Vasoactive intestinal peptide enhancement of antigen-induced differentiation of a cultured line of mouse thymocytes

^a Departments of Medicine and Microbiology, University of California, San Francisco, California 94143–0711, USA
^b Howard Hughes Medical Institute, University of California, San Francisco, California 94143–0711, USA
^c The Scripps Research Institute, La Jolla, California 92037, USA

	ABSTRACT

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The prominence of vasoactive intestinal peptide (VIP) in rodent thymic neurons suggested that this potent mediator of T cell functions may alter developmental responses of thymocytes to T cell receptor (TCR) -dependent stimulation. CD4⁺8⁺ DPK cells derived from a thymic lymphoma of a TCR transgenic mouse respond to pigeon cytochrome C (PCC) antigen in association with distinct I-E MHC II haplotypes on antigen-presenting cells (APCs) by differentiating into CD4⁺8^- T cells. The specific recognition of VIP by two types of homologous G-protein-coupled receptors (VIPR1 and VIPR2) on DPK cells was attributable predominantly to VIPR1 before and to VIPR2 after exposure to APCs and PCC, as assessed by quantification of the respective mRNAs. PCC-evoked differentiation of DPK cells was enhanced significantly by 1 to 100 nM VIP after 3 to 4 days. The effects of VIP analogs with VIPR type selectivity implied that VIP enhancement of differentiation of DPK cells was mediated principally by VIPR2. Differential reduction in the expression of each type of VIPR by transfection of DPK cells with plasmids encoding the respective antisense mRNAs confirmed the central role of VIPR2 in VIP-enhanced conversion to CD4⁺8^- T cells. The suppression of DPK cell differentiation by inhibitors of adenylyl cyclase and protein kinase A suggested a transductional role for VIP-elicited increases in [cAMP]_i. That the changes in frequency of CD4⁺8⁺ and CD4⁺8^- DPK cells reflected principally differentiation was supported by the lack of consistent differences between the two subsets in the effects of VIP and VIPR2 agonist on cell number, viability, apoptosis, and proliferation. VIP may be one endogenous mediator that explains the unique thymic microenvironment for topographically specific development of T cells.—Pankhaniya, R., Jabrane-Ferrat, N., Gaufo, G. O., Sreedharan, S. P., Dazin, P., Kaye, J., Goetzl, E. J. Vasoactive intestinal peptide enhancement of antigen-induced differentiation of a cultured line of mouse thymocytes. FASEB J. 12, 119–127 (1998)

Key Words: neuropeptide • immunology • T lymphocyte • receptor • apoptosis

	INTRODUCTION

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VASOACTIVE INTESTINAL PEPTIDE (VIP)² 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). 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">, 20–22). Comprehensive investigations of the distinctive activities of VIPR1 and VIPR2 have been limited by the lack of sufficiently potent antagonists and type-specific agonists. In some model systems that permit studies of functions of T cells expressing only one type of VIPR, VIPR1 and VIPR2 appear to mediate different or even opposing effects (13, 22).

Although there is some understanding of the effects of VIP on mature T cells, little is known of how the high levels of VIP in thymic tissues (23) may influence T cell differentiation. A model system devised for studies of antigen-initiated conversion of mouse CD4⁺8⁺ thymocytes to CD4⁺ T cells (24, 25) has now been used to elucidate mechanisms of developmental determination of expression of VIPR1 and VIPR2. In this system, VIP is demonstrated to enhance the extent of differentiation of CD4⁺8⁺ thymocytes to CD4⁺ T cells by a VIPR2-dependent mechanism that is independent of any preferential alterations in proliferation or apoptosis of either subset of T cells.

	MATERIALS AND METHODS

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Cell culture, differentiation, purification, and transfection
DPK cells were derived originally from a spontaneous thymic lymphoma of a transgenic mouse expressing a V11/Vß3 T cell receptor (TCR) specific for a carboxyl-terminal fragment of pigeon cytochrome C (PCC) presented by I-E MHC II of the k, b, or s haplotypes (24, 25). DPK cells of the C7.C4.2G9 clone were cultured in EHAA (Click's) medium (Irvine Scientific, Santa Ana, Calif.) containing 12% heat-inactivated fetal bovine serum (Hyclone, Logan, Utah), 3 mM L-glutamine, 0.55 µM ß-mercaptoethanol, 100 U/ml of penicillin, and 100 µg/ml of streptomycin at densities of 2 x 10⁵/ml initially up to 2 x 10⁶/ml before dilution. Monolayers of the antigen-presenting DCEK cells, a line of murine fibroblasts doubly transfected with class II MHC E^k and type 1 intercellular adhesion molecule (ICAM-1) (24, 25), were maintained in RPMI 1640 medium with 10% fetal bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and (every other passage) 200 µg/ml of geneticin, 6 µg/ml of mycophenolic acid, 0.25 mg/ml of xanthine, and 15 µg/ml of hypoxanthine.

Differentiation was evoked in replicate aliquots of 0.5 to 1 x 10⁶ DPK cells by coculture with monolayers of DCEK cells at 30% to 80% confluency, for different experiments, in 1 ml of complete EHAA medium with 0.7 µM PCC in 24-well culture plates for 1 to 4 days. In the standard protocol, each aliquot of DPK cells was preincubated for 2 h with VIP, an analog of VIP, or medium alone before addition to a layer of DCEK cells. 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 differentiated CD4⁺CD8^- from CD4⁺CD8⁺ DPK cells and to isolate subsets of T cells from suspensions of spleen and thymus mononuclear leukocytes of 6-wk-old female Balb/c mice. The purity of each set of T cells used for studies of apoptosis and proliferation and for quantification of mRNA encoding VIP receptors was at least 95%, as assessed by flow cytometry. Lipotransfections of replicate aliquots of 1 x 10⁷ DPK cells were carried out in 1 ml of protein-free Opti-MEM (Gibco-BRL, Grand Island, N.Y.) with 16 µg of antisense plasmid DNA, 0.6 µg of DNA of the REP 4 plasmid (InVitrogen, San Diego, Calif.) encoding hygromycin-resistance, and 40 µl of Lipofectamine (Gibco-BRL) for 6 h at 37°C, followed by addition of fetal bovine serum to a final concentration of 1%. After a further 16 h of incubation at 37°C, the DPK cells were transferred to complete EHAA medium with 800 µg/ml of hygromycin and cultured for 5 more days before analyses and differentiation in the standard protocol.

Chemical reagents and antibodies
The amino acid 88 to 104 carboxyl-terminal peptide of PCC and VIP were synthesized by Dr. Christoph W. Turck (Howard Hughes Medical Institute core protein structure laboratory, U.C.S.F.), who used a standard solid-phase system (Model 433 peptide synthesizer, Perkin Elmer, AB Division, Foster City, Calif.) with fluorenyl-methoxycarbonyl (FMOC) -protected amino acids (Bachem, Torrance, Calif.). Side chain deprotection and cleavage of the peptide from the resin were completed in trifluoroacetic acid: anisole:dimethylsulfide (9:0.5:0.5, v:v), and the peptide was purified by high-performance liquid chromatography and its sequence confirmed by mass spectrometry with an LCQ iontrap (Finnigan MAT, San Jose, Calif.). The VIPR1-preferential peptide analog Ac-[K12,NL17,A19,25,L26,K27,28, A29–31]-VIP, which binds with 30- to 50-fold higher affinity to VIPR1 than VIPR2, and the VIPR2-selective cyclic peptide analog Ac-[E8,OCH3-Y10,K12,NL17,A19,D25,L26,K27,28]-VIP-cyclo (21–25), which binds with 300- to 1000-fold higher affinity to VIPR2 than VIPR1 (26), were obtained from Dr. David Bolin of Hoffman-LaRoche (Nutley, N.J.). A VIPR1-directed antisense plasmid was constructed by inserting a cDNA encoding the amino-terminal 140 amino acids of human VIPR1 into pRc/CMV expression vector (InVitrogen) in the antisense orientation with respect to the CMV promoter. Similarly, a VIPR2 antisense plasmid was derived by insertion of a full-length coding cDNA for human VIPR2 into the pCR3.1 expression vector (InVitrogen) in the antisense orientation relative to the CMV promoter. The antibodies used for flow cytometry were fluorescein isothiocyanate-conjugated rat monoclonal IgG2a/k anti-mouse CD4 (L3T4) and R-phycoerythrin-conjugated rat monoclonal IgG2a/k anti-mouse CD8a (Ly-2) (Pharmingen, San Diego, Calif.).

Flow cytometry and FACS
After 1–4 days of incubation of DPK cells with DCEK cells and PCC, their state of differentiation was assessed by flow cytometry without or after resolution of the CD4⁺8^- from CD4⁺8⁺ populations by immunomagnetic bead selection. Control and experimental samples were always analyzed concurrently. Replicate suspensions of 1 to 2 x 10⁶ DPK cells in 0.2 ml of protein-free RPMI-1640 medium with dissociating buffer (20:1, v:v) were incubated at 4°C separately with 500 ng of each of the fluorescently labeled monoclonal antibodies for 60 min and diluted to 1 ml with 1 ng/ml of propidium iodide in phosphate-buffered saline (PBS). Flow cytometry was performed with a FACScan system (Becton-Dickinson, San Jose, Calif.), using the CELL QUEST software program. The separate sets of propidium iodide-negative DPK cells were identified and their frequency quantified in 5000 cells per sample to permit calculation of the percentage conversion to CD4⁺CD8^- cells. FACS resolution of the CD4⁺8^- and CD4⁺8⁺ subsets of differentiated DPK cells for studies of their VIPRs and cellular responses was conducted sterilely with a FACStarPlus system (Becton-Dickinson) after preparation and antibody labeling as for flow cytometry.

Assessment of mRNA encoding VIPRs by RT-PCR
Total cellular RNA was prepared from each DPK, thymus, and spleen population of T cells by the TRIzol method (Gibco-BRL), and a SuperScript kit (Gibco-BRL) was used for reverse transcription (RT) synthesis of cDNAs. Oligonucleotide primers were: 5'-dCCTGGCCAAGGTCATCCATGACAAC and 5'-dTGTCATACCAGGAAATGAGCTTGAC for the internal standard glyceraldehyde 3-phosphate dehydrogenase (G3PDH); 5'-dAGTCCTCAAATCATCCCACATCTGC and 5'-dAAGTGGCACTTCCTGTCTCGTAATC for VIPR1; and 5'-dTCCCAGCAGGTGTTTCCTGGCCTAC and 5'-dCGAGCCTCTTGTACTGTGACTGGTC for VIPR2. Two µCi of [-^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/100 ml 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 ß-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 and Schuell, Keene, N.H.). In previous 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 usually used yielded proportionately altered uptake of ³²P into each of the respective products.

Initially, two different-sized portions of each pool of cDNA templates from each type of leukocyte were selected for PCR amplification of G3PDH, based on the number of leukocytes 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 then was 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 (27). Levels of [cAMP]_i were quantified by ELISA, as described (20).

Quantification of apoptosis and cellular proliferation
The CD4⁺8^- and CD4⁺8⁺ DPK cells from 3-day differentiation mixtures were resolved by FACS or immunomagnetic bead selection. Replicate suspensions of 2 x 10⁵ of each in 0.2 ml of EHAA complete medium were incubated in 96-well culture plates for an additional 3 days without and with VIP or a VIPR subtype-selective synthetic peptide. Cell density and viability were determined daily by microscopic counting of trypan blue-stained samples. Apoptosis was quantified on days 2 and 3 by labeling of newly generated 3'-OH ends of nucleosomal fragments of DNA, as described (28). A 25% portion of each suspension was fixed in 100 µl of 4% neutral-buffered formalin and dried on a glass slide, which was washed twice in PBS and incubated sequentially with terminal deoxynucleotidyl transferase and digoxigenin-dUTP, peroxidase-conjugated anti-digoxigenin antibody, and chromogenic diaminobenzidine in hydrogen peroxide for brown staining of apoptotic cells (Apoptag, Oncor, Gaithersburg, Md.). Two hundred cells per sample were examined for calculation of percentage apoptosis. On day 3, 1 µCi of [³H]thymidine was added to some suspensions; 18 h later cellular uptake of radioactivity was quantified by ß-scintillation counting of the cells recovered with a Model 200A multiwell PHD cell harvester (Cambridge Technology). Each measurement of DPK cell proliferation was expressed as a stimulation index calculated from the ratio of uptake of radioactivity in the presence of VIP or a VIP analog to that in medium alone.

	RESULTS

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Sets of mouse native thymocytes purified by an immunomagnetic bead method express both VIPR1 and VIPR2, as demonstrated by the results of RT-PCR analyses. VIPR1 mRNA was more prominent than VIPR2 mRNA in CD4^-8^- thymocytes and CD4^-8⁺ thymic T cells ( Fig. 1, Table 1). In contrast, the relative level of VIPR2 mRNA was higher than that of VIPR1 mRNA in CD4⁺8⁺ thymocytes and CD4⁺8^- thymic T cells. For splenic T cells, both single-positive subsets had a relatively higher level of VIPR1 mRNA than VIPR2 mRNA, and the mRNAs encoding both types of VIPRs were more prominent in CD4⁺8^- than CD4^-8⁺ T cells ( Fig. 1, Table 1), as previously reported in other systems (21). As for isolated mouse native thymocytes, DPK cells in both the undifferentiated state and after exposure to antigen-dependent differentiating conditions contain mRNAs encoding both VIPR1 and VIPR2 ( Fig. 2, Table 2). VIPR1 mRNA is more prominent than VIPR2 mRNA in DPK cells before differentiation, whereas after exposure to differentiating conditions, both CD4⁺8⁺ DPK cells and DPK-derived CD4⁺8^- T cells express relatively more VIPR2 mRNA than VIPR1 mRNA, as was found for the corresponding sets of native cells.

View larger version (64K): [in this window] [in a new window]   Figure 1. RT-PCR analysis of the expression of VIPR1 and VIPR2 by mouse thymocytes and T cells. The quantity of each sample was selected to provide similar levels of intensity of G3PDH cDNA (upper band in each frame). a) VIPR1 (lower band). b) VIPR2 (lower band). Lane 1 = CD4-8- thymocytes, 2 = CD4+8+ thymocytes, 3 = CD4+8- thymic T cells, 4 = CD4-8+ thymic T cells, 5 = negative control incubation without any cDNA, but with an amount of RNA used for RT of samples in lanes 1 (a) and 2 (b), 6 = CD4+8- splenic T cells, 7 = CD4-8+ splenic T cells, 8 = a 100 bp DNA ladder for mol. wt. determination; the hatch lines at the right of each frame mark the 200 and 400 bp standards in panel a, and the 300 and 400 bp standards in panel b. Radioactive quantification of cDNAs is reported in Table 1.

View larger version (69K): [in this window] [in a new window]   Figure 2. RT-PCR analysis of the effects of differentiation and transfected antisense plasmids on expression of VIPR1 and VIPR2 by mouse DPK cells. The basis for selecting the quantity of each PCR mixture for electrophoresis was the same as in the studies of Fig. 1. a) VIPR1. b) VIPR2. Lane 1 = undifferentiated DPK cells, 2 = CD4+8+ DPK cells after exposure to PCC antigen and DCEK cells, 3 = CD4+8- DPK cells after exposure to PCC antigen and DCEK cells, 4 = negative control incubation without any cDNA, 5 = DPK cells transfected with plasmid containing cDNA encoding VIPR1 in the antisense orientation, 6 = DPK cells transfected with plasmid containing cDNA encoding VIPR2 in the antisense orientation, 7 = 100 bp DNA ladder for mol. wt. determination; the hatch lines at the right of each frame mark the 200 and 400 bp standards in panel a, and the 300 and 400 bp standards in panel b. Radioactive quantification of cDNAs is reported in Table 2.

Incubation of DPK cells on 30% confluent monolayers of DCEK antigen-presenting cells confirmed the known PCC antigen dependence of differentiation to CD4⁺8^- T cells. A progressive increase in differentiation was observed from 2 to 4 days, and a maximal mean increment was attained at 4 days when 23% of the total DPK T cells were CD4⁺8^- in the presence of PCC, as contrasted with 5.5% in the absence of PCC. At the same low 30% confluence of DCEK cells in one typical experiment ( Fig. 3), the stimulatory effects of 10 nM VIP and 10 nM VIPR2-selective agonist Ro 25–1392 on PCC-induced differentiation of DPK cells were apparent by day 2 and increased substantially on day 4. The conversion to CD4⁺8^- DPK T cells increased from 14, 17, and 23% with PCC alone on days 2, 3, and 4, respectively, to 24, 29, and 49% with 10 nM VIP and to 21, 37, and 73% with 10 nM VIPR2 agonist ( Fig. 3). When the confluence of DCEK cells was increased to 70%, the peak of conversion to CD4⁺8^- DPK T cells was reached by day 3 in most experiments. In one representative study of VIP concentration dependence at this density of DCEK cells, the conversion to CD4⁺8^- T cells was 31% with PCC alone and increased to a maximum of 45% at 10 nM VIP ( Fig. 4a). As both VIPR1 and VIPR2 are expressed by DPK cells before and during PCC-induced differentiation ( Fig. 2, Table 2), the possibility that signaling by one type of VIPR might predominate was examined with the highly selective VIPR2 agonist and a partially preferential VIPR1 agonist ( Fig. 4b). At the same 10 nM concentration, the VIPR2 agonist stimulated the greatest conversion to 70% CD4⁺8^- T cells, as compared with 44% for VIP, whereas the VIPR1 agonist elicited only a marginal increase to 34% relative to 30% for PCC alone. More detailed analyses of these variables in three additional experiments at 70% confluency of DCEK cells demonstrated maximal enhancement of PCC-induced differentiation of DPK cells by 10 nM VIPR2 agonist on day 3 and by 100 nM VIPR2 agonist on days 2 and 4 ( Table 3). VIP elicited less enhancement than the VIPR2 agonist, with maximal effects by 10 nM VIP on day 2 and by 100 nM VIP on days 3 and 4. The VIPR1 agonist only evoked significant increases in conversion to CD4⁺8^- DPK T cells at 100 nM on days 2 and 3.

View larger version (28K): [in this window] [in a new window]   Figure 3. Time course of enhancement of antigen-induced differentiation of DPK cells by VIP and the VIPR2 agonist Ro 25–1392. The frames display representative images of the development of CD4+8- T cells after 2 (A, D), 3 (B, E), and 4 (C, F) days of incubation of DPK cells with 10 nM VIP (A–C) or 10 nM VIPR2 agonist (D–F) and 1 µM PCC on a 30% confluent monolayer of DCEK cells. The percentage of total DPK cells shown to be converted to CD4+8- T cells on days 2, 3, and 4 with VIP were 24, 29, and 49%, respectively, and with VIPR2 agonist were 21, 37, and 73%, which may be compared to levels of 14, 17, and 23% concurrent conversion in the absence of either stimulus.

View larger version (68K): [in this window] [in a new window]   Figure 4. VIP concentration dependence and VIPR type selectivity of enhancement of antigen-induced differentiation of DPK cells. a) Concentration dependence. The frames display representative images of the development of CD4+8- DPK T cells after 3 days of incubation on 70% confluent DCEK cells with PCC alone (A), and with VIP at 1 nM (B), 10 nM (C), and 100 nM (D). The levels of conversion of DPK cells to CD4+8- DPK T cells at the respective concentrations of VIP were 41, 45, and 43%, as compared to 31% in the absence of VIP. b) VIPR type selectivity. The frames display representative images of the level of development of CD4+8- DPK T cells after 3 days of incubation of DPK cells with PCC on 70% confluent DCEK cells without VIP (A, 30% CD4+8-) and with 100 nM VIP (B, 44% CD4+8-), 100 nM VIPR1-preferential agonist (C, 34% CD4+8-) and 100 nM VIPR2-selective agonist (D, 70% CD4+8-).

To further define the relative contributions of DPK cell VIPR1 and VIPR2 in VIP enhancement of their differentiation to CD4⁺8^- T cells, replicate suspensions of DPK cells were transfected separately, with plasmids encoding antisense mRNA specific for the respective VIPRs and enriched by hygromycin selection for 5 days before addition to DCEK cells with PCC antigen. Both types of antisense mRNA suppressed DPK cell expression of the corresponding VIPR preferentially by approximately 50%, as assessed by standardized RT-PCR analyses, with a concurrent increase in VIPR2 mRNA after introduction of the VIPR1 antisense plasmid ( Fig. 2, Table 2). The suppression of VIPR2s on undifferentiated DPK cells reduced significantly the extent of enhancement of antigen-induced differentiation by 10 nM VIP and eliminated the enhancement attributable to 100 nM VIP and to 10 nM and 100 nM VIPR2 agonist ( Table 4). In contrast, a similar decrease in expression of VIPR1 by DPK cells had only a modest inhibitory effect on the enhancement of differentiation by 10 nM VIP, without affecting the enhancement evoked by 100 nM VIP or either concentration of VIPR2 agonist ( Table 4). DCEK cells also express VIPR1 and VIPR2 with approximately equal density. Preincubation of DPK cells with VIP before addition to the DCEK cell monolayers in the standard protocol was intended to minimize any effects mediated by DCEK cell VIPRs. Two approaches failed to identify any effect of VIP on antigen presentation by DCEK cells sufficient to alter the extent of differentiation of DPK cells. First, preincubation of DCEK cell monolayers with 10 nM and 100 nM VIP and VIPR2-selective agonist for 1 and 3 days before addition of DPK cells and PCC did not modify the extent of conversion of DPK cells to CD4⁺8^- T cells after 3 days. Second, antisense plasmids reduced DCEK cell expression of VIPR1s and VIPR2s by a mean of 61 and 54% (n=2), respectively, in protocols similar to those used for DPK cells, as assessed by RT-PCR radioquantification of the respective mRNAs, but did not alter significantly the degree of VIP enhancement of DPK cell differentiation.

The intracellular concentrations of cAMP ([cAMP]_i) without VIP were 1.6 ± 0.4 pmol (mean±SD) per 10⁶ undifferentiated DPK cells and 4.3 ± 0.7 pmol per 10⁶ CD4⁺8⁺ DPK cells exposed to DCEK cells and PCC for 3 days. Incubation with 1, 10, and 100 nM VIP for 5 min resulted in respective mean increases in [cAMP]_i of 3.3-, 6.5-, and 7.3-fold (n=3) for undifferentiated DPK cells and of 3.0-, 3.9-, and 4.5-fold for CD4⁺8⁺ DPK cells exposed to differentiating conditions. As intracellular cAMP is the major transducer of VIPR signaling, a known inhibitor of adenylyl cyclase, termed MDL-12, and the protein kinase A inhibitor H-89 were next applied to evaluate further the mechanisms of enhancement of DPK cell differentiation by VIP. Concentrations of MDL-12 and the optimal level of H-89 both suppressed significantly the enhancement of conversion of DPK cells to CD4⁺8^- T cells by 10 nM VIPR2 agonist. However, only the two concentrations of MDL-12, but not H-89, reduced significantly the enhancement of conversion of DPK cells to CD4⁺8^- T cells by 10 nM VIP after 3 days ( Table 5). Increases in the intracellular concentration of cAMP and subsequent phosphorylation events thus appear to be required for VIP enhancement of antigen-induced conversion of DPK cells to CD4⁺8^- T cells. Neither inhibitor alone suppressed the level of antigen-induced differentiation of DPK cells in the absence of VIP.

We investigated the possibility that the relative frequencies of CD4⁺8⁺ and CD4⁺8^- DPK cells found after antigen stimulation might be attributable in part to differential effects of VIP on survival or proliferation of one of the two subpopulations. After 3 days of exposure of DPK cells to an optimal concentration of PCC and antigen-presenting DCEK cells, the CD4⁺8⁺ and CD4⁺8^- subsets were resolved by FACS and incubated separately for an additional 3 days without and with several concentrations of VIP and VIPR2 agonist. The number of DPK cells in both subpopulations increased similarly in the absence of VIP to a mean of twofold higher on day 3 than on day 0 ( Fig. 5). Except for one, each concentration of VIP and the VIPR2-selective agonist similarly increased the mean total number of DPK cells in the CD4⁺8⁺ (DP) and CD4⁺8^- (SP) subsets on day 3 to a maximum of threefold higher than on day 0. The increase in number of CD4⁺8⁺ (DP) DPK cells relative to the initial number on day 0 was greater than that of the CD4⁺8^- (SP) DPK cells at 10^-9 M VIP ( Fig. 5), but the relative increase was never significantly greater for the CD4⁺8^- (SP) DPK cells. The viability of both the CD4⁺8⁺ DPK cells and the CD4⁺8^- DPK cells, as assessed by trypan blue dye exclusion, was also increased similarly by all but one concentration of VIP and the VIPR2 agonist ( Fig. 5). As for cell number, the increase in viability of CD4⁺8⁺ (DP) DPK cells, relative to viability on day 0, was greater than that of the CD4⁺8^- (SP) DPK cells at 10^-9 M VIP ( Fig. 5), but the relative increase was never significantly greater for the CD4⁺8^- (SP) DPK cells.

View larger version (53K): [in this window] [in a new window]   Figure 5. Effects of VIP and the VIPR2-selective agonist Ro25–1392 on survival, apoptosis, and proliferation of CD4+8+ (dp) and CD4+8- (sp) DPK cells. Each bar and bracket depicts the mean ± SD of the results of three different studies. The statistical significance of each effect of an agonist compared to the respective control was calculated by the paired Student's t test. The levels of significance are represented by: +P < 0.05; *P < 0.01.

The principal determinant of the capacity of VIP to improve survival of both subpopulations of DPK cells was a reduction in apoptosis. Apoptosis of CD4⁺8⁺ and CD4⁺8^- DPK cells was decreased by VIP and VIPR2 agonist with similar or greater significance for DP than SP cells. In contrast, neither subset exhibited a proliferative response to PCC alone or with VIP or VIPR2 agonist ( Fig. 5). In summary, the enhancement of antigen-induced differentiation of DPK cells by VIP, reflected in greater increases in the relative percentage of CD4⁺8^- (SP) DPK cells with VIP and especially the VIPR2 agonist Ro25–1392, cannot be attributed to either selective increases in survival or selective stimulation of proliferation of the SP over the DP subset.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The ability of a neural mediator to influence differentiation, tissue distribution, and specific functions of immune cells depends on both the expression of immune cellular receptors specific for the mediator and the local attainment of concentrations of the mediator that are significant in relation to receptor affinity. Although thymic tissue content of VIP has not been quantified, immunohistochemical studies in rodents have shown levels higher than those in the central nervous system with the predominant localization in nerve fibers and apparent neural storage varicosities among thymocytes (23). The developmental and immunological events responsible for VIP release into thymic extracellular fluid also have not been characterized, but are likely to include secretion of cytokines by T lymphocytes and nonimmune thymic cells, as such cytokines alter the release of many neuropeptides in other systems (7, 29). Mouse thymocytes at the CD4⁺8⁺ stage of differentiation and DPK cells express both VIPR1 and VIPR2 ( Figs. 1 and 2, Tables 1 and 2). The expression of VIPR1 exceeds that of VIPR2 in DPK cells before the introduction of antigen, whereas VIPR2 predominates over VIPR1 in CD4⁺8⁺ DPK cells after exposure to antigen, CD4⁺8^- T cells derived from antigen-stimulated DPK cells, and the corresponding sets of native thymic cells. As DPK cells at each stage express both VIPR1 and VIPR2, studies have been designed to identify the functionally dominant type of VIPR that transduces enhancement of antigen-induced development of CD4⁺8^- T cells from CD4⁺8⁺ thymocytes.

The enhancing effect of VIP was detected at 1 nM and was maximal at 10 nM ( Fig. 4a, Table 3). Although the stimulatory effects of VIP and the VIPR2 agonist showed similar concentration dependence and time course, at each point the VIPR2 agonist promoted much greater differentiation to CD4⁺8^- DPK T cells than did VIP ( Fig. 3, Fig. 4b; Table 3). In contrast, the VIPR1 agonist had no significant effects or only marginal activity at the highest concentration tested ( Fig. 4b, Table 3). To confirm the predominant role of VIPR2s, DPK cells were transfected with expression plasmids encoding antisense mRNA specific for each type of VIPR under conditions that decreased by approximately 50% their respective levels of expression before addition to antigen-presenting DCEK cells with PCC ( Fig. 2). As a result of suppression of VIPR2, enhancement of development of CD4⁺8^- T cells by VIP and VIPR2 agonist was reduced by 2/3 or eliminated ( Table 4). In contrast, antisense suppression of VIPR1 reduced significantly the effect of only the lower concentration of VIP.

The greater activity of the VIPR2 agonist than VIP may reflect an inhibitory influence of VIP exerted through VIPR1s, as has been observed for T lymphocyte chemotaxis and secretion of MMPs (22). Although concurrent engagement of VIPR1s by native VIP but not VIPR2 agonist could elicit a negative signal that accounts for the lower differentiation responses to VIP than the VIPR2 agonist, this possibility was not supported by the results of antisense studies. Antisense suppression of VIPR1 did not enhance the effect of VIP ( Table 4). The logical experimental extensions would be further analyses of VIP effects in DPK cells expressing predominantly VIPR1s as a result of treatment with VIPR2-specific antisense plasmids ( Table 4) or application of higher concentrations of VIPR1 agonist in combination with VIPR2 agonist. That neither protocol showed inhibitory effects of VIPR1 may be attributable to the low rate of differentiation under these conditions after suppression of VIPR2s and to the relatively low selectivity of the VIPR1 agonist that mediates VIPR2 effects at concentrations over 30–100 nM ( Table 3).

The preincubation of DPK cells with VIP and VIPR-selective agonists in standard protocols minimizes any VIPR-mediated effects on the antigen-presenting DCEK cells. Additional controls supported the lack of functionally significant effects of VIP on DCEK cells. Introduction of VIP and VIPR2 agonist to DCEK cells 1 and 3 days before PCC and DPK cells had no effect on the development of CD4⁺8^- DPK T cells. Antisense suppression of VIPR1 and VIPR2 in DCEK cells before addition of DPK cells and PCC also had no effect on appearance of CD4⁺8^- DPK T cells. That VIP evoked increases in [cAMP]_i in DPK cells, and that inhibitors of both adenylyl cyclase and protein kinase A suppressed significantly the level of conversion of DPK cells to CD4⁺8^- DPK T cells, suggested the involvement of G-protein-coupled pathways in VIPR2 mediation of antigen-evoked differentiation of thymocytes. Although increases in [cAMP]_i may either initiate or inhibit apoptosis of lymphocytes in different circumstances (28, 30), these effects were modest and very similar in magnitude for CD4⁺8⁺ and CD4⁺8^- DPK cells ( Fig. 5). The slightly greater reduction in apoptosis of CD4⁺8⁺ DPK cells than of CD4⁺8^- DPK-derived T cells would lead to underestimations of the enhancement of differentiation of thymocytes by VIP. Finally, the lack of alteration of proliferation of DPK cells by VIP at either stage of maturity again supports the interpretation that observed increases in percentage frequency of CD4⁺8^- DPK T cells are attributable to enhanced differentiation ( Fig. 5).

That T cell receptor-dependent interaction of DPK cells with thymic epithelial MHC II proteins in vivo and in vitro without PCC antigen initiates conversion to CD4⁺8^- T cells, whereas non-thymic antigen-presenting cells require PCC for the same activity, suggested the possibility of dependence on other factors in the thymic milieu (31). T cell receptors were found concurrently to mediate up-regulation of selected surface receptors and intracellular proteins critical for thymocyte survival and differentiation (32). The potential significance of interactions of T cell receptors with receptors for other thymic mediators was recognized in a hypothesis that intrathymic development of CD4⁺ and CD8⁺ sets of T cells is conditioned not only by the efficiency of MHC antigen presentation, but also by pathways that regulate the sensitivity and magnitude of responsiveness of CD4⁺8⁺ thymocytes (33). Thus, VIP released in the thymus by cytokines and other mediators appears to be one potent factor that can set the threshold of sensitivity of CD4⁺8⁺ thymocytes to T cell receptor signals resulting from interactions with MHC proteins without and with antigens (24). Other potent mediators shown to be generated intrathymically and postulated to function in a manner similar to VIP include eicosanoids, such as thromboxane A2 and prostaglandin E2, and corticosteroids (28, 34). There are likely to be circumstances when a mixture of these mediators act concertedly to set thymocyte thresholds and response ranges to T cell receptor-dependent stimulation. Such potent mediators also act directly on thymocytes and on pre-T cells at more mature stages to alter generation and secretion of cytokines central to development of the CD4⁺ set and its terminally differentiated subsets (35). Of the many mediators in the thymic milieu that may influence thymocyte responsiveness to T cell receptor-dependent stimulation, VIP and possibly other neuropeptides are distinguished by their rapid and spatially prescribed release from a preformed reservoir. It remains to be determined whether other components of the nervous system can condition the size and responsiveness of the thymic reservoir of neuropeptides.

	ACKNOWLEDGMENTS

 
Supported by grants AI29912 and AI34570 from the National Institutes of Health.

	FOOTNOTES

 
¹ Correspondence: Immunology and Allergy, University of California, Room UB8B, Box 0711, 533 Parnassus, San Francisco, CA 94143–0711, USA.

² Abbreviations: FACS, fluorescence-activated cell sorting; FMOC-, fluorenyl-methoxycarbonyl-; ICAM-1, type 1 intercellular adhesion molecule; PBS, phosphate-buffered saline; PCC, pigeon cytochrome C; PCR, polymerase chain reaction; RT, reverse transcription; TCR, T cell receptor; VIP, vasoactive intestinal peptide; VIPR1, type I VIP receptor; VIPR2, type II VIP receptor.

Received for publication July 31, 1997. Accepted for publication October 13, 1997.

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