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Departments of Medicine and Microbiology-Immunology, University of California Medical Center, San Francisco, California 94143-0711, USA
1Correspondence: University of California, UB8B, Box 0711, 533 Parnassus at 4th, San Francisco, CA 94143-0711, USA. E-mail : egoetzl{at}itsa.ucsf.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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after TCR activation. VPAC2 R TG mice consequently have significant elevations of blood IgE, IgG1, and eosinophils. VPAC2 R TG mice also show increased IgE antibody responses, which mediate heightened cutaneous allergic reactions, and have depressed delayed-type hypersensitivity. VIP enhancement of the ratio of Th2 cell to Th1 cell cytokines thus evokes an allergic state in normally nonallergic mice, which suggests the possibility of neuropeptide contributions to immune phenotypic alterations in human hypersensitivity diseases.—Voice, J. K., Dorsam, G., Lee, H., Kong, Y., Goetzl, E. J. Allergic diathesis in transgenic mice with constitutive T cell expression of inducible vasoactive intestinal peptide receptor.
Key Words: neuropeptide • IgE • cytokines • eosinophils • hypersensitivity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. VIP has potent effects on T cell differentiation, migration, and generation of diverse cytokines (4
5
6
7
8
9
10
11
12)
. Production of cytokines by helper T (Th) cells in vitro is regulated differentially by VIP with inhibition of some from type 1 Th (Th1) cells, which mediate cellular immunity to infections, and enhancement of some from type 2 Th (Th2) cells that mediate hypersensitivity reactions such as allergy. Although VIP suppression of interleukin 2 (IL-2) and enhancement of IL-5 generation by Th cells have been observed consistently, effects of VIP on other cytokines have depended on the source and mechanism of activation of the T cells. High levels of type I G-protein-coupled VIP receptors (VPAC1 Rs) and far fewer of the homologous VPAC2 Rs are expressed by unstimulated Th cells in blood and spleen (13
14
15
16
17
18)
. However, VPAC2 Rs are up-regulated to high levels and VPAC1 Rs down-regulated by Th cell stimulation, suggesting that VPAC2 Rs are the dominant transducer of effects of VIP on activated Th cells (19
20
21
22)
. Investigations of the capacity of VIP to enhance Th2-mediated hypersensitivity reactions in vivo through VPAC2 Rs have been hampered by the lack of effective pharmacological agents. We now describe a transgenic (TG) model in which inducible VPAC2 Rs are constitutively expressed in CD4+ (helper-inducer) T cells of the Th1 cell-dominant C57BL/6 strain of mice, at levels greater than those observed at the peak of hypersensitivity responses, so that endogenous VIP suppresses Th1 cells and activates Th2 cells. These mice have elevated blood immunoglobulin E (IgE), IgG1, and eosinophils with increased IgE antibody responses that heighten cutaneous allergic reactions. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and a 4.6 kb minigene encoding the lck promoter 5' to the huVPAC2 R (LCK-huVPAC2) was liberated with NcoI and PvuII (Promega). C57BL/6 oocytes were injected with LCK-huVPAC2 and introduced into pregnant Balb/c mice. A male founder C57BL/6 pup expressing huVPAC2 in tail DNA was bred with normal C57BL/6 females to establish a huVPAC2 R TG colony. Levels of huVPAC2 R-specific DNA in tail tissue of TG mice increased from a mean (±SD) of 3.2 ± 1.2 copies in F2 mice (n=23) to 7.1 ± 4.7 in F3 mice (n=17) and 6.2 ± 1.9 in F4 mice (n=6, range=2.6 to 10), as quantified by real-time/TaqMan PCR.
Real-time PCR and Western blot quantification of huVPAC2 R
RNA from 20–100 µg snap-frozen fragments of tissues and suspensions of 5 to 8 x 106 immunomagnetically purified immune cells (Miltenyi Biotec, Auburn, CA) was extracted with TRIzol (Life Technologies, Gibco-BRL, Grand Island, NY) for amplification of mRNA encoding huVPAC2, murine (m)VPAC2, mVPAC1, and mGAPDH [Perkin-Elmer/Applied Biosystems PRISM 7700 Sequence Detector (Foster City, CA); standard TaqMan PCR reagent kit, ABI User Bulletin #2, PRISM 7700 Sequence Detection System, 1997]. The sequences of the huVPAC2 primers were 5'-CTGCCAATGTGGGAGAGACC-3' and 5'-TCTGGGAACGTCTCTGACCAT-3', and of the TaqMan probe 5'-FAM-ACGGTGCCCTGCCCAAAAGTCTTC-TAMRA-3'; those of mVPAC2 were 5'-AAGCAGCCAAACGGAGAATC-3' and 5'-GGCAGGGCACTGTGACAGTT-3', and of the TaqMan probe 5'FAM-CTGCAGCGGTGTCTGGGACAACA-TAMRA-3'; those of mVPAC1 were 5'-AACTTTAAGGCCCAGGTGAAAAT-3' and 5'-CCTGCA-CCTCGCCATTG-3' and, of the TaqMan probe, 5'FAM-TTGTGGTGGCCATCC-TCTACTGCTTCC-TAMRA-3'; and those of mGAPDH primers were 5'-TGCACC-ACCAACTGCTTAG-3' and 5'-GGATGCAGGGATGATGTTC-3' and, of the TaqMan probe, fluorescein-AGAAGACTGTGGATGGCCCCTC-TAMRA-3'. The TaqMan probe end-labels were 5- (and 6-)carboxy-tetramethylrhodamine (TAMRA) (quencher fluorochrome) conjugated to the 3'-terminal nucleotide and 6-carboxyfluorescein (FAM) or fluorescein (reporter fluorochrome) linked to the 5'-terminal nucleotide (Integrated DNA Technologies, Coralville, FL). The CT difference derived by subtracting the threshold cycle (CT) value for mGAPDH from the corresponding CT value for human or murine VPAC2 was expressed as a ratio relative to the CT difference for liver of organ extracts and macrophages for spleen cell extracts, which were set at 1.0. Western blots of 10 µg of each protein extract were performed as described and developed with rabbit polyclonal anti-amino-terminal peptide antibodies to huVPAC1 (amino acids 122–134) and huVPAC2 (amino acids 105–122) (24
, 25)
.
Determination of serum concentrations of immunoglobulins and blood eosinophil counts
Serum Ig concentrations and blood levels of eosinophils were measured in tail vein blood of 8- to 9-wk-old mice anesthetized with methoxyflurane (Metofane, Schering-Plough Animal Health Division, Union, NJ). ELISA kits were for total IgG (Cygnus Technologies, Plainville, MA), IgG1, IgG2a, IgA, IgM (Bethyl Laboratories, Montgomery, TX), and IgE (Crystal Chemical, Chicago, IL). Eosinophils in heparinized tail vein blood diluted 1:5 (v:v) with a mixture of 1 g of phloxine B, 500 ml of propylene glycol (Sigma Fine Chemicals, St. Louis, MO), and 500 ml of distilled water were counted microscopically and expressed as eosinophils/ mm3. Tail vein serum IgE and IgG concentrations were determined by ELISA before (day 0) and on day 14 after two intraperitoneal (i.p.) injections of 100 µg of rat IgG1 anti-IL-4 monoclonal antibody (11B11, BD PharMingen, La Jolla, CA) plus 100 µg of rat IgG2b anti-IL-13 monoclonal antibody (MAB413, R&D Systems, Minneapolis, MN) on days 0 and 7.
Assessment of cutaneous delayed-type hypersensitivity
Groups of 12-wk-old mice were immunized subcutaneously in each flank with 1 mg per site of 4-hydroxy-3-nitrophenylacetyl-hydroxysuccinimide ester (NP OSu; Biosearch Technologies, Novato, CA) in 40 µl of dimethyl sulfoxide, followed by 200 µl of 0.05 M sodium borate-buffered 0.1 M NaCl (pH=8.6) in the dorsal midline skin. Six days later, one rear footpad was challenged with 40 µg of NP OSu in 1 µl of dimethyl sulfoxide:10 µl of PBS and the opposite rear footpad received 11 µl of dimethyl sulfoxide:PBS alone. Footpad thickness was quantified before and 24 and 48 h after the challenge injection, using a calibrated digital read-out micrometer with 0.025 mm resolution (Fisher Scientific, Boston, MA).
Assessment of serum concentration of IgE anti-TNP antibodies and active cutaneous anaphylaxis
Groups of 8- to 12-wk-old huVPAC2 R TG mice and normal control mice were immunized i.p. with 10 µg of trinitrophenyl-derivatized keyhole limpet hemocyanin (TNP-KLH; Biosearch Technologies) adsorbed to 0.2 mg of Al(OH)3. Each mouse was bled 14 days later (primary response), boosted with 10 µg of TNP-KLH i.p., and bled 1 wk after the booster dose (secondary response). IgE anti-TNP antibody levels in tail vein sera were quantified by ELISA using 0.5 µg of TNP chicken gamma globulin (CGG) (Biosearch Technologies) to coat each well of 96-well plates, blocking each well with 0.2 ml of 1 g BSA/100 ml, then incubating sequentially with 0.1 ml of serum diluted 1/10, 1/30, and 1/100, 0.1 ml of a 1:20,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgE (Bethyl Labs., Montgomery, TX) and finally standard development reagents for peroxidase. Values of absorbancy were converted to ng/ml using a curve generated with monoclonal IgE anti-TNP (clone C38–2; BD PharMingen). IgG1 anti-TNP antibody levels were quantified similarly but using 1/300, 1/1000 and 1/3000 dilutions of sera, a 1:40,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG1 (Bethyl Labs., Montgomery, TX), and monoclonal IgG1 anti-TNP antibody (BD PharMingen) as a standard. Groups of immunized normal and huVPAC2-TG mice were challenged 2 wk after primary immunization and 1 wk after the booster dose with 1.25 µg of TNP-KLH in 5 µl of PBS in one rear footpad. The opposite rear footpad received 5 µl of PBS alone. Footpad thickness was quantified before and 1 and 6 h after the challenge injection, using a calibrated digital read-out micrometer with 0.025 mm resolution (Fisher Scientific, Boston, MA). Antigen-induced and PBS-induced increases in footpad thickness are expressed as a percentage of that before injection. The significance of differences between swelling evoked by TNP-CGG and by PBS alone in each group of mice was calculated with a standard Student’s paired t test. One group of mice also were challenged 2 wk after primary immunization and 1 wk after the booster dose with 0.5 µg and 2.0 µg of TNP-CGG in 5 µl of PBS in multiple cutaneous sites on the flanks and PBS alone in several sites within 5 min of 0.4 ml of 0.5 g% of i.v. Evans blue. Mice were killed 30 min later and flank skin was reflected to allow measurement of the mean diameter of extravasated Evans blue; the diameter of TNP-induced reactions was corrected for that of PBS control sites.
Production and quantification of cytokines by splenic CD4+ T cells
Replicate suspensions of 107 nonadherent splenic mononuclear leukocytes in 400 µl of 0.02 M sodium phosphate-buffered 0.13 M NaCl (pH 7.3) with 2 mM EDTA and 0.5 g/100 ml of fatty acid-free bovine serum albumin (Calbiochem-Novabiochem Corp., La Jolla, CA) were incubated sequentially with 20 µg of biotin-conjugated anti-CD4 monoclonal mouse antibody (BD PharMingen) for 60 min at 4°C and 25 µl of streptavidin-conjugated metallic beads (Miltenyi Biotec, Auburn, CA) for 30 min at 4°C or with anti-CD4 monoclonal antibody-conjugated metallic beads directly. Two cycles of magnetic column chromatography yielded CD4+ T cells of > 96% purity, as assessed by analytical flow cytometry. Replicate 0.5 ml aliquots of 3 x 105 CD4+ T cells in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml of penicillin G, and 100 µg/ml of streptomycin were stimulated in 24-well plates with 0.5 µg/well each of adherent anti-CD3 and anti-CD28 mouse monoclonal antibodies (PharMingen) without and with preincubation for 15 min with 10-9 M to 10-6 M purified synthetic VIP or VPAC1- or VPAC2-selective peptide analogs of VIP. The VPAC1-selective peptide [K15,R16,L27]VIP(1-7)/GRF(8-27), and the VPAC2-selective peptide Ac-[E8, OCH3-Y10, K12, NL (17)
, A19, D25, L26, K27,28]-VIP cyclo (21
22
23
24
25)
were synthesized and purified as described (26
, 27)
. After 24 and 96 h at 37°C, plates were centrifuged and supernatant media were harvested for ELISA assays of IL-4, IL-5, and interferon (IFN-; Endogen, Cambridge, MA). An index describing composite changes in levels of the predominant IgE-determining, T cell-derived cytokines IL-4 (Th2 cells) and IFN- (Th1 cells) was calculated from the ratio of percentage increase in IL-4 to the percentage decrease in IFN-.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). The highest levels of human VPAC2 R mRNA were detected in thymic and splenic tissues. Of the splenic cells, T cells had much higher levels of huVPAC2 R mRNA than B cells and macrophages (Fig. 1A
). The mean level of huVPAC2 R mRNA was 
25-fold higher for CD4+ (helper/inducer) T cells than for CD8+ (suppressor/cytotoxic) T cells. Western blot analyses of huVPAC2 R protein extracted from the same tissues and immune cells confirmed a much higher level of expression in spleen than in liver or brain (Fig. 1B
). Similarly, the expression of huVPAC2 R protein was much higher in T cells than in B cells or macrophages and in CD4+ than CD8+ T cells. The levels of endogenous mVPAC2 R in spleen and unstimulated splenic immune cells were all 1/100 or less of those for huVPAC2 R in TG mice (Fig. 1A
). After 24 h of stimulation of normal CD4+ T cells by adherent monoclonal anti-CD3 + anti-CD28 antibodies, the level of mRNA encoding mVPAC2 R increased to 1/5 of that of huVPAC2 in the TG mice (Fig. 1A
). No tissues or cells of normal C57Bl/6 mice had detectable huVPAC2 R mRNA or protein, and huVPAC1 R mRNA was not found in any tissues or immune cells of huVPAC2 R TG mice. The levels of mRNA encoding mVPAC1 R were the same in spleens of wild-type and TG mice. The TG expression of huVPAC2 R also did not significantly alter the number or composition of splenic T cells (Table 1
).
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Isolated constitutive expression of huVPAC2 Rs by CD4+ T cells of TG mice at a level similar to that of mVPAC2 Rs on activated CD4+ T cells of normal mice was predicted to suppress activities of Th1 cells, promote those of Th2 cells, and enhance immediate-type hypersensitivity mechanisms. The profile of serum immunoglobulins found in huVPAC2 R TG mice was distinctively different from that of normal C57BL/6 mice. Concentrations of IgE and IgG1 that mediate immediate-type hypersensitivity reactions were significantly higher in the TG mice than in normal mice (Fig. 2
). In contrast, there were no differences between TG and normal mice in the serum concentrations of total IgG, IgG2a, IgA, or IgM, which usually have no role in immediate hypersensitivity. Blood levels of eosinophils, a hallmark of allergy, were significantly higher in the TG mice than in age- and sex-matched normal mice (Fig. 2)
, whereas blood basophil counts were the same. These findings are most consistent with an endogenous VIP/TG huVPAC2 R-directed increase in the effective ratio of functional Th2- to Th1-type T cells.
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The generation of several cytokines that define Th subsets was determined to assess relative levels of functional Th1 and Th2 cells in splenic CD4+ T cells. When exposed to adherent antibodies to the TCR for 24 h to recruit memory T cells and for 96 h to activate effector T cells, capable of far greater production of each cytokine, purified splenic CD4+ T cells from normal C57BL/6 mice produced far greater quantities of IFN-, typical of Th1 cells, than of IL-4 and Il-5, typical of Th2 cells (Fig. 3
A). Similarly stimulated splenic CD4+ T cells of the huVPAC2 R TG mice produced significantly more IL-4 and IL-5 and less IFN- than the normal CD4+ T cells at both 24 and 96 h. This alteration in cytokine profile of CD4+ T cells from TG mice is consistent with an increase in effective Th2/Th1 ratio under the influence of endogenous VIP.
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The addition of equipotent concentrations of exogenous VIP or a VPAC2 R-selective agonist to cultures of splenic CD4+ T cells before TCR stimulation resulted in further deviation of the relative levels of production of IFN- and IL-4 by CD4+ T cells from TG mice (but not normal mice) at 96 h and for some conditions at 24 h (Fig. 3B
). The generation of IFN- by CD4+ T cells from TG mice at 24 and 96 h was suppressed by up to a mean of 95% by VIP and up to a mean of 78% by the VPAC2 R-selective agonist. In contrast, generation of IFN- by CD4+ T cells of normal C57Bl/6 mice at 24 and 96 h did not respond significantly to VIP or the VPAC2 R-selective agonist. The generation of IL-4 after 96 h by CD4+ T cells of TG mice was enhanced significantly by VIP and the VPAC2 R-selective agonist, but there was no effect on that of normal CD4+ T cells (Fig. 3B
). For CD4+ T cell production of IL-4 at 24 h, only the VPAC2 R-selective agonist was effective with moderately greater enhancement for TG than normal Th cells. IL-5 production by both TG and normal CD4+ T cells was enhanced only by the VPAC2 R-selective agonist, with no significant differences (Fig. 3B
). The baseline level at both 24 and 96 h was much higher for TG CD4+ T cells (Fig. 3A
). The VPAC1 R-selective agonist had no significant effect on production of any of the cytokines by any CD4+ T cells (Fig. 3B
). The ratio of increase in IL-4 to decrease in IFN- is a sensitive index of the augmentation of Th2 over Th1 cytokines and was calculated for the results of each study (Fig. 3C
). At 24 and 96 h, VIP strikingly augmented the ratio of Th2 to Th1 cytokines secreted by TCR-stimulated CD4+ T cells of TG mice, but not normal mice (Fig. 3C
). The VPAC2 R-selective agonist, but not the VPAC1 R agonist, had a similar but less prominent enhancing effect at both times for CD4+ T cells of TG but not normal mice.
The central role of Th2 cytokines in augmenting blood levels of IgE and eosinophils in human VPAC2 R transgenic mice was examined in vivo by administering courses of neutralizing monoclonal antibodies specific for IL-4 and the similarly active IL-13. The serum level of IgE in F4 TG mice was strikingly higher than in matched normal C57BL/6 mice and was suppressed a mean of 40% by sustained immunoneutralization of IL-4 and IL-13 (Fig. 4
). In contrast, the much lower level of IgE in normal serum was unaffected by the same neutralizing antibodies. The elevated levels of blood eosinophils in this same group of TG mice, which depend on IL-5, were not reduced by specific immunoneutralization of IL-4 and IL-13.
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Levels of antigen-specific IgE antibody attained during primary and secondary responses to TNP-KLH were significantly higher in VPAC2 R TG mice than normal mice (Fig. 5
). In contrast, the level of IgG1 anti-TNP antibody was only marginally higher in VPAC2 R TG mice than normal mice in the primary response and no different in the secondary response. An immediate-type hypersensitivity reaction was elicited in rear footpads of immunized VPAC2 R TG and normal mice to investigate whether the greater elevations in blood IgE antibody and eosinophils were sufficient to mediate enhanced responses. As expected in this nonallergic strain, antigen challenge did not elicit footpad swelling greater than the saline vehicle after primary sensitization of normal mice and resulted in only marginally significant swelling after secondary sensitization (Fig. 6
). In contrast, antigen challenge evoked significantly greater swelling of the footpads of VPAC2 R TG mice after primary and secondary sensitization with peak responses at 1 h. Standard active cutaneous anaphylaxis reactions were also elicited in flank skin of one of the groups of secondarily immunized VPAC2 R TG and normal mice. Challenge with 0.1 µg of TNP-CGG elicited cutaneous reactions at 30 min of 0.86 ± 0.40 mm diameter (mean±SD) in normal mice and 3.86 ± 0.88 mm in VPAC2 R TG mice (P<0.01); 1.0 µg of TNP-CGG gave cutaneous reactions of 2.20 ± 0.48 mm in normal mice and 8.80 ± 1.73 mm in VPAC2 R TG mice (P<0.01). In contrast, antigen-specific delayed-type hypersensitivity was depressed in VPAC2 R TG mice vs. normal mice. Intracutaneous immunization of groups of six 12-wk-old mice with NP OSu followed by intradermal challenge with NP OSu in rear footpads, resulted in increases in footpad thickness of 15% ± 6% (mean±SD) in normal mice and 6.7% ± 4.2% in VPAC2 R TG mice at 24 h (P<0.05), and of 14% ± 5% in normal mice and 6.0% ± 4.3% in VPAC2 R TG mice at 48 h (P<0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 12
, 19)
. Although in vitro studies have shown numerous effects of several neuropeptides on T cell migration, proliferation, and synthetic functions, results have often depended so much on experimental conditions and composition of the T cells as to raise doubts about their in vivo significance. VIP is the quantitatively most prominent neuroendocrine factor in the immune system and strikingly affects many T cell activities (3
, 12
, 19)
. However, there are no potent and bioavailable VPAC R-specific VIP antagonists for delineation of the in vivo contributions of VIP to immunity and hypersensitivity. The major regulatory effects of VIP on generation and secretion of diverse cytokines by T cells in vitro may be transduced by one or both VPAC Rs, depending on the target subset of T cells and their state of activation (12
, 19)
. The exact roles of each VPAC R have not been defined in vivo. The principal feature of the new TG model described is persistent and isolated constitutive expression of the usually inducible VPAC2 R selectively on CD4+ T cells at levels characteristic of those in normal T cells stimulated maximally through the TCR (Fig. 1A
). Our findings in this model are increases in blood concentrations of IgE, IgG1, and eosinophils in vivo, which are directly attributable to persistent VPAC2 R-mediated elevation of the levels of Th2-derived cytokines and concomitant suppression of Th1 cell-derived cytokines (Figs. 2
3
4)
. The resultant abnormalities in IgE and eosinophils are coupled to enhanced immediate-type hypersensitivity in vivo (Figs. 5
, 6)
. The elevated ratio of Th2/Th1 cytokines also leads to depressed delayed-type hypersensitivity.
Several elements of the immune phenotype of VPAC2 R TG mice could have been predicted from the results of previous in vitro studies. T cell-dependent production of IgE and class switching to IgE were both stimulated by VIP in two independent analyses (28
, 29)
. However, in similar investigations, the level of IgA was increased and that of total IgG was decreased, but neither effect was observed in the TG mice. T cell production of IL-5 was found previously to be increased by VIP (4)
, as noted in the present study. T cell generation of IL-4 often is decreased by VIP in vitro, through IL-2-dependent posttranslational mechanisms, and effects of VIP on T cell generation of IFN- have been inconsistent (12
, 19
, 20)
. In contrast, the endogenous VIP-VPAC2 R axis clearly and significantly increases IL-4 and decreases IFN- in vivo in the huVPAC2 R-TG mouse model (Fig. 3)
. The possible role of endogenous mVPAC1 R in these differences between in vitro and in vivo effects of VIP on cytokine production cannot be resolved pharmacologically at this time, but future plans to answer this question include crossing the VPAC2 R-TG mouse with a VPAC1 R-null C57Bl/6 mouse now in development. The relative contributions of VIPergic nerves in the T cell corridors and of Th2 cells themselves in supplying endogenous VIP necessary for signaling through the TG VPAC2 R also have not been delineated. Neural sources are presumed to dominate, as the amounts available from Th2 cells are almost always far less than from neural sources and of greatest relevance in noninnervated structures such as new granulomas and transplanted organs.
Future investigations of the allergic diathesis and depressed delayed-type hypersensitivity of huVPAC2 R-TG mice will encompass both analyses of altered expression of compartmental hypersensitivity and inflammation and of modified resistance to selected infections in which host defense is differentially dependent on Th1 or Th2 cells. The findings of this research are more compelling as a result of parallel studies of recently devised VPAC2-null C57BL/6 mice, which possess an inverse immune phenotype to that of VPAC2 R TG mice (30)
. The VPAC2 R-null mice have enhanced delayed-type hypersensitivity and decreased immediate-type hypersensitivity because of greater production of IFN- and lesser generation of IL-4 by CD4+ T cells. These new data predict clinical significance of the VIPergic system in human immunology, including the possibility that blood CD4+ T cells from patients with severe allergies and asthma will have higher levels of VPAC2 R than CD4+ T cells of nonallergic subjects. However, such predictions require several untested assumptions, including similarity of the VIP/VPAC2 R system among different species and CD4+ T cells from different tissue sources and a constancy of expression and influence of the VIP/VPAC2 R system in CD4+ T cells required for allergies and asthma. Therefore, studies of VPAC2 Rs in allergic diseases will include not only profiling of expression of VPAC Rs, but also assessment of the potencies of VPAC2-selective agonists relative to VIP and VPAC1-selective agonists in evoking functional responses and alterations in cytokine generation by CD4+ T cells throughout the full course of such diseases.
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ACKNOWLEDGMENTS |
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Received for publication August 15, 2001. Accepted for publication September 11, 2001.
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REFERENCES |
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