Immunological role of neuronal receptor vanilloid receptor 1 expressed on dendritic cells

Abstract

Capsaicin (CP), the pungent component of chili pepper, acts on sensory neurons to convey the sensation of pain. The CP receptor, vanilloid receptor 1 (VR1), has been shown to be highly expressed by nociceptive neurons in dorsal root and trigeminal ganglia. We demonstrate here that the dendritic cell (DC), a key cell type of the vertebrate immune system, expresses VR1. Engagement of VR1 on immature DCs such as by treatment with CP leads to maturation of DCs as measured by up-regulation of antigen-presenting and costimulatory molecules. This effect is present in DCs of VR1^+/+ but not VR1^–/– mice. In VR1^+/+ mice, this effect is inhibited by the VR1 antagonist capsazepine. Further, intradermal administration of CP leads to migration of DCs to the draining lymph nodes in VR1^+/+ but not VR1^–/– mice. These data demonstrate a powerful influence of a neuroactive ligand on a central aspect of immune function and a commonality of mechanistic pathways between neural and immune functions.

The perception of heat by the nervous system is understood in broad outlines. Neurons that “sense” temperature are present in the skin, and the stimuli received by them travel to the spinal chord/brainstem and synapse with the second neuron, which travels to the thalamus. The information is then relayed to the sensory cortex. The first step in this process, i.e., stimulation of the sensory neuron, is mediated by a family of receptors, the transient receptor potential channel vanilloid subfamily (TRPV). In mammals, this family of thermosensitive molecules consists of six members, including TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, and TRPA1. Of these, TRPV1 is specifically activated by temperatures >43°C. The temperatures for activation of three other channels range from warm (TRPV3 and TRPV4) to extremely hot (TRPV2). TRPM8 and TRPA1 are activated by cold temperatures (1–4). In addition to certain sensory neurons, selected TRPV members have been reported to be expressed on mast cells (5) and in the gastric mucosa (6).

Although heat has an equally profound effect on the immune system, its perception by the immune system is poorly understood. An increase in temperature of infected lizards by 2°C increases survival from 25% to 67% (7), and housing infected mice at 38°C increased their survival rate from 0% to 85% (8). In humans, fever causes rapid neutrophil migration and secretion of antibacterial chemicals and T cell proliferation, etc. (8–12). Heat is arguably the primordial defense mechanism conserved in fishes, amphibians, reptiles, birds, and mammals. A small number of preliminary observations have explored the mechanism of the effects of heat. Kluger and coworkers (7, 13–15) have identified the beneficial effects of fever in response to infection, the factors that are responsible for the cause and maintenance of fever, and the endogenous mechanisms that control high body temperature. Repasky and coworkers (16–19) have shown that hyperthermia regulates lymphocyte delivery to high endothelial venules and plays a role in migration of Langerhans cells to lymph nodes. In a previous study, we observed that heat causes maturation of dendritic cells (DCs), and that heat-matured DCs are as effective in stimulating potent T cell responses as DCs caused to mature by more traditional biological means such as cytokines, heat-shock proteins, and LPS (20). We have examinined the mechanism through which DCs perceive heat, and our attention has been drawn to the mechanisms used by sensory neurons for such perception.

We report here on the expression and role of the TRPV1 [also known as vanilloid receptor 1 (VR1) and hereafter referred to as such] molecules of the TRPV family on DCs. Although VR1 molecules do not appear to be responsible for perception of heat by DCs, our studies show that DCs, like the sensory neurons, express VR1, and that VR1 ligand capsaicin (CP), which has been shown previously to engage VR1 and transmit the perception of pain, similarly engages VR1 on the DCs and transmits the immunological inflammatory signals. These observations uncover yet another commonality between the neural and immune circuits and bear on the mechanisms of neuro–immune interactions.

Materials and Methods

Mice, Cells, and Reagents. C57BL/6 (VR1^+/+) and VR1^–/– mice were obtained from The Jackson Laboratory. Bone marrow-derived DCs were generated from the femurs and tibia of mice. The bone marrow was flushed out and the leukocytes obtained and cultured for 6 days as described (21). CP was purchased from Sigma–Aldrich. Abs against CD86 (B7-2), CD11c, MHCII for FACS analysis, Rab IgG, and anti-rabbit-FITC were purchased from Pharmingen. Abs for VR1 (P-19) and PA1-747 and their neutralizing peptides were purchased from Santa Cruz Biotechnology and Affinity BioReagents (Golden, CO), respectively.

RNA Isolation and RT-PCR. Total RNA was isolated from day 6 DCs by using RNeasy kit (Qiagen, Valencia, CA). Total RNA was reverse-transcribed by using a reverse transcription system (Promega) with oligo(dt) primers for 1 h at 42°C. The reaction was terminated by incubating the mixture at 99°C for 5 min. The resulting cDNA was used as a template for PCR amplification. PCR amplification of cDNA was performed according to The Jackson Laboratory genotyping protocol, in brief: 3 min at 94°C followed by 35 cycles of 94°C for 30 sec, 64°C for 1 min, and 72°C for 1 min, followed by 72°C for 2 min by using the following primers: oIMR0297, 5′-CACGAGACTAGTGAGACGTG-3′; oIMR1561, 5′-CCTGCTCAACATGCTCATTG-3′; and oIMR1562, 5′-TCCTCATGCACTTCAGGAAA-3′.

Immunoblotting for VR1. Day 6 DCs from VR1^+/+ mice were lysed by using detergent. Lysates of equal cell number were resolved on SDS/PAGE and immunoblotted by using VR1 Ab P19 with or without (≈70-fold excess) P19-specific neutralizing peptide or OVA19 irrelevant peptide.

Intracellular Staining of CD11c⁺ Cells. CD11c cells are positively selected from day 6 bone marrow cultures by using the magnetic-activated cell sorting columns and CD11c Ab, supplied as CD11c MicroBeads (Miltenyi Biotec, Auburn, CA), according to the manufacturer's protocols. After selection, cells were fixed with 4% paraformaldehyde for 15 min on ice, washed with PBS, and then permeabilized with 0.1% Triton X-100 at room temperature for 10 min. Cells were washed and incubated with 10% goat serum for 30 min at room temperature and then incubated for 40 min at room temperature with different dilutions of VR1 Ab (PA1-747) alone or with PA1-747-specific neutralizing peptide, irrelevant peptide OVA19 mer, or rabbit IgG as control. Cells were washed after incubation and stained with anti-rabbit-FITC for 20 min at room temperature, washed, and analyzed by FACS.

CP Treatment for DC Maturation. Day 6 DCs were harvested and washed once with OPTI MEMI medium (GIBCO). Cells were resuspended in OPTI MEMI medium and treated with CP (freshly prepared in ethanol) for 16 h at 37°C. For capsazepine treatment, DCs were pretreated with capsazepine for 10 min at 37°C and then incubated with CP for 16 h at 37°C. At the end of incubation period, cells were washed and analyzed.

CP Treatment for DC Migration. VR1^+/+ and VR1^–/– mice were injected intradermally with 200 μg (in 200 μl) of CP in vehicle containing 10% Tween 80, 10% ethanol, and 80% PBS. Control mice were injected with equal volume of vehicle. Lymph node cells were harvested after 4 h of injection, and DCs were enriched by using Opti-Prep (Axis-Shield, Oslo), according to the manufacturer's protocol. The enriched cells were stained with Abs to CD11c, IA^b, CD86, CD11b, and CD205 and analyzed.

Adoptive Transfer of OT1 T Cells and Tetramer Staining. Spleen cells from OT1-T cell antigen receptor transgenic mice were purified by positive selection by using magnetic-activated cell sorting (MACs) anti-CD8α MicroBeads (Miltenyi Biotec), according to the manufacturer's protocol. Then, 0.5 × 10⁶ CD8⁺ T cells purified from OT1 mice were injected i.v. in a volume of 200 μl of PBS into VR1^+/+ mice. Antigen was injected in a 200-μl volume intradermally after 2 days of adoptive transfer. After 3 days of antigen injection, lymph nodes and spleens of mice were harvested and stained with CD8-FITC/K^b-SIINFEKL-PE or CD8-FITC/CD45.1-PE.

Labeling of 5,6-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE). Splenocytes were resuspended in 10⁶ cells per ml concentration in PBS. Cells were labeled with CFSE (Molecular Probes) (5 mM stock) by diluting CFSE stock 1,000-fold into the cell suspension (5 μM final concentration for CFSE^high cells). CFSE^low cells were labeled in the same way, except the CFSE stock used in this case was 0.5 mM, such that the final concentration in the cell suspension was 0.5 μM. Cells were incubated with CFSE at 37°C for 10 min. After incubation of the cells, FCS was added, and cells were immediately centrifuged, washed with PBS, and counted.

In Vivo Cytotoxicity Assay. Splenocytes from C57BL/6 mice were pulsed with PBS or with 1 μM SIINFEKL peptide for 1 h at 37°C. Cells were washed and labeled with high CFSE fluorescence intensity (for peptide pulsed cells) and with low CFSE fluorescence intensity (for unpulsed cells). For i.v. injection, 5 × 10⁶ of each population were mixed in 200 μl of PBS. Specific in vivo cytotoxicity was determined by harvesting lymph node cells after 16 h of target cell transfer and detecting the differentially labeled fluorescent target cell populations by flow cytometry. The ratio r between the percentages of unpulsed vs. pulsed (CFSE^low/CFSE^high) was calculated to obtain a numerical value of cytotoxicity.

Results

VR1 Is Expressed in DCs. On day 6, bone marrow-derived DCs were tested for expression of CP receptor VR1. Total RNA from day 6 cultures of bone marrow cells (BMCs) was reverse-transcribed and subjected to PCR in the presence of primers for the mouse VR1 receptor (described in Materials and Methods). Fig. 1A depicts the PCR products from day 6 BMCs of WT (VR1^+/+) mice. VR1-specific PCR product was detected with an expected size of 984 bp. The expression of VR1 receptor protein in DCs was further examined by using an Ab that recognizes the N terminus of the CP receptor. Immunoreactive VR1 with a molecular mass of ≈100 kDa was detected when proteins isolated from DCs were separated by SDS/PAGE (Fig. 1B). The binding of the Ab to the VR1 protein is specific; it could be competed off by the P19 neutralizing peptide but not by an irrelevant peptide OVA19. Because both the N and C termini of VR1 protein are intracellular, the specificity of binding of anti-VR1 Ab to DCs was tested by intracellular staining. Purified CD11c⁺ (≥98%) cells (Fig. 1C) were stained by using various doses of anti VR1 Ab (Fig. 1D Left) along with VR1 specific neutralizing peptide and an irrelevant peptide to compete anti-VR1 binding (Fig. 1D Right). The stained cells were analyzed by FACS. A specific staining of DCs by anti VR1 Ab is shown.

Fig. 1.

Expression of VR1 on mouse bone marrow-derived DCs. (A) RNA isolated from mouse BMC (day 6) was subjected to RT-PCR for mouse VR1. PCR product was separated on 1% agarose gel and visualized with ethidium bromide. Lane 1, 100-bp DNA ladder; lane 2, VR1 PCR product. (B) VR1 protein expression was determined by loading ≈0.1 × 10⁶ BMC (day 6) lysate per lane and separating on a 10% SDS/PAGE. VR1 immunoreactivity was determined by using anti-VR1 Ab alone (lane 1), anti-VR1 Ab with irrelevant peptide (IP) (lane 2), or anti-VR1 Ab with neutralizing peptide (NP) (lane 3). (C and D) Intracellular staining of VR1 protein. (C) CD11c⁺ cells were purified from day 6 BMCs (≈98%), as described in Materials and Methods.(D) The purified cells were permeabilized and incubated with anti-VR1 Ab or isotype control rabbit IgG at different dilutions (Left) or anti-VR1 Ab (1:2,500) with or without neutralizing peptide or irrelevant peptide (Right). After incubation with primary Ab cells were washed and stained with FITC-conjugated goat anti-rabbit Ab and analyzed by FACs.

CP Can Cause Maturation of DCs from VR1^+/+ but Not VR1^–/– Mice. VR1^+/+ and VR1^–/– bone marrow derived DCs were tested for expression of VR1. Total RNA from day 6 cultures of BMCs was reverse-transcribed and subjected to PCR in the presence of primers for the mouse VR1 receptor. Fig. 2A shows the location of primers with respect to VR1 gene. Fig. 2B shows the PCR products from day 6 BMCs of VR1^+/+ and VR1^–/– mice. Primer oIMR1562 lies within the next exon downstream of the deleted sequence in VR1-targeted mutant allele. With primer oIMR0297 (pgk promoter/neo primer), it amplifies a 600-bp fragment from the RNA of disrupted allele (VR1^–/–), but no product is formed with VR1^+/+ RNA. From the same RNA samples, using primer oIMR1561 (that lies within the deleted exon of the VR1-targeted mutant allele) and primer oIMR1562, a VR1-specific PCR product was detected in VR1^+/+ RNA with an expected size of 984 bp but not in the VR1^–/– RNA. Because the VR1^–/– mice have been created by insertional inactivation rather than deletion of the gene, they still express the product of the gene, which is still recognizable by the Ab against VR1 (data not shown). Hence, the absence of a functional VR1 gene cannot be demonstrated by protein-based structural means such as immunoblotting or FACS analysis. To test the functionality of the VR1 receptor, immature DCs from day 6 granulocyte/macrophage colony-stimulating factor-stimulated BMCs from VR1^+/+ and VR1^–/– mice were exposed to increasing concentrations of CP for 16 h at 37°C. Increase in IA^b intensity on DCs was monitored by FACS. A titratable and specific increase in intensity of IA^b was observed. For maturation controls, DCs from both strains of mice were exposed to high temperature, i.e., 41°C/6 h (22, 23) or LPS (1 μg/ml) at 37°C. DCs from VR1^–/– mice did not express increased level of IA^b when exposed to CP but did so in presence of high temperature or LPS. In contrast, VR1^+/+ DCs responded to CP in a dose-dependent manner as well as to high temperature and LPS. The addition of VR1 antagonist capsazepine inhibited the increased expression of surface IA^b induced by CP (Fig. 2C). The CP-treated MHCII-positive DCs also expressed elevated levels of CD86 on their surface (data not shown).

Fig. 2.

CP mediates maturation of VR1^+/+ DCs but not VR1^–/– DCs. (A) Schematic representation of VR1 protein structure in VR1^+/+ and VR1^–/– mice including the position of primers used to detect VR1 mRNA in DCs by RT-PCR. (B) RNA isolated from VR1^+/+ and VR1^–/– bone marrow culture (day 6) was subjected to RT-PCR for mouse VR1. PCR product was separated on 1% agarose gel and visualized with ethidium bromide. Lanes 1 and 2, PCR product from VR1^+/+ and VR1^–/– DCs by using forward primer 297 and reverse primer 1,562; lanes 3 and 4, PCR product by using forward primer 1,561 and reverse primer 1,562 (in A). (Because the VR1^–/– mice have been created by insertional inactivation rather than deletion of the VR1 gene, they still express the product of the gene, which is still recognizable by the Ab against VR1; therefore, the lack of functional VR1 cannot be shown by using Ab-based methods.) (C) Day 6 BMCs from VR1^+/+ (Left) and VR1^–/– mice were cultured with different doses of CP with or without CP as indicated or with LPS (1 μg/ml) for 20 h at 37°C or heat shocked at 41°C for 6 h and allowed to recover for 16 h at 37°C. Cells were analyzed for expression of IA^b by using FACS. Increase in mean fluorescence intensity values as compared with vehicle-treated controls are plotted for IA^b.

Skin DCs Migrate to Draining Lymph Nodes upon Administration of CP. The preceding data suggest a relation between VR1 receptor expression by bone marrow-derived DCs and their maturation by CP in vitro. To test the effect in vivo of CP on DCs, both VR1^+/+ mice and VR1^–/– mice were injected with CP intradermally. The cells from the draining lymph nodes from injected mice were harvested after 4 h of injection and tested for CD11c and MHCII expression by FACs (Fig. 3A). The CD11c⁺ cells from the lymph node of VR1^+/+ mice injected with CP had very high expression of MHCII compared with the vehicle-injected mice, but the CD11c⁺ cells from VR1^–/– mice were unresponsive to CP injection in vivo (Fig. 3B). We analyzed the phenotype of the CD11c⁺ cells that migrated to the lymph nodes in VR1^+/+ mice by three-color flow cytometry (Fig. 3C). After immunizing with CP, the number of CD11c⁺ cells increased from 1% to 2.4% of total lymph node cells and the cells were larger in size than in the control mice. These CD11c⁺ cells were also MHCII^highCD86⁺CD11b⁺ and CD205⁺.A large proportion (75%) of the high-FSC/CD11c⁺ cells were CD205⁺, whereas 25% of the low-FSC/CD11c⁺ cells were CD205⁺. This phenotype is analogous to activated skin-derived DCs (24).

Fig. 3.

Injection of CP leads to maturation and migration of skin DCs in a VR1-dependent manner. (A) Experimental design. (B) Lymph node cells from vehicle- and CP-injected mice (see A) were stained for CD11c and IA^b and analyzed by flow cytometry. Increase in MFI values as compared with vehicle-treated controls for surface marker IA^b on CD11c⁺ cells is plotted. (C) Analysis of CD11c⁺ cells (R1) in the draining lymph nodes of vehicle- and CP-injected VR1^+/+ mice with respect to size, surface expression of IA^b (high, R2; and low, R3), CD86, CD11b, and CD205 by flow cytometry.

CP Induces Antigen-Specific T Cell Proliferation in Vivo. The potent effect of CP in activation of skin DCs in vivo led us to determine whether it can induce antigen-specific T cell proliferation. An adoptive transfer model in which ovalbumin (OVA)-specific B6-Ly5.1 CD8⁺ T cells (OT1 cells) are transferred to naive B6-Ly5.2 mice (VR1^+/+) was used. Expansion of OT1 cells in the draining lymph nodes or spleen of recipient mice was identified by staining with Abs to CD8/Ly5.1 or CD8/K^b tetramer associated with the OVA-derived peptide SIINFEKL. The fate of OT1 cells in the recipient mice after injection with soluble OVA (100 μg) and CP (200 μg) (2 h apart) was detected 3 days later. A small percentage (≈0.5%) of OT1 cells could be detected in the PBS-injected mice. Treatment with CP alone did not stimulate the OT1 cells nonspecifically. However, when OVA was injected with CP, there was a significant expansion of OT1 cells in spleen and lymph node as compared with the OVA and vehicle-injected control mice (Fig. 4 A–D). As proliferation of OT1 cells may not be an accurate reflection of effector T cell response, an in vivo CTL assay was performed. Differentially CFSE-labeled target cells from naive mice were pulsed with (CFSE^high) or without SIINFEKL (CFSE^low) and injected i.v. into experimental animals. After 18 h of target cell transfer, CTL responses to labeled targets in the lymph node and spleen were examined. All of the groups that showed significant OT1 proliferation also showed strong SIINFEKL-specific effector CTL response in vivo (Fig. 4E).

Fig. 4.

Adjuvanticity of CP. (A) Experimental design. (B) Expansion of OT1 cells in the draining lymph node of VR1^+/+ mice after 3 days of injection of OVA and CP was determined by flow cytometry by using K^b-SIINFEKL tetramer staining. (C) Proportion of CD8⁺ T cells that are K^b-SIINFEKL⁺ in the spleen and draining lymph node of VR1^+/+ mice after 3 days of injection. (D) Clonal expansion of OVA-specific CD8⁺ T cells in the draining lymph node of VR1^+/+ mice 3 days after injection by using CD45.1 as a clonotypic marker. (E) In vivo effector function of OT1 cells was determined by CTL assay in vivo. C57BL/6 splenocytes from naive mice were pulsed with OVA 8 mer. The peptide-pulsed cells were labeled with CFSE^high, and unpulsed cells were labeled with CFSE^low. Both cell types were transferred i.v. to day 3 OVA/CP-injected mice. Specific cytotoxic effector function of OT1 cells was determined by monitoring the elimination of peptide-pulsed targets. The ratios of percentages of unpulsed vs. pulsed targets is shown.

Discussion

Our studies were initiated with intent to identify the heat receptor on DCs that is responsible for the heat-induced maturation of DCs, as reported (22). Clearly VR1 is not such a receptor, because DCs from VR1^–/– mice undergo maturation in response to heat in the same way that the DCs from VR1^+/+ mice do (Fig. 2). We are in the process of investigating the other members of the TRPV family for that role. Regardless of that quest, the unique observations described here reveal significantly that (i) a potent neuroactive ligand is also a potent immunoactive ligand (Figs. 2, 3, 4); (ii) this exogenous ligand, CP, is engaged by sensory neurons and DCs alike through the VR1 receptors of the TRPV family (Figs. 2 and 3); and (iii) engagement of CP by the nervous system leads to perception of pain and temperature, whereas a similar engagement by the immune system leads to innate and adaptive immune responses (Figs. 3 and 4). These observations highlight “a certain symmetry” between ligands and mechanisms of their engagement between the neurological perceptions of pain/temperature and the immunological perception of inflammation.

This symmetry has certain implications and predictions. At the broadest level, the observations of symmetry strengthen the premise that the neural and immune systems share a common phylogenetic origin, a premise based on disperse but persistent and rigorous studies by a small number of investigators (25–30). Note, in this regard, that receptors for SEMA4D, Slit2, Eph, semaphorin 3A, etc., are expressed on DCs as well as on neurons but are not otherwise widely distributed (28, 31–37). For example, the neuronal receptor neuropilin-1, involved in axon guidance, is expressed on human DCs and is essential for initiation of the primary immune response (33). One may deduce from such examples the possibility, as highlighted by our results, that neuroactive and immunoactive ligands exert cross-system effects. More specifically, our data suggest that other neuroactive ligands such as Substance P, bradykinins, enkephalins, and other opioids may have unknown immunological effects. Indeed, the potent neuroactive opoid enkephalin has been reported by several investigators to be expressed in an antigen-specific inducible manner by some but not other T lymphocytes (38), and the immunomodulatory activity of opoids has been attributed to μ but not δ or κ receptors (39). It is also noteworthy in this regard that Substance P and bradykinins have been shown to mediate cytoplasmo-nuclear translocation of NFκB, a transcription factor and a mechanism central to innate immune responses (40, 41). Endogenously produced substance P has been shown to contribute to lymphocyte proliferation and T cell antigen receptor ligation (34). The possibility that these molecules may act as immunological adjuvants, as shown in these experiments for CP, deserves experimental enquiry. Conversely, the possibility that immunological ligands such as heat-shock proteins, LPS, or toll-like receptors ligands in general, may have unknown neurological properties, needs to be examined. Note, in this regard, that the immunological adjuvant muramyl dipeptide has a potent activity as a slow-wave sleep factor (42), and indeed, sleep has been described as a neuroimmune phenomenon (43).

The identity of the internal ligand of VR1 (or an “internal CP”) merits thought. N-arachidonoyldopamine, lipoxygenase products of arachidonic acid and anandamide, have been suggested to be candidates for endogenous vanilloids (44). Of these, arachidonoyldopamine is the closest to CP in certain assays of potency. Our studies suggest that these or other endovanilloids possess immunomodulatory properties and potentially mediate crosstalk between neural and immunological activities in vivo. It is interesting in this regard that Ananadamide, a candidate endovanilloid, has been detected in human DCs (45).

Heat (or cold) are the only common ligands of members of the TRPV family. It is reasonable to speculate that similar to the situation with VR1, candidate endogenous chemical ligands for these other receptors shall be identified in due course. The mechanisms through which these receptors may have a common responsiveness for a chemical signal (endogenous ligand) and a physical signal (heat) deserves contemplation. The molecular details involved in the two interactions are still unknown, but plausible mechanisms may be imagined. The TRPV receptors are ion channels, and one can imagine them being opened by a ligand as also by discrete changes in membrane fluidity brought about by temperature changes. In addition, temperature changes (or changes in the pH in a discrete microenvironment) may bring about changes in proton transport across the plasma membrane and these in turn, may open the ion channel.

Our results also suggest a nexus between nutrition and immunity. CP is a common component of staple foods of many cultures, and although its adjuvanticity has been demonstrated in the present studies through the parenteral rather than the enteral route, the question of interaction of CP (and like compounds) with the enteric immune system, as well as with the enteric nervous system, is worthy of investigation.