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(The FASEB Journal. 1999;13:347-353.)
© 1999 FASEB

RESEARCH COMMUNICATION

Enhancement by vasoactive intestinal peptide of {gamma}-interferon production by antigen-stimulated type 1 helper T cells

NABILA JABRANE-FERRATa ,1 , DEBRA BLOOMb ,1 , ADRIAN WUc , LI LIc , DAVID LOc , SUNIL P. SREEDHARANa , CHRISTOPH W. TURCKa and EDWARD J. GOETZLa , 2


a Departments of Medicine and Microbiology-Immunology, University of California Medical Center, San Francisco, California 94143-0711, USA;

b Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, USA; and

c Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vasoactive intestinal peptide (VIP) is a neuroendocrine mediator in immune tissues that affects many T cell functions through two homologous high-affinity G-protein-coupled receptors, termed VIPR1 and VIPR2. Antigen-stimulated secretion of {gamma}-interferon (IFN-{gamma}) by sperm whale myoglobin-specific Th1 cells of DBA/2 mouse I-Ed-restricted clones, which express VIPR1 and VIPR2, was enhanced by 10-10 M to 10-7 M VIP. Enhancement of IFN-{gamma} secretion reached a mean maximum of fourfold for VIP and threefold for a VIPR2-selective agonist, without any effect of a VIPR1-selective agonist. Secretion of IFN-{gamma} by PMA and ionomycin-stimulated clones of Th1 cells was not altered by VIP. Antigen-stimulated secretion of IFN-{gamma} by T cell receptor-transgenic, influenza hemagglutinin-specific, and cytokine-differentiated mouse lymph node Th1 cells, which also express VIPR1 and VIPR2, was enhanced by 10-10 M to 10-8 M VIP. Enhancement of IFN-{gamma} secretion increased to a maximum of 14-fold for VIP, 14-fold for the VIPR2-selective agonist, and 20-fold for the VIPR1-selective agonist. In contrast to VIP suppression of interleukin production and lack of effect on IFN-{gamma} production by T cells stimulated with anti-CD3 antibody or a mitogenic lectin, generation of IFN-{gamma} by antigen-stimulated T cells is enhanced significantly by physiological concentrations of VIP.—Jabrane-Ferrat, N., Bloom, D., Wu, A., Li, L., Lo, D., Sreedharan, S. P., Turck, C. W., Goetzl, E. J. Enhancement by vasoactive intestinal peptide of {gamma}-interferon production by antigen-stimulated type 1 helper T cells.


Key Words: neuropeptide • receptors • G-proteins • cytokines • lymphocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VASOACTIVE INTESTINAL PEPTIDE (VIP)3 is a 28-amino acid neuromediator that is delivered to primary immune organs and lymphoid tissues in the gastrointestinal tract, lungs, and skin by several types of neurons 1-5) . VIP has many potent effects on smooth muscle, epithelial and endothelial cells, glands, and neurons (6 , 7 ). At concentrations attained after antigen challenge 8,9) , VIP alters a wide range of T cell and macrophage functions including adhesion, migration, secretion of matrix metalloproteinases, production of numerous cytokines, and interactions with other cells (7 , 9-19 ). Analyses of VIP effects on production and secretion of cytokines have focused on murine thymocytes and on murine and human blood T cells, using anti-CD3 monoclonal antibodies with or without phorbol esters and mitogenic lectins as the stimuli. In these systems, VIP suppresses production of interleukin 2 (IL-2) and IL-10 by transcriptional mechanisms and IL-4 by posttranscriptional effects (20 , 21 ). In two studies of similarly stimulated mixed T cells, VIP enhanced production of IL-5 and IL-6 (22 , 23 ). VIP was shown not to affect {gamma} interferon (IFN-{gamma}) production by mouse splenocytes or human blood T cells stimulated with concanavalin A, anti-CD3 antibody, or phorbol myristate acetate (PMA) plus anti-CD3 antibody (24) .

Many T cells and macrophages, as well as nonimmune target cells, express two different types of G-protein-coupled receptors, termed VIPR1 and VIPR2, which are homologous in structure and similar in use of signal transduction pathways (9 , 13 , 25-27 ). T helper type 1 (Th1) cells have been shown to express both VIPR1 and VIPR2, but there has been no investigation of the effects of VIP on antigen-stimulated cytokine secretion by this T cell subset. We now describe the ability of concentrations of VIP attained in immune tissues in vivo to increase strikingly the secretion of IFN-{gamma} by antigen-stimulated Th1 cells of stable clones and cytokine-differentiated lymph node T cells with T cell receptors of defined specificities.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media, chemical reagents, and peptides
The sources of materials were: RPMI-1640 medium, Dulbecco's modified minimal essential medium (DMEM), heat-treated fetal bovine serum (FBS), penicillin and streptomycin concentrate, nonessential amino acids (UCSF Cell Culture Facility), PMA, ionomycin and hypoxanthine, aminopterin, and thymidine (HAT) medium supplement (100 µM hypoxanthine, 0.4 µM aminopterin, and 16 µM thymidine) (Sigma Chemical Co., St. Louis, Mo.), thapsigargin, BAPTA-AM, the adenylyl cyclase inhibitor MDL-12,330A, and the protein kinase A inhibitors H89 and KT5720 (Calbiochem-Novabiochem Corp., LaJolla, Calif.). VIP 1-28, the VIPR1-selective agonist [K15, R16, L27] VIP [1-7]/GRF[8-27] (28) , the amino acid 110 to 121 substituent peptide of sperm whale myoglobin (SWMP), and the amino acid 110 to 119 substituent peptide of influenza hemagglutinin (HAP) were synthesized using a standard solid-phase system (Model 433 peptide synthesizer, Perkin Elmer, AB Division, Foster City, Calif.) with fluorenyl-methoxycarbonyl-protected amino acids (Bachem, Torrance, Calif). Side chain deprotection and cleavage of the peptides from the resin were completed in trifluoroacetic acid:anisole:dimethylsulfide (9:0.5:0.5, v:v), the peptides purified by high-performance liquid chromatography and their sequence confirmed by mass spectrometry with an LCQ iontrap (Finnigan MAT, San Jose, Calif.). The VIPR2-selective agonist Ro25-1392 (29) was a gift from Dr. David Bolin of Hoffmann-LaRoche, Inc. (Nutley, N.J.). Rat monoclonal IgG anti-IL-12 (clone C.17.8.20) was a generous gift from Dr. G. Trinchieri.

Cell culture, Th1 cell differentiation, and generation of {gamma}-interferon (IFN-{gamma})
Metallic microbeads derivatized with monoclonal anti-mouse CD4 or anti-mouse CD8 and columns in a magnetic field (Miltenyi Biotec, Inc., Auburn, Calif.) were used to resolve and isolate subsets of T cells from suspensions of spleen nonadherent mononuclear leukocytes of 6-wk-old female Balb/c mice. The purity of each set of T cells used for quantification of mRNA encoding VIP receptors was at least 95%, as assessed by flow cytometry (30) . SWMP-specific mouse Th1 cells of DBA/2 I-Ed-restricted clones 11.3 and 8.2, which have been described (31 , 32 ), were cultured in RPMI-1640 with 10% FBS, 100 U/ml of penicillin and 100 µg/ml of streptomycin, and restimulated with IL-2 and purified the day before each experiment, as described (31 , 32 ). The antigen-presenting (AP) B7-1-transfected RT 7.7 L cells, which have also been described (33) , were cultured in DMEM with 10% FBS, 100 U/ml of penicillin, 100 µg/ml of streptomycin, nonessential amino acids, and HAT and relayered in 24-well culture plates the day before each experiment. Replicate 1 ml suspensions of 3–4 x 105 Th1 cells were preincubated for 2 h at 37°C with inhibitors and/or VIP or analogs of VIP, then added to RPMI-1640-washed monolayers of 0.6–1 x 105 AP cells with 10 µM SWMP or 0.5 µM PMA plus 0.1 µM ionomycin, followed by incubation for 24 h.

Lymph node cells were isolated from Balb/c mice transgenic for a T cell receptor (TcR) specific for an influenza hemagglutinin substituent peptide (HAP) of amino acids 110-119 presented on I-Ed (34) . CD4+ Mel-14hi cells were isolated by fluorescence-activated cell sorting and cultured with a fivefold greater number of Balb/c splenocytes, previously irradiated with 2500 rads, and 0.5 µg/ml of HAP. For Th1 cell generation, 20 ng/ml of IL-12 (R & D Systems, Inc., Minneapolis, Minn.) was added; for Th2 generation, 25 ng/ml of IL-4 (Pepro Tech, Inc., Rocky Hill, N.J.) and 100 µg/ml of anti-IL-12 (monoclonal rat IgG, clone C17.8.20) were added together. Media were changed every 2–3 days and, from day 3 on, 50 U/ml of IL-2 (Pepro Tech) was also added to the cultures. T cells were harvested and used for study on day 7. The levels of mRNA encoding VIP receptors were assessed in Th1 and Th2 cells. Replicate suspensions of 1 to 2 x 105 viable Th1-enriched lymph node cells in 1 ml of RPMI-1640-FBS-penicillin-streptomycin were preincubated with VIP or a VIP analog for 2 h at 37°C, added to layers of 0.5–1 x 106 Balb/c mouse adherent splenocytes that had been irradiated with 3000 rads 3–4 h before in 24-well plates with 2.5 µM HAP, and incubated for an additional 24 h.

The suspensions of Th1 clones and Th1-enriched lymph node cells were then recovered, the total number of cells and viable cells were counted, and each suspension was centrifuged at 400 x g for 10 min at 4°C. The supernatants were frozen in 200 µl aliquots and RNA was extracted from the pellets for reverse transcription-polymerase chain reaction (RT-PCR) analysis. IFN-{gamma} in the supernatants was quantified by enzyme-linked immunoassay according to a standardized procedure (Endogen, Inc., Woburn, Mass.).

Assessment of mRNA encoding VIPRs by RT-PCR
Total cellular RNA was prepared from each Th1 cell clone and set of Th1-enriched lymph node T cells, before and after stimulation, by the TRIzol method (GIBCO-BRL, Grand Island, N.Y.) and a SuperScript kit (GIBCO-BRL) was used for RT synthesis of cDNAs. Oligonucleotide primers were: 5'-dCCTGGCCAAGGTCATCC-ATGACAAC and 5'-dTGTCATACCAGGAAATGAGCTTGAC for the internal standard glyceraldehyde 3-phosphate dehydrogenase (G3PDH), 5'-dAGTCCTCAAATCATCCC-ACATCTGC and 5'-dAAGTGGCACTTCCTGTCTCGTAATC for VIPR1 and 5'-dTCC-CAGCAGGTGTTTCCTGGCCTAC and 5'-dCGAGCCTCTTGTACTGTGACTGGTC for VIPR2. Two µCi of [alpha-32P] dCTP were added to each standard reaction mixture. After a `hot-start' at 94°C for 3 min, Taq DNA polymerase was added and polymerase chain reaction (PCR) amplification was carried out with 32 cycles of 30 s at 94°C, 2 min at 55°C, and 1 min at 72°C. PCR products were resolved by electrophoresis in a 2 g% agarose gel at 105 v for 1 1/2 h. The intensity of each band visualized by ethidium bromide staining was quantified by densitometric analysis of autoradiographs and by beta-scintillation counting of bands cut from the gels and solubilized in 0.5 ml of sodium perchlorate at 55°C for 1 h (Elu-Quick, Schleicher & Schuell, Inc., Keene, N.H.). In prior optimization of PCR efficiency, it was found that each pair of 25-base primers had very similar temperature dependence for annealing with the highest specificity and catalyzed similar linear kinetics for up to 34 cycles. Amounts of cDNA two- and fourfold higher and threefold lower than those normally used yielded proportionately altered uptake of 32P into each respective product.

Initially, two different-sized portions of each pool of cDNA templates from each source of Th1 cells were selected for PCR amplification of G3PDH, based on the number of T cells from which RNA was prepared, in order to establish the volumes that would result in G3PDH bands of equal intensity for each sample in every set. This volume of each pool of cDNAs was then used for PCR amplification of VIPR1 and VIPR2 messages in each sample separately, in parallel with the G3PDH message. The relative quantity of message in each VIPR1 and VIPR2 band is expressed as the ratio of radioactivity to that in the corresponding G3PDH band, as described (35) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The relative levels of expression of VIPR1 and VIPR2 by sets of purified CD4+ and CD8+ splenic T cells, Th1 and Th2 clones, and sets of Th1 and Th2 lymph node cells differentiated in culture were determined semiquantitatively by RT-PCR (Fig. 1 ). VIPR1 and VIPR2 were more prominent in CD4+ than CD8+ splenic T cells and in both cytokine-differentiated Th2 cells and Th2 clones than in the corresponding Th1 cells. These patterns of preferential expression of VIPR1 and VIPR2 were confirmed by radioactive analyses of PCR cDNA products (Table 1 ). Incubation of the Th1-enriched lymph node cells with antigen-presenting cells (APCs) and HAP antigen, and clones of Th1 cells with APCs plus SWMP or with a combination of PMA and ionomycin, at concentrations identical to those used for stimulation of IFN-{gamma} production, had no effect on the levels of mRNA encoding VIPR1 or VIPR2 (Fig. 2, Table 2 ).



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  		Figure 1. RT-PCR semiquantification of VIPR1 (A; upper frame) and VIPR2 (B; lower frame) in mouse T cells. The amount of the cDNA pool from each source to be amplified by PCR was chosen, based on the results of preliminary PCR analyses, to ensure that the G3PDH cDNA (highest band in each lane) would be of the same intensity as that of every other source. Of the doublet found for VIPR1 in mouse T cells, the denser and lower of these cDNAs was found by sequence analysis to be the true component of VIPR1. Lanes 1 and 2: isolated native CD4+ and CD8+ spleen cells, respectively; lanes 3 and 4: primary cultured lymph node Th1 and Th2 cells, respectively; lanes 5 and 6: stably cultured clones of Th1 cells (11.3) and Th2 cells, respectively; lane 7: no cDNA control; lane 8: m.w. marker.


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  		Table 1. Relative frequency of expression of VIP receptor subtypes by mouse T cellsa

The amount of IFN-{gamma} secreted by clone 11.3 Th1 cells incubated with APCs in the absence of SWMP was barely detectable, but increased to respective means of 292 ng/ml and 432 ng/ml after addition of SWMP and PMA plus ionomycin (Fig. 3 ). VIP further increased the level of IFN-{gamma} evoked by SWMP, with a mean maximal rise of fourfold at 10-9 M. As for VIP, the VIPR2-selective agonist Ro25-1392 enhanced the effect of SWMP on clone 11.3 Th1 cells up to a maximal increase of threefold, whereas the same concentrations of the VIPR1-selective agonist had no significant activity. In two of the studies performed in duplicate, IFN-{gamma} generation was increased a mean of 1.2-fold by 10-11 M VIP and 1.5- and 1.9-fold, respectively, by 10-11 M and 10-10 M VIPR2-selective agonist. Neither VIP nor either VIPR-selective agonist enhanced the stimulatory effect of PMA plus ionomycin on IFN-{gamma} generation by clone 11.3 Th1 cells (Fig. 3) . Th1 cells of clone 8.2 showed results similar to those with clone 11.3 in two separate studies.



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  		Figure 3. Enhancement by VIP and VIP analogs of antigen-induced, but not PMA and ionomycin-induced, secretion of IFN-{gamma} by cloned Th1 cells. Each bar and bracket depicts the mean ±SD of the results of three separate studies, where values were calculated as the percentage of IFN-{gamma} secretion by cloned Th1 cells in medium alone. The results of statistical analyses of the significance of increases above control values by a paired Student's t test are shown by X = P <0.05. The mean control levels (100%) of secretion of IFN-{gamma} were 292 ng/ml and 432 ng/ml for antigen and PMA plus ionomycin, respectively, from a mean of 3.6 x 105 Th1 cells per well.

VIP enhanced significantly the generation of IFN-{gamma} by Th1-enriched lymph node T cells stimulated with HAP and APCs, with mean maximal increases of 14-fold at 10-10 M to 10-8 M VIP (Fig. 4 ). In one of these studies and an additional experiment, IFN-{gamma} production was increased a mean of eightfold by 10-7 M VIP. Similar enhancement was attained by the optimal concentrations of 10-8 M and 10-7 M VIPR2-selective agonist. In one of these studies and one additional experiment, IFN-{gamma} production was increased a mean of three- and ninefold, respectively, by 10-10 M and 10-9 M VIPR2-selective agonist. An equal or greater level of enhancement of HAP-elicited secretion of IFN-{gamma} by Th1-enriched lymph node T cells was observed with the VIPR1-selective agonist up to a maximum increase of 20-fold (Fig. 4) . In one of these studies and one additional experiment, IFN-{gamma} production was increased a mean of 4-and 11-fold, respectively, by 10-10 M and 10-9 M VIPR1-selective agonist. This finding contrasts with the lack of effect of the VIPR1-selective agonist on Th1 clone cells (Fig. 3) .



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  		Figure 4. Enhancement by VIP and VIP analogs of antigen-induced secretion of IFN-{gamma} by primary cultures of mouse lymph node Th1 cells. Each bar and bracket depicts the mean ±SD of the results of two separate studies performed in duplicate, where values were calculated as the fold increase in IFN-{gamma} secretion above that seen with Th1 cells in medium alone. The results of statistical analyses of the significance of increases above control values by a paired Student's t test are shown by X = P <0.05 and * = P <0.01. The mean control level (100%) of secretion of IFN-{gamma} was 22 ng/ml from a mean of 1.2 x 105 Th cells per well.

VIP and the VIPR-selective agonists had no effect on the number of Th1 cells in clones or in Th1-enriched lymph node cells or on their viability as assessed by trypan blue exclusion after any period of incubation. A preliminary examination of the biochemical prerequisites for VIP enhancement of antigen-stimulated secretion of IFN-{gamma} by clone 11.3 Th1 cells was conducted with pharmacological inhibitors of selected pathways relevant to VIP actions. A role for intracellular calcium was detected by the capacity of 1 µM thapsigargin and 10 µM BAPTA-AM to inhibit by respective means ±SD. (n=3) of 83% ±23% and 87% ±13% (P<0.05) the increases in IFN-{gamma} generation by 10-8 M VIP. In contrast, increases in intracellular cAMP appear not to be required, as neither the adenylyl cyclase inhibitor MDL-12,330A (250 µM) nor the protein kinase A inhibitors H89 (1 µM) or KT5720 (0.1 µM) had significant effects on VIP enhancement of IFN-{gamma} generation in the same studies.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent elucidation of the genes encoding VIPR1 and VIPR2 has allowed determination of their separate expression by T cells purified and cloned from transgenic mice with TcRs of known peptide antigen specificity. Concurrent development of stable VIP analogs with agonist activity and VIPR subtype selectivity, which bind to VIPR1 or VIPR2 with an affinity 300- to 1000-fold higher than to the other VIPR, now permits investigations of the separate roles of each VIPR in T cell functions. VIPR1 and VIPR2 were both found in subsets of lymph node T cells from mice with TcRs specific for an influenza hemagglutinin peptide (HAP), after cytokine treatments and flow-cytometric selection for Th1 and Th2 enrichment (Fig. 1 , Table 1 ). Several clones of Th1 and Th2 cells from mice with TcRs specific for a SWMP also expressed both VIPR1 and VIPR2 (Fig. 1 , Table 1 ). VIPR1 and VIPR2 appeared to be more abundant in Th2 than Th1 cells in both enriched and cloned T cell subsets. Incubation of T cells of two of the Th1 clones with SWMP and APCs or with PMA plus ionomycin (to evoke optimal generation of IFN-{gamma}) did not affect the expression of VIPR1 or VIPR2 (Fig. 2 , Table 2 ). This result differs from previous findings of stable T cell expression of VIPR1 but increased expression of VIPR2 after incubation with anti-CD3 antibody plus PMA or VIP (36) .



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  		Figure 2. RT-PCR semiquantification of the changes in levels of VIPR1 (A; upper frame) and VIPR2 (B; lower frame) in mouse Th1 clone 11.3. Selection of the amount of cDNA pool from each source was based on the same method used in the studies depicted in Fig. 1 . The largest cDNA in each lane, as in Fig. 1 , is for the G3PDH standard, and the lower VIPR1 cDNA of the doublet is the authentic message. The contents of each lane are: 1 = no cDNA control, 2 = incubation of Th1 cell clone in medium alone for 6 h, 3 = incubation with SWMP and APCs, 4 = incubation with PMA plus ionomycin.


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  		Table 2. Lack of effect of antigen-dependent and -independent stimulation of clone 11.3 Th1 cells on expression of VIPR1 and VIPR2a

This first investigation of the VIPR-selective regulation of antigen-elicited production of IFN-{gamma} by enriched and cloned Th1 cells showed striking stimulation by physiological concentrations of VIP and by a VIPR2-selective agonist, but stimulation by a VIPR1-selective agonist only for the enriched population and not the Th1 clones (Figs. 3 and 4) . The most likely explanation is that all Th1 cells of the clones express levels of VIPR1 too low to transduce VIPR1-selective responses, whereas a subset of the enriched Th1 lymph node cells must contribute sufficient IFN-{gamma} to alter the net output of the total population and also express VIPR1 at a level endowing susceptibility to the stimulatory effect of the VIPR1-selective agonist.

The lack of effect of VIP and either VIPR-selective agonist on Th1 cell generation of IFN-{gamma} evoked by PMA plus ionomycin is consistent with the lack of effect of VIP on IFN-{gamma} previously reported from mixed T cells stimulated with anti-CD3 plus PMA or a mitogenic lectin (24) . Other costimulatory events elicited by antigen and APCs, but not by anti-CD3 antibody alone, might explain the discrepant responses of IFN-{gamma} to VIP. In one limited study of lymph node-derived Th1 cell generation of IFN-{gamma} induced by anti-CD3 plus costimulatory anti-CD28 antibodies, 10-9 M and 10-8 M VIP increased IFN-{gamma} release by 3.4- and 3.7-fold, respectively. This finding suggests that enhancing effects of VIP on antigen-induced generation of IFN-{gamma} are in part attributable to the more complete stimulatory effect of antigen than to CD3-directed activation alone. Although we did not examine IL-2 secretion, one published study of neuropeptide modulation of antigen-evoked IL-2 secretion by a murine T cell hybridoma showed enhancement by VIP, as well as substance P and somatostatin (37) . However, a significant effect was limited to 10-14 M to 10-11 M VIP and disappeared at 10-10 M to 10-7 M, and IL-2 was measured solely by bioassay. That the same VIP concentration dependence of enhancement of IL-2 production was observed with mixed mononuclear leukocytes from normal human colonic tissues suggested possible in vivo significance (37) .

Several critical questions remain unanswered, including the basis for the much greater VIP enhancement of IFN-{gamma} generation elicited by antigen challenge of Th1-enriched lymph node cells than of cloned Th1 cells, the mechanisms of signal transduction from VIPRs to pathways of antigen-driven production and secretion of IFN-{gamma}, and the determinants of responsiveness of Th1-enriched lymph node cells, but not cloned Th1 cells, to VIPR1-selective agonists despite apparently similar levels of expression of VIPR1 and VIPR2 by both Th1 cell populations. It is clear, however, that neuropeptide regulation of cytokine production and possibly other functions of T cells depend on the primary immunological stimulus. The results of in vitro studies of functions of T cells stimulated with mitogens, antibodies to membrane receptors or other surface proteins, phorbol esters, and ionophores may differ completely from those of studies of specific antigenic stimulation.


   ACKNOWLEDGMENTS
 
These studies were supported by grant AI29912 from the National Institutes of Health. The authors are grateful to Bethann Easterly for expert preparation of graphics.


   FOOTNOTES
 
2 Correspondence: Immunology and Allergy, UB8B, Box 0711, University of California Medical Center, 533 Parnassus, San Francisco, CA 94143-0711 USA. E-mail: egoetzl{at}itsa.ucsf.edu

1 These scientists contributed equally to the experimental results reported in this manuscript.

3 Abbreviations: VIP, vasoactive intestinal peptide; IFN-{gamma}, {gamma}-interferon; IL, interleukin; SWM, sperm whale myoglobin; SWMP, synthetic substituent peptide of SWM; DMEM, Dulbecco's modified essential medium; AP, antigen-presenting; APCs, AP cells; HAP, influenza hemagglutinin peptide; HAT, hypoxanthine, aminopterin, and thymidine; FBS, fetal bovine serum; G3PDH, glyceraldehyde 3-phosphate dehydrogenase; PMA, phorbol myristate acetate; RT-PCR, reverse transcription-polymerase chain reaction; TcR, T cell receptor; Th1, T helper type 1.

Received for publication August 5, 1998. Revision received October 26, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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