FACS analysis.
Nonimmune DBA/2 mice were used as a source for peritoneal macrophages. Mice were injected i.p. with 2 ml of 3% thioglycollate broth (Difco, Detroit, MI), and 4 days later, the animals were killed, injected i.p. with 5 ml of cold DMEM, and the peritoneal fluid was harvested. The peritoneal exudate cells were washed and treated with anti-Thy-1 and anti-B220 mAbs followed by complement-mediated lysis to remove contaminating B and T cells. The surviving cells were >96% macrophages as judged by Mac-1+ expression (FACS analysis) and by morphologic and phagocytic criteria.
Purified peritoneal macrophages (1 x 106 cells/ml) were allowed to adhere for 24 h, followed by removal of possible nonadherent contaminants by washing, and cultured in the presence or absence of different concentrations of VIP or PACAP, for 22 h at 37°C. After washing, the VIP-/PACAP-treated macrophages (1 x 105 cells) were added to the Ag-primed CD4+ T cells (4 x 105 cells/well) in the presence of soluble KLH (10 µg/ml). T cell proliferation was determined as described below.
In vivo Ag stimulation of T cells
C57BL/6 mice were immunized i.p. with 50 µg of KLH in monophosphoryl lipid a + synthetic trehalose dicorynomycolate adjuvant (RIBI Immunochemical Research, Hamilton, MT) as previously described (18, 19). Two weeks later, the mice were challenged i.p. with 100 µg of KLH, in PBS. One group of mice was bled immediately before Ag challenge for serum Ig measurements. VIP or PACAP (5 nmol/mouse, 30 µg neuropeptide/kg) and/or anti-B7.1, anti-B7.2, or isotype-matched control Abs (100 µg Ab/mouse) were injected i.p. simultaneously with the Ag challenge. Two weeks later, serum samples were obtained and assayed for anti-KLH Abs. In some experiments, splenocytes and peritoneal cells were obtained 2 weeks after the Ag challenge, stimulated in vitro with soluble Ag (50 µg KLH), and subjected to enzyme-linked immunospot (ELISPOT) analysis.
FACS analysis
DBA/2 mice were injected i.p. with medium alone or with VIP or PACAP (5 nmol/mouse, 30 µg neuropeptide/kg). After 8 h, macrophages were purified from the peritoneal exudate as indicated above. Cells were washed twice with PBS containing 0.1% sodium azide plus 2% heat-inactivated FCS and incubated with anti-B7.1 or anti-B7.2 mAbs (2.5 µg/ml) at 4°C for 1 h. Isotype-matched Abs were used as controls, and IgG block (purchased from Sigma) was used to block the nonspecific binding to Fc receptors. The cells were washed and further stained with FITC-conjugated goat F(ab')2 anti-rat IgG (Sigma) (2.5 µg/ml) for 30 min at 4°C. After extensive washing, the cells were fixed in 1% buffered paraformaldehyde. The stained macrophages, gated according to scatter characteristics, were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Fluorescence data are expressed as mean channel fluorescence and as percentage of positive cells after subtraction of background isotype-matched values.
Induction of cytokine synthesis in CD4+ T cells
Purified KLH-primed CD4+ T cells were
incubated in 96-well plates (final volume, 150 µl of complete DMEM)
at a concentration of 4 x 105 cells/well
with various numbers of syngeneic macrophages preincubated with VIP or
PACAP for 18 h; soluble KLH (10 µg/ml) was added to the
cultures. Samples were performed in quadruplicate. OVA (10 µg/ml) was
used as an irrelevant Ag. Culture supernatants were harvested on day 2
or 4 as indicated and assayed for levels or IL-2, IL-4, IL-5, and
IFN- by ELISA. In some experiments, the T cells were recovered after
4 days of culture and restimulated at a concentration of 4 x
105 cells/well with fresh normal peritoneal
macrophages (1 x 105 cells/well) and Ag
(KLH, 10 µg/ml). Supernatants were harvested 24 h after
restimulation.
Cytokine ELISA
The content of cytokines in the supernatants was determined by
specific sandwich ELISAs as previously described. Briefly, 96-well
plates were coated overnight with primary anti-cytokine capture Ab.
The plates were washed and blocked, and dilutions of supernatants or
standards were added. Dilutions of culture supernatants were incubated
overnight at 4°C, and after washing, the wells were incubated with
biotin-conjugated anti-cytokine detecting mAbs. After a 2-h
incubation, the plates were washed and a horseradish
peroxidase-streptavidin conjugate was added. The plates were incubated
for an additional hour, and after washing,
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate was
added. After developing, the OD was determined at 405 nm. The cytokine
amount in each supernatant was extrapolated from the standard curve.
The Ab pairs used were as follows, listed by capture/biotinylated
detection Abs: IL-4, BVD4–1D11/BVD6–24G2; IFN-, R4–6A2/XMG1.2;
IL-5, TRFK5/TRFK4; IL-2, and JES6–1A12/JES6–5H4.
Assay of the macrophage costimulatory activity
The proliferation of the KLH-specific T cells was determined by incubating the KLH-primed CD4+ T cells in flat-bottom 96-well plates (4 x 105 cells/well) with KLH (10 µg/ml) and varying numbers of macrophages, in 150 µl of complete DMEM. Samples were assayed in quadruplicate. OVA (10 µg/ml) was used as an irrelevant Ag. The cultures were pulsed with 0.5 µCi [3H]TdR (specific activity, 97 Ci/mmol; Amersham, Arlington Heights, IL) for the last 16 to 18 h of a 4-day culture, and the proliferation was determined by measuring [3H]TdR incorporation in a ß-scintillation counter (Beckman, Palo Alto, CA). Results are expressed as the mean cpm ± SD of triplicate assays.
ELISPOT assay
The frequency of Ag-specific T cells producing IFN- or IL-4
was determined by the ELISPOT technique according to the supplier’s
protocol (PharMingen). Groups of four C57BL/6 mice were immunized and
challenged with KLH as described above. Fourteen days after the Ag
challenge, 5 x 107/ml splenocytes or 1
x 107/ml peritoneal cells were stimulated ex
vivo with 50 µg/ml KLH in complete DMEM for 24 h. Viable cells
were recovered by passage through a Histopaque 1007 density gradient
(Sigma). The viable cells were washed, serially diluted, and seeded in
nitrocellulose-bottomed 96-well Milititer HA plates (Millipore,
Bedford, MA) precoated with anti-IL-4 (BVD4-1D11) or
anti-IFN- (R4-6A2) capture mAbs (5 µg/ml in 0.1 M bicarbonate
buffer, pH 8.2, for 24 h at 4°C). The cells were cultured for
20 h at 37°C in 5% CO2. After washing
with PBS (three times), followed by 0.05% (v/v) Tween-20/PBS (three
times), the cytokines captured on the cellulose ester membranes were
detected with biotinylated anti-IL-4 (BVD6–24G2) or
anti-IFN- (XMG1.2) mAbs. Spots, representing single IL-4- or
IFN--producing cells were visualized using avidin-peroxidase and
5-bromo-4-chloro-3-indolyl-phosphate/nitroblue tetrazolium and were
counted using a dissecting microscope. Data represent total
cytokine-producing cells/105 splenocytes or
peritoneal cells, calculated from the serially diluted samples. No
spots were detected in unstimulated cultures without KLH or in cultures
stimulated with an irrelevant Ag (OVA, 100 µg/ml).
Determination of Ab responses
Specific anti-KLH Ab responses in the KLH-immunized C57BL/6 mice were determined by ELISA. Briefly, Maxisorb plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 100 µl of soluble KLH (10 µg/ml) in 0.1 M bicarbonate buffer, pH 9.6. After washing with PBS containing 0.05% v/v Tween-20, the plates were blocked with 3% BSA and incubated with serial dilutions of serum for 2 h at 37°C. After washing, biotinylated anti-IgG1 (2.5 µg/ml) and anti-IgG2a (2.5 µg/ml) (Serotec, Oxford, U.K.) were added for 1 h at 37°C. The plates were washed, followed by incubation with streptavidin-horseradish peroxidase. The bound enzyme was detected with the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate, and quantitated at OD405 in an ELISA reader (Bio-Tek Intruments, Winooski, VT). A standard curve was constructed for each assay by coating wells with an isotype-specific anti-mouse Ig followed by addition of known concentrations of the mouse Ig isotype as previously described (20). The remaining steps for the ELISA were identical with those used for the KLH-coated wells. The values for the experimental samples were interpolated using the standard curve.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have recently reported that VIP and PACAP enhance the
costimulatory activity of macrophages for naive T cells stimulated with
anti-CD3 or in allogeneic reactions (36). In this study, we
investigate whether VIP and PACAP regulate the costimulatory capacity
of macrophages for the proliferation of Ag-primed T cells. DBA/2
peritoneal macrophages were treated with VIP or PACAP for 24 h,
washed, and added to the responder T cells in the presence of soluble
KLH. The responder T cells were obtained from the draining lymph nodes
of DBA/2 mice injected with KLH in CFA 2 weeks previously. The
proliferation of the responder T cells after exposure to macrophages
and soluble Ag was measured through [3H]TdR incorporation.
Macrophages cultured with medium alone had a low stimulatory activity
for the proliferation of CD4+ T cells. However,
VIP and PACAP significantly up-regulated the costimulatory capacity of
macrophages (Fig. 1
). The rate of
proliferation increased with the number of VIP-/PACAP-treated
macrophages. The ability of VIP and PACAP to induce the costimulatory
activity of macrophages was dose dependent, with maximal effects in the
10-7 to 10-9 M
concentration range (Fig. 1).
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In a previous study, we showed that VIP and PACAP up-regulate
expression of B7.2 but not B7.1 on macrophages over an extended time
period (from 12 to 72 h) (36). Recent studies indicated that
expression of B7, and especially of B7.2, is essential for the
development of Th2 effector cells. Therefore, we asked whether the
cytokine profile of the Ag-primed CD4+ T cells
cultured in the presence of VIP- or PACAP-pretreated macrophages would
be altered. Peritoneal macrophages obtained from DBA/2 mice were
cultured in vitro with VIP or PACAP for 18 h, washed, and added to
syngeneic CD4+ T cells purified from lymph nodes
of KLH-primed mice in the presence of soluble Ag. Stimulation with
macrophages pretreated with medium alone resulted in the development of
effector T cells producing high levels of IFN- and low levels of
IL-4 (Fig. 2). However, pretreatment of
macrophages with either VIP or PACAP led to the inhibition of IFN-
and to significant increase in the IL-4 production (Fig. 2). In
addition to the effect on IL-4 and IFN- production, macrophages
pretreated with VIP or PACAP developed an increased capacity to induce
another Th2 cytokine, IL-5, and a reduced capacity to induce IL-2 (Fig. 3). No cytokine production by T cells was
detectable in the absence of macrophages (data not shown) or in the
presence of an unrelated Ag (see Fig. 2 legend). Thus, pretreatment of
macrophages with VIP or PACAP enhances their capacity to preferentially
induce Th2-cytokine synthesis and inhibits Th1-cytokine synthesis.
These results suggest that Ag-specific T cells stimulated with VIP- or
PACAP-treated macrophages preferentially differentiate into Th2
effector cells. The change in cytokine profile induced by VIP and PACAP
was even more pronounced when the T cells were restimulated after 4
days with normal APCs and KLH (Fig. 3, lower panels
).
This was to be expected whether costimulation with VIP-/PACAP-treated
macrophages led to an increased number of Th2 effector cells.
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After activation by Ag, naive CD4+ T cells
differentiate into Th1 and/or Th2 effector cells. To identify the
differentiation pathway that is predominantly activated by VIP and
PACAP after immunization, we analyzed the number of cells that release
Th1 vs Th2 cytokines. To raise a sufficient number of Ag-reactive T
cells, C57BL/6 mice were immunized with KLH emulsified in adjuvant and
challenged with soluble KLH 2 weeks later. VIP and PACAP were injected
simultaneously with the Ag challenge. Two weeks after the Ag challenge,
splenocytes and peritoneal cells were harvested and restimulated in
vitro for 24 h with KLH, presented by spleen or peritoneal APCs.
The live cells were recovered, and the frequency of Ag-reactive Th1 and
Th2 cells was assessed on the basis of IFN-- and IL-4-secreting
cells, respectively, using ELISPOT assays. Mice injected with the Ag in
the absence of VIP/PACAP developed high numbers of IFN--producing
Th1 cells and very low numbers of IL-4-producing Th2 cells (Fig. 4). In contrast, mice injected with VIP
or PACAP generated few IFN--specific spots, whereas they developed
high numbers of IL-4-secreting Th2 cells (Fig. 4). The results obtained
by this approach mirror the in vitro changes seen by the analysis of
the Th1 and Th2 cytokine profile and confirm that VIP and PACAP shift
the T cell response to Th2 differentiation and/or proliferation, in
vivo as well as in vitro.
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Th1 and Th2 lymphocytes differentially affect the shift from the T
cell-independent IgM isotype to IgG isotypes in activated B
lymphocytes, inducing a switch to IgG2a/b and IgG1, respectively
(21). Because VIP/PACAP favor the differentiation into Th2
effector cells, we expected a switch in anti-KLH Abs toward the
IgG1 isotype. C57BL/6 mice were immunized with KLH emulsified in
adjuvant, followed by a challenge with free KLH, and the isotype of the
serum anti-KLH Abs was determined 2 weeks later. The mice injected
with KLH in the absence of VIP/PACAP produced IgG2a, but not IgG1
anti-KLH Abs (Fig. 5). In contrast,
mice injected with VIP or PACAP simultaneously with the second Ag
challenge produced IgG1 (but not IgG2a) anti-KLH Abs (Fig. 5). This
remarkable IgG isotype composition indicates a nearly exclusive
activation of Th2 cells, in agreement with the results obtained by
ELISPOT.
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As a result of LPS and/or IFN- activation, B7.2 expression on
macrophages occurs earlier and at higher levels than B7.1. In a
previous study, we determined that VIP and PACAP induce B7.2, but not
B7.1, expression in macrophages, at least during an initial period of
up to 72 h (36). Here, we determined whether VIP and PACAP
up-regulate B7.2 expression in vivo and whether the expression of B7.2
is responsible for the preferential development of a Th2 response.
DBA/2 mice were injected i.p. with 5 nmol VIP or PACAP (30 µg
neuropeptide/kg), and B7 expression was analyzed on purified peritoneal
macrophages 8 h later by flow cytometry. As expected, VIP and
PACAP increased in vivo B7.2, but not B7.1, expression on macrophages
(Fig. 6A). In addition, the in
vivo treatment with VIP or PACAP enhanced the macrophage costimulatory
activity for Ag-primed CD4+ T cells (Fig. 6B, top panel). Moreover, macrophages from VIP-
and PACAP-treated mice preferentially induced a Th2 cytokine profile in
the Ag-primed CD4+ T cells (Fig. 6B,
middle and bottom panels).
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production were assayed by ELISPOT. Anti-B7.2, but not
anti-B7.1 Abs, reversed the VIP effect, increasing IFN- and
reducing IL-4 production (Fig. 7A).
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and IL-4 were determined by
ELISA in the culture supernatants. Again, anti-B7.2, but not
anti-B7.1 Abs, reversed the effects of VIP, by increasing IFN-
and decreasing IL-4 production (Fig. 7
B). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2. Previous reports from our laboratory indicated that VIP and PACAP inhibit the production of IL-2, IL-4, and IL-10 from naive T cells stimulated with Con A or anti-CD3 (11, 22, 23, 24). The inhibitory effect of VIP on IL-4 production by naive T cells is in apparent contradiction with the results presented in this study, in which VIP and PACAP promote IL-4 production. However, the two experimental systems are different. In the first system we investigated the effect of VIP directly on naive T cells stimulated with Con A or anti-CD3 mAbs. Moreover, we concluded that the effect of VIP on IL-4 production by T cells is indirect, being mediated through the reduction in available IL-2 (23). Here, we investigate the effects of VIP/PACAP on macrophages and indirectly on the differentiation of Ag-primed Th cells. The possibility that VIP/PACAP could be carried over from the macrophage to the T cell cultures is extremely unlikely, because the pretreated macrophages were washed extensively, and the neuropeptides have a very short life span in culture.
In our experiments we used thioglycollate-injected mice to increase the number of harvested peritoneal macrophages. Although thioglycollate-induced macrophages are not identical with quiescent cells, they express low levels of B7.2 and no B7.1. In contrast, LPS-activated macrophages express B7.1 and up-regulate significantly the expression of B7.2 (reviewed in Ref. 25). In preliminary experiments, we determined that VIP and PACAP affect quiescent macrophages the same way as thioglycollate-induced ones, by up-regulating B7.2, but not B7.1.
Because the Ag-primed T cells exposed to VIP-/PACAP-treated macrophages
secrete primarily Th2-type cytokines, we propose that the
VIP-/PACAP-treated macrophages preferentially induced differentiation
into Th2 effector cells. This conclusion is supported by the ELISPOT
experiments after the in vivo administration of VIP or PACAP in mice
immunized and challenged with Ag. Similar to the in vitro experiments,
which indicated an increase in IL-4 production and a decrease in
IFN- production, the in vivo inoculation of VIP and PACAP led to
significantly reduced numbers of IFN--secreting cells and enhanced
numbers of IL-4-secreting cells.
One of the consequences of the shift in cytokine profile is the change
in the Ig isotype. Cytokines such as IFN- and IL-4 regulate the Ig
isotype switch. In mice, IL-4 promotes IgG1 (26, 27) and
inhibits IgG2a (28), whereas IFN- has the opposite
effects (28). Therefore, we expected that the in vivo
treatment with VIP or PACAP would induce a shift in the anti-KLH Ab
isotype, with an increase in IgG1 and a decrease in IgG2a. This was
indeed the case.
One of the most interesting questions is the nature of the mechanism by
which VIP and PACAP enable macrophages to induce Th2 differentiation.
We have recently shown that VIP and PACAP specifically up-regulate the
expression of B7.2 on peritoneal macrophages and that the enhanced B7.2
expression is responsible for the increase in the macrophage
stimulatory activity for naive T cells (36). Similar findings are
reported in this study. VIP and PACAP were found to up-regulate B7.2
expression on macrophages in vivo, at least in the short term, and to
enhance both their costimulatory activity and their subsequent capacity
to increase IL-4 and decrease IFN- production. The direct
involvement of B7.2 in the switch in cytokine production is supported
by the fact that the in vivo administration of anti-B7.2 Abs
reversed the effect of VIP on both cytokine production and number of
cytokine-secreting cells. Although the role of B7.1 vs B7.2 in
selectively activating the Th1- or the Th2-type response is
controversial, several in vivo disease models suggest that B7.2 plays
the essential role in the differentiation of Th2 cells
(29, 30, 31, 32). This is supported by the different cytokine
profile exhibited by B7.1-/- and
B7.2-/- deficient mice in a model of murine
schistosomiasis. Whereas the B7.1-/- mice do
not differ significantly from the wild type, the
B7.2-/- mice express high levels of IFN- and
low levels of IL-4 and IL-10, suggesting the involvement of B7.2 in Th2
differentiation (33). However, the double
B7.1-/B7.2-deficient mice exhibit even more pronounced changes in the
cytokine profile, with very high levels of IFN- and no IL-4 or IL-10
(33). The most direct interpretation is that, although
B7.2 alone induces Th2 differentiation whereas B7.1 does not, together
the two isoforms act in a complementary manner.
In addition to the MHC haplotype, the dose and nature of the Ag, the
route of Ag administration, and the availability of IFN- and IL-12,
as opposed to IL-4, are decisive for the differentiation to Th1 and
Th2, respectively (1, 34). We have recently reported that
VIP and PACAP inhibit IL-12 production by activated macrophages, with
the subsequent inhibition of IFN- production by T cells
(35). The neuropeptide-induced absence of IL-12 and
IFN-, two cytokines necessary for development of Th1 cells, may
further amplify the bias toward Th2 differentiation.
In conclusion, our observations suggest that pretreatment of
macrophages with VIP/PACAP enhances their costimulatory capacity for
both naive and Ag-primed T cells, through the up-regulation of B7.2
expression. The VIP/PACAP treatment enables macrophages to initiate a
predominant Th2 response after Ag stimulation. In addition, the
regulatory effect of VIP/PACAP on IL-12 production by macrophages and
subsequently on IFN- production by activated T cells could further
promote Th2 differentiation and inhibit the initiation of Th1
responses. Because Th1-dominated responses are associated with
inflammatory reactions typical of cell-mediated immunity, the VIP/PACAP
inhibition of Th1 development may represent an additional mechanism for
the general anti-inflammatory activity of the two
neuropeptides.
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Footnotes |
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2 Address correspondence and reprint requests to Mario Delgado, Rutgers University, Department of Biological Sciences, 101 Warren Street, Newark, NJ 07102. E-mail address:
3 Abbreviations used in this paper: VIP, vasoactive intestinal peptide; PACAP, pituitary adenylate cyclase-activating polypeptide; KLH, keyhole limpet hemocyanin; ELISPOT, enzyme-linked immunospot.
Received for publication April 28, 1999. Accepted for publication July 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 switch regions in activated murine B cells by IL-4. EMBO J. 8:483.[Medline]
and B cell stimulating factor-1 reciprocally regulate Ig-isotype production. Science 236:944. synthesis by T cells. J. Neuroimmunol. 96:167.[Medline]
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E. Gonzalez-Rey, A. Fernandez-Martin, A. Chorny, J. Martin, D. Pozo, D. Ganea, and M. Delgado Therapeutic Effect of Vasoactive Intestinal Peptide on Experimental Autoimmune Encephalomyelitis: Down-Regulation of Inflammatory and Autoimmune Responses Am. J. Pathol., April 1, 2006; 168(4): 1179 - 1188. [Abstract] [Full Text] [PDF]
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 V. Sharma, M. Delgado, and D. Ganea Granzyme B, a New Player in Activation-Induced Cell Death, Is Down-Regulated by Vasoactive Intestinal Peptide in Th2 but Not Th1 Effectors J. Immunol., January 1, 2006; 176(1): 97 - 110. [Abstract] [Full Text] [PDF]
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 D. Miotto, P. Boschetto, I. Bononi, E. Zeni, G. Cavallesco, L.M. Fabbri, and C.E. Mapp Vasoactive intestinal peptide receptors in the airways of smokers with chronic bronchitis Eur. Respir. J., December 1, 2004; 24(6): 958 - 963. [Abstract] [Full Text] [PDF]
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S. Kodali, W. Ding, J. Huang, K. Seiffert, J. A. Wagner, and R. D. Granstein Vasoactive Intestinal Peptide Modulates Langerhans Cell Immune Function J. Immunol., November 15, 2004; 173(10): 6082 - 6088. [Abstract] [Full Text] [PDF]
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 D. POZO and M. DELGADO The many faces of VIP in neuroimmunology: a cytokine rather a neuropeptide? FASEB J, September 1, 2004; 18(12): 1325 - 1334. [Abstract] [Full Text] [PDF]
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 H. Keino, T. Kezuka, M. Takeuchi, N. Yamakawa, T. Hattori, and M. Usui Prevention of Experimental Autoimmune Uveoretinitis by Vasoactive Intestinal Peptide Arch Ophthalmol, August 1, 2004; 122(8): 1179 - 1184. [Abstract] [Full Text] [PDF]
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Y. Zafirova, M. Yordanov, and R. Kalfin Antiarthritic effect of VIP in relation to the host resistance against Candida albicans infection Int. Immunol., August 1, 2004; 16(8): 1125 - 1131. [Abstract] [Full Text] [PDF]
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 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]
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 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]
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E. Kuroda and U. Yamashita Mechanisms of Enhanced Macrophage-Mediated Prostaglandin E2 Production and Its Suppressive Role in Th1 Activation in Th2-Dominant BALB/c Mice J. Immunol., January 15, 2003; 170(2): 757 - 764. [Abstract] [Full Text] [PDF]
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 A. Metwali, A. M. Blum, D. E. Elliott, and J. V. Weinstock IL-4 inhibits vasoactive intestinal peptide production by macrophages Am J Physiol Gastrointest Liver Physiol, July 1, 2002; 283(1): G115 - G121. [Abstract] [Full Text] [PDF]
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 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]
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M. Delgado and D. Ganea Cutting Edge: Is Vasoactive Intestinal Peptide a Type 2 Cytokine? J. Immunol., March 1, 2001; 166(5): 2907 - 2912. [Abstract] [Full Text] [PDF]
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