Augmentation of Cutaneous Immune Responses by ATPS: Purinergic Agonists Define a Novel Class of Immunologic Adjuvants1
http://www.100md.com
免疫学杂志 2005年第12期
Abstract
Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X1, P2X7, P2Y1, P2Y2, P2Y4, P2Y9, and P2Y11 receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.
Introduction
Recent years have seen great interest in vaccine development. This has resulted in part from the clinical need for better ways to prevent infection, but there has also been much interest in the development of therapeutic vaccines for tumors and for the treatment of HIV infections (1, 2). New approaches to the production of vaccines include the use of synthetic peptides, DNA, RNA, and protein subunits. These approaches offer a number of advantages, including increased specificity and reduced toxicity (1, 2). However, many are poorly immunogenic when administered alone (1, 2, 3). Currently used adjuvants include mineral salts, immunostimulatory cytokines, lipid particles, microparticulates, and so-called mucosal adjuvants (1, 2, 3). Although the mechanisms of action for some of these, such as immunostimulatory cytokines, may be inferred from what is known of their physiologic activities, the mechanisms of action of many of these are poorly understood. Preclinical and clinical trials have been performed with a variety of adjuvants, and although some have been immunologically effective, most have been too toxic for routine use. At the current time, the only adjuvants proved for use by the U.S. Food and Drug Administration are aluminum-based mineral salts (alum) (1). Despite decades of use, the mechanism of action of alum remains obscure. Recent studies suggest that alum up-regulates costimulatory molecules on APCs and promotes the expression of IL-4. Alum has been associated with local reactions in some individuals and is believed to be a relatively weak adjuvant for cell-mediated immunity and Ab induction to protein subunits. Thus, the development of safer and more effective adjuvants is of great importance.
Langerhans cells (LCs)3 are an important subclass of dendritic cells that reside in the epidermis (4). Unlike other dendritic cells, they have a distinctive intracellular organelle (the Birbeck granule), and express the protein langerin (5). Because of their location in the skin, they are in a position to initiate an immune response to pathogens and vaccines that enter through the skin. Thus, they are a population that merits special attention from those who are seeking to administer vaccines by the cutaneous route. The dermis also contains populations of dendritic APCs (6). These are heterogeneous and generally less well-characterized than LCs; however, they are capable of presenting protein Ags, peptide Ags, and tumor Ags in experimental systems (7, 8, 9). Given their location, dermal dendritic cells are also probably important for presentation of Ags administered intradermally or s.c. LCs express high levels of cell surface ATPase (CD39), suggesting that they may be sensitive to ATP (10). Therefore, we hypothesized that agonists of purinergic receptors may augment cutaneous immunity and serve as adjuvants. Our hypothesis also suggests that ATP lost from cells subsequent to trauma or infection may serve as a local signal to enhance the immune response, i.e., an endogenous adjuvant. There are several precedents for this possibility. Monocyte-derived dendritic cells have been shown to express a number of P2Rs of both the P2X and the P2Y series (11, 12). Exposure of monocyte-derived dendritic cells to ATP results in up-regulation of CD54 and MHC class II molecules, induces secretion of IL-12, and augments stimulatory capacity for allogeneic T cells (11, 13). Exposure of LPS-matured dendritic cells to ATP induces the release of IL-1 and TNF- (12). In combination with TNF-, ATP has been shown to increase the expression of CD80, CD83, and CD86 in monocyte-derived dendritic cells (11). Furthermore, the ability of dendritic cells derived from murine fetal skin to present Ag was reduced by oxidized ATP, an irreversible inhibitor of P2XRs, and clones of dendritic cells selected for lack of P2X7Rs also had decreased Ag-presenting ability, suggesting a role for this receptor in Ag presentation (14).
We report that exogenous administration of a purinergic agonist can enhance cutaneous immunity and provide evidence that it does so by augmenting the ability of LCs to present Ag. These data demonstrate that ATP is an endogenous adjuvant and that the release of ATP during infection or trauma to the skin may result in enhanced cutaneous immunity as a homeostatic protective response. These results also suggest that agonists of P2Rs represent a potentially useful class of therapeutic adjuvants.
Materials and Methods
The significance of differences among groups was measured by Student’s two-tailed t test for unpaired samples (Excel software; Microsoft).
Results
ATPS enhances the induction of CHS
To determine whether P2Rs modulate the immune response, we determined the effects of purinergic agonists on CHS. Groups of BALB/c mice were injected intradermally on the dorsum with vehicle alone or 30 nmol of ATPS, a stable analog of ATP that interacts with purinergic receptors (18). Fifteen minutes later, these mice were immunized by careful application of DNFB at the injected site or, in some experiments, at a site contralateral to that of the injection. Nonimmunized control mice were similarly injected with ATPS or medium alone, but were not immunized. Seven days later, all mice were challenged on the ears by application of 0.2% of DNFB in acetone, and 24 h ear swelling was assessed as a measure of the CHS response. Intradermal administration of ATPS at the site of immunization (but not at a distant site) significantly increased the CHS response (Fig. 1), supporting the hypothesis that P2 agonists enhance immune responsiveness.
ATPS augments the induction of DTH to a model tumor vaccine
To examine whether ATPS enhances immunity induced by a vaccine, we immunized CAF1 mice by intradermal injection of either TAA or TAA containing 30 nmol of ATPS. Negative control mice were not immunized. One week after the last immunization, each mouse was challenged in a hind footpad with TAA, and footpad swelling was measured as an indication of immunological responsiveness. ATPS significantly enhanced the DTH response at 24 h (Fig. 2), again supporting the hypothesis that P2 agonists enhance immune responsiveness. Assessment of the DTH response at 48 h yielded the same results (data not shown).
Treatment of epidermal LCs with ATPS in vitro augments their ability to present Ag to HDK-1 cells
The increase in immune responsiveness could occur by several mechanisms. One possibility is ATPS enhances Ag presentation in the skin. To determine whether ATPS increases the Ag-presenting ability of LCs, we examined whether ATPS would enhance Ag presentation to the T cell clone HDK-1. This cloned line responds to presentation of KLH by I-Ad with production of IFN-. We cultured a population of LCs enriched to >95% in medium alone or in medium containing graded concentrations of ATPS. After 3 h, KLH was added, and 2 h later, cells were gamma irradiated and washed three times to remove any residual ATPS. LCs were then cocultured with HDK-1 cells. After 72 h, we harvested supernatants and measured IFN- content by ELISA. Exposure to ATPS resulted in a dose-dependent increase in IFN- release (Fig. 3). When LCs were incubated with ATPS in the absence of KLH, only background levels of IFN- were produced. Thus, ATPS enhanced Ag presentation. At concentrations >10–6 M, the response began to decrease, possibly due to induction of apoptosis, because concentrations >10–5 M induce apoptosis in the LC-like cell line XS106. After 24 h of exposure to 10–4 M ATPS, 3–4% of XS106 cells were dead (by trypan blue exclusion testing), and 13–21% were killed with a concentration of 10–3 M, compared with 2–3% dead when cultured in medium without ATPS.
Although LCs were washed extensively to remove ATPS before being added to HDK-1 cells, we performed additional experiments to test whether residual ATPS might be acting directly on HDK-1 cells. When HDK-1 cells (rather than LCs) were pretreated with ATPS, there was no increase in IFN- release (data not shown).
ATPS augments the ability of LCs to present alloantigen to T cells in vitro
To determine the effect of ATPS on presentation to naive T cells, we examined the ability of ATPS to modulate the presentation of alloantigens by an unseparated population of ECs to naive allogeneic T cells (i.e., the mixed epidermal cell-lymphocyte reaction). ATPS (10–6 M) enhanced the ability of ECs to present alloantigens to allogeneic T cells (Fig. 4). Thus, activation of P2Rs on ECs also enhances Ag presentation to naive T cells.
ATP agonists augment the expression of I-A, CD80, and CD86 on XS106 cells
To explore mechanisms by which ATP agonists enhance Ag presentation, we studied the effect of ATPS on XS106, a cloned cell line that was derived from neonatal A/J epidermis and has many of the functional and phenotypic characteristics of LCs (15). To stimulate the expression of I-A, CD80, and CD86, XS106 cells were cultured in medium containing GM-CSF and LPS in the presence of graded concentrations of ATPS. As indicated by FACS analysis, expression of three surface molecules that are important for Ag presentation (I-A, CD80, and CD86) was enhanced by exposure to 10–4 M ATPS (Fig. 5). Because this is a cell line, any effects we observed were presumably due to direct effects of ATPS on the cells. We conclude that ATPS can directly modulate LC maturation.
P2 agonists enhance the secretion of IL-1 and IL-12 while reducing the release of IL-10
We used a similar strategy to determine ATPS-modulated cytokine production. XS106 cells were cultured in medium containing GM-CSF and LPS in the presence or the absence of increasing concentrations of ATPS. After 24 h, we harvested supernatants and measured cytokine content by ELISA. ATPS significantly increased the release of IL-1 and IL-12 while decreasing the release of IL-10 from GM-CSF/LPS-stimulated XS106 cells (Fig. 6). In the absence of LPS and GM-CSF, ATPS induced IL-1 release, but to a much lesser degree than in the presence of LPS and GM-CSF (data not shown). The combination of ATPS and GM-CSF or LPS resulted in greater IL-1 production than ATPS alone, but still considerably less than when ATPS was combined with both LPS and GM-CSF (data not shown). ATPS alone did not induce a detectable level of IL-12 release (data not shown), but it did induce some release in the presence of LPS or GM-CSF (data not shown). However, as shown in Fig. 6b, in the presence of both LPS and GM-CSF, ATPS greatly augments IL-12 production. Thus, the effects of ATP agonists on immune responsiveness may be explained in part by the regulation of cytokine expression by APCs in the skin, but a detailed understanding of how signaling by ATPS interacts with signaling by LPS and GM-CSF will require additional work.
As a pharmacological test for the involvement of cell surface P2Rs in modulating LC function, we pretreated with agents that are antagonists of these receptors and then measured the effect of ATPS on cytokine production. XS106 cells were treated with four different P2 antagonists (oxidized ATP, suramin, or PPNDS) for 1 h before addition of ATPS. Supernatants were harvested for measurement of IL-1 content. All inhibitors tested significantly reduced the response (Fig. 7a). When XS106 cells were washed three times after exposure to the inhibitors, but before addition of ATPS, only oxidized ATP inhibited the response (Fig. 7b). Because oxidized ATP is an irreversible inhibitor of P2Rs and the other agents are competitive inhibitors, this observation also supports the role of P2Rs in the responses to ATP.
XS106 cells express mRNA for P2Rs
All these experiments are consistent with our hypothesis that ATP acts as an adjuvant by binding to cell surface P2Rs. As a direct test for the presence of these receptors, we used RT-PCR. XS106 cells were found to express mRNA for P2X1, P2X7, P2Y1, P2Y2, P2Y4, P2Y9, and P2Y11 receptors (Fig. 8). In each case the PCR product was of the predicted length and was dependent on the presence of RT. Thus, LCs express several different P2Rs, but it is not clear which of these receptors or which combinations of these receptors mediate the effects of ATP on Ag presentation or immunity.
Discussion
We have demonstrated that ATP agonists have significant immunostimulatory effects on cell- mediated immunity within the skin. Correlative experiments have demonstrated that these agonists enhance the LC Ag-presenting ability in vitro as well as the expression of MHC class II molecules, CD86, CD80, and proinflammatory cytokines while decreasing the expression of IL-10. These data strongly suggest that the mechanism of action of ATP agonists in vivo relates to immunostimulatory effects on APCs. The ability of P2R antagonists to block the effect of ATPS on induction of IL-1 release demonstrates that, at least for this end point, the effect observed is dependent on signaling through a P2R. Although the various inhibitors we used have different capacities to block different P2Rs (19), in the absence of detailed dose-response experiments with each inhibitor, it is premature to speculate on which receptor(s) is relevant to the effects we have observed. We have recently obtained preliminary data showing that Abs to the P2X7R can inhibit some responses to ATP, but additional work needs to be performed to establish the importance of this receptor. It also remains possible that ATPS may signal through non-P2Rs. Of course, the experiments examining the effects of ATPS on MHC, CD86, CD80, and cytokine expression were performed on XS106 cells, rather than fresh LCs; therefore, the results must be interpreted cautiously.
These observations have several important implications. First, ATP is most likely an endogenous regulator of LC function within the skin. Damaged cells are known to release ATP into the extracellular environment (10), and we hypothesize that the release of ATP from keratinocytes after injury or irritation of the skin leads to ATP-induced up-regulation of LC function as a homeostatic protective mechanism. This observation may also explain why contact allergens are invariably also contact irritants; irritation may be necessary for up-regulation of APC function and subsequent induction of immunity to the allergen (20). In this regard, purinergic agonists may represent a class of danger signals that alert the immune system to dying cells in a manner analogous to that recently proposed for uric acid (21). Interestingly, it appears that the stimulatory effect of P2 agonists is maximal in the presence of additional stimulatory signals. In our experiments, LPS and GM-CSF provided these signals. Physiologically, in the setting of infection, such a signal(s) could be derived from a pathogen. The results seen with our CHS and DTH experiments demonstrate that immunity induced with an epicutaneously applied hapten or our intradermally administered vaccine is significantly boosted without an additional signal being supplied, or that the immunogen itself supplies a second signal or induces the release of such a signal endogenously. Second, and most importantly, the immunostimulatory properties of ATP agonists may be exploited for adjuvant activity to enhance the efficacy of vaccines. As stated above, there is great interest in the development of new adjuvants for use with vaccines. All currently available adjuvants have limitations, and the development of novel, more effective, and safer adjuvants could have practical benefits for increasing the efficacy and utility of both prophylactic and therapeutic vaccines. Although additional work remains to be performed, understanding the molecular and cellular mechanisms by which purinergic agonists exert their immunostimulatory effects and the range of immune responses that may be enhanced is of considerable importance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by National Institutes of Health Grant AI59226 (to R.D.G. and J.A.W.), a grant from the Abercrombie Foundation, a grant from the Edith C. Blum Foundation, contributions from the Ann L. and Herbert J. Siegel Philanthropic Fund, a gift from the Jacob L. and Lillian Holtzmann Foundation, contributions from Mr. and Mrs. Blair O’Connor, and a gift from Call on an Angel.
2 Address correspondence and reprint requests to Dr. Richard D. Granstein, Department of Dermatology, Weill Medical College of Cornell University, 1300 York Avenue, Room F-342, New York, NY 10021. E-mail address: rdgranst{at}med.cornell.edu
3 Abbreviations used in this paper: LC, Langerhans cells; CHS, contact hypersensitivity; CM, complete medium; DNFB, dinitrofluorobenzine; DTH, delayed-type hypersensitivity; EC, epidermal cell; KLH, keyhole limpet hemocyanin; PPNDS, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate); TAA, tumor-associated Ag.
Received for publication October 19, 2004. Accepted for publication April 12, 2005.
References
Singh, M., D. T. O’Hagan. 2002. Recent advances in vaccine adjuvants. Pharm. Res. 19: 715-728.
Hunter, R. L.. 2002. Overview of vaccine adjuvants: present and future. Vaccine 20: S7-S12.
Alving, C. R.. 2002. Design and selection of vaccine adjuvants: animal models and human trials. Vaccine 20: S56-S64.
Streilen, J. W., P. R. Bergstresser. 1984. LC: APC of the epidermis. Immunobiology 186: 285-300.
Valladeau, J., O. Ravel, C. Dezutter-Dambuyant, K. Moore, M. Kleijmeer, Y. Liu, V. Duvert-Frances, C. Vincent, D. Schmitt, J. Davoust, et al 2000. Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules. Immunity 12: 71-81.
Nestle, F. O., X. G. Zheng, C. B. Thompson, L. A. Turka, B. J. Nickoloff. 1993. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets. J. Immunol. 151: 6535-6545.(Richard D. Granstein2,*, )
Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X1, P2X7, P2Y1, P2Y2, P2Y4, P2Y9, and P2Y11 receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.
Introduction
Recent years have seen great interest in vaccine development. This has resulted in part from the clinical need for better ways to prevent infection, but there has also been much interest in the development of therapeutic vaccines for tumors and for the treatment of HIV infections (1, 2). New approaches to the production of vaccines include the use of synthetic peptides, DNA, RNA, and protein subunits. These approaches offer a number of advantages, including increased specificity and reduced toxicity (1, 2). However, many are poorly immunogenic when administered alone (1, 2, 3). Currently used adjuvants include mineral salts, immunostimulatory cytokines, lipid particles, microparticulates, and so-called mucosal adjuvants (1, 2, 3). Although the mechanisms of action for some of these, such as immunostimulatory cytokines, may be inferred from what is known of their physiologic activities, the mechanisms of action of many of these are poorly understood. Preclinical and clinical trials have been performed with a variety of adjuvants, and although some have been immunologically effective, most have been too toxic for routine use. At the current time, the only adjuvants proved for use by the U.S. Food and Drug Administration are aluminum-based mineral salts (alum) (1). Despite decades of use, the mechanism of action of alum remains obscure. Recent studies suggest that alum up-regulates costimulatory molecules on APCs and promotes the expression of IL-4. Alum has been associated with local reactions in some individuals and is believed to be a relatively weak adjuvant for cell-mediated immunity and Ab induction to protein subunits. Thus, the development of safer and more effective adjuvants is of great importance.
Langerhans cells (LCs)3 are an important subclass of dendritic cells that reside in the epidermis (4). Unlike other dendritic cells, they have a distinctive intracellular organelle (the Birbeck granule), and express the protein langerin (5). Because of their location in the skin, they are in a position to initiate an immune response to pathogens and vaccines that enter through the skin. Thus, they are a population that merits special attention from those who are seeking to administer vaccines by the cutaneous route. The dermis also contains populations of dendritic APCs (6). These are heterogeneous and generally less well-characterized than LCs; however, they are capable of presenting protein Ags, peptide Ags, and tumor Ags in experimental systems (7, 8, 9). Given their location, dermal dendritic cells are also probably important for presentation of Ags administered intradermally or s.c. LCs express high levels of cell surface ATPase (CD39), suggesting that they may be sensitive to ATP (10). Therefore, we hypothesized that agonists of purinergic receptors may augment cutaneous immunity and serve as adjuvants. Our hypothesis also suggests that ATP lost from cells subsequent to trauma or infection may serve as a local signal to enhance the immune response, i.e., an endogenous adjuvant. There are several precedents for this possibility. Monocyte-derived dendritic cells have been shown to express a number of P2Rs of both the P2X and the P2Y series (11, 12). Exposure of monocyte-derived dendritic cells to ATP results in up-regulation of CD54 and MHC class II molecules, induces secretion of IL-12, and augments stimulatory capacity for allogeneic T cells (11, 13). Exposure of LPS-matured dendritic cells to ATP induces the release of IL-1 and TNF- (12). In combination with TNF-, ATP has been shown to increase the expression of CD80, CD83, and CD86 in monocyte-derived dendritic cells (11). Furthermore, the ability of dendritic cells derived from murine fetal skin to present Ag was reduced by oxidized ATP, an irreversible inhibitor of P2XRs, and clones of dendritic cells selected for lack of P2X7Rs also had decreased Ag-presenting ability, suggesting a role for this receptor in Ag presentation (14).
We report that exogenous administration of a purinergic agonist can enhance cutaneous immunity and provide evidence that it does so by augmenting the ability of LCs to present Ag. These data demonstrate that ATP is an endogenous adjuvant and that the release of ATP during infection or trauma to the skin may result in enhanced cutaneous immunity as a homeostatic protective response. These results also suggest that agonists of P2Rs represent a potentially useful class of therapeutic adjuvants.
Materials and Methods
The significance of differences among groups was measured by Student’s two-tailed t test for unpaired samples (Excel software; Microsoft).
Results
ATPS enhances the induction of CHS
To determine whether P2Rs modulate the immune response, we determined the effects of purinergic agonists on CHS. Groups of BALB/c mice were injected intradermally on the dorsum with vehicle alone or 30 nmol of ATPS, a stable analog of ATP that interacts with purinergic receptors (18). Fifteen minutes later, these mice were immunized by careful application of DNFB at the injected site or, in some experiments, at a site contralateral to that of the injection. Nonimmunized control mice were similarly injected with ATPS or medium alone, but were not immunized. Seven days later, all mice were challenged on the ears by application of 0.2% of DNFB in acetone, and 24 h ear swelling was assessed as a measure of the CHS response. Intradermal administration of ATPS at the site of immunization (but not at a distant site) significantly increased the CHS response (Fig. 1), supporting the hypothesis that P2 agonists enhance immune responsiveness.
ATPS augments the induction of DTH to a model tumor vaccine
To examine whether ATPS enhances immunity induced by a vaccine, we immunized CAF1 mice by intradermal injection of either TAA or TAA containing 30 nmol of ATPS. Negative control mice were not immunized. One week after the last immunization, each mouse was challenged in a hind footpad with TAA, and footpad swelling was measured as an indication of immunological responsiveness. ATPS significantly enhanced the DTH response at 24 h (Fig. 2), again supporting the hypothesis that P2 agonists enhance immune responsiveness. Assessment of the DTH response at 48 h yielded the same results (data not shown).
Treatment of epidermal LCs with ATPS in vitro augments their ability to present Ag to HDK-1 cells
The increase in immune responsiveness could occur by several mechanisms. One possibility is ATPS enhances Ag presentation in the skin. To determine whether ATPS increases the Ag-presenting ability of LCs, we examined whether ATPS would enhance Ag presentation to the T cell clone HDK-1. This cloned line responds to presentation of KLH by I-Ad with production of IFN-. We cultured a population of LCs enriched to >95% in medium alone or in medium containing graded concentrations of ATPS. After 3 h, KLH was added, and 2 h later, cells were gamma irradiated and washed three times to remove any residual ATPS. LCs were then cocultured with HDK-1 cells. After 72 h, we harvested supernatants and measured IFN- content by ELISA. Exposure to ATPS resulted in a dose-dependent increase in IFN- release (Fig. 3). When LCs were incubated with ATPS in the absence of KLH, only background levels of IFN- were produced. Thus, ATPS enhanced Ag presentation. At concentrations >10–6 M, the response began to decrease, possibly due to induction of apoptosis, because concentrations >10–5 M induce apoptosis in the LC-like cell line XS106. After 24 h of exposure to 10–4 M ATPS, 3–4% of XS106 cells were dead (by trypan blue exclusion testing), and 13–21% were killed with a concentration of 10–3 M, compared with 2–3% dead when cultured in medium without ATPS.
Although LCs were washed extensively to remove ATPS before being added to HDK-1 cells, we performed additional experiments to test whether residual ATPS might be acting directly on HDK-1 cells. When HDK-1 cells (rather than LCs) were pretreated with ATPS, there was no increase in IFN- release (data not shown).
ATPS augments the ability of LCs to present alloantigen to T cells in vitro
To determine the effect of ATPS on presentation to naive T cells, we examined the ability of ATPS to modulate the presentation of alloantigens by an unseparated population of ECs to naive allogeneic T cells (i.e., the mixed epidermal cell-lymphocyte reaction). ATPS (10–6 M) enhanced the ability of ECs to present alloantigens to allogeneic T cells (Fig. 4). Thus, activation of P2Rs on ECs also enhances Ag presentation to naive T cells.
ATP agonists augment the expression of I-A, CD80, and CD86 on XS106 cells
To explore mechanisms by which ATP agonists enhance Ag presentation, we studied the effect of ATPS on XS106, a cloned cell line that was derived from neonatal A/J epidermis and has many of the functional and phenotypic characteristics of LCs (15). To stimulate the expression of I-A, CD80, and CD86, XS106 cells were cultured in medium containing GM-CSF and LPS in the presence of graded concentrations of ATPS. As indicated by FACS analysis, expression of three surface molecules that are important for Ag presentation (I-A, CD80, and CD86) was enhanced by exposure to 10–4 M ATPS (Fig. 5). Because this is a cell line, any effects we observed were presumably due to direct effects of ATPS on the cells. We conclude that ATPS can directly modulate LC maturation.
P2 agonists enhance the secretion of IL-1 and IL-12 while reducing the release of IL-10
We used a similar strategy to determine ATPS-modulated cytokine production. XS106 cells were cultured in medium containing GM-CSF and LPS in the presence or the absence of increasing concentrations of ATPS. After 24 h, we harvested supernatants and measured cytokine content by ELISA. ATPS significantly increased the release of IL-1 and IL-12 while decreasing the release of IL-10 from GM-CSF/LPS-stimulated XS106 cells (Fig. 6). In the absence of LPS and GM-CSF, ATPS induced IL-1 release, but to a much lesser degree than in the presence of LPS and GM-CSF (data not shown). The combination of ATPS and GM-CSF or LPS resulted in greater IL-1 production than ATPS alone, but still considerably less than when ATPS was combined with both LPS and GM-CSF (data not shown). ATPS alone did not induce a detectable level of IL-12 release (data not shown), but it did induce some release in the presence of LPS or GM-CSF (data not shown). However, as shown in Fig. 6b, in the presence of both LPS and GM-CSF, ATPS greatly augments IL-12 production. Thus, the effects of ATP agonists on immune responsiveness may be explained in part by the regulation of cytokine expression by APCs in the skin, but a detailed understanding of how signaling by ATPS interacts with signaling by LPS and GM-CSF will require additional work.
As a pharmacological test for the involvement of cell surface P2Rs in modulating LC function, we pretreated with agents that are antagonists of these receptors and then measured the effect of ATPS on cytokine production. XS106 cells were treated with four different P2 antagonists (oxidized ATP, suramin, or PPNDS) for 1 h before addition of ATPS. Supernatants were harvested for measurement of IL-1 content. All inhibitors tested significantly reduced the response (Fig. 7a). When XS106 cells were washed three times after exposure to the inhibitors, but before addition of ATPS, only oxidized ATP inhibited the response (Fig. 7b). Because oxidized ATP is an irreversible inhibitor of P2Rs and the other agents are competitive inhibitors, this observation also supports the role of P2Rs in the responses to ATP.
XS106 cells express mRNA for P2Rs
All these experiments are consistent with our hypothesis that ATP acts as an adjuvant by binding to cell surface P2Rs. As a direct test for the presence of these receptors, we used RT-PCR. XS106 cells were found to express mRNA for P2X1, P2X7, P2Y1, P2Y2, P2Y4, P2Y9, and P2Y11 receptors (Fig. 8). In each case the PCR product was of the predicted length and was dependent on the presence of RT. Thus, LCs express several different P2Rs, but it is not clear which of these receptors or which combinations of these receptors mediate the effects of ATP on Ag presentation or immunity.
Discussion
We have demonstrated that ATP agonists have significant immunostimulatory effects on cell- mediated immunity within the skin. Correlative experiments have demonstrated that these agonists enhance the LC Ag-presenting ability in vitro as well as the expression of MHC class II molecules, CD86, CD80, and proinflammatory cytokines while decreasing the expression of IL-10. These data strongly suggest that the mechanism of action of ATP agonists in vivo relates to immunostimulatory effects on APCs. The ability of P2R antagonists to block the effect of ATPS on induction of IL-1 release demonstrates that, at least for this end point, the effect observed is dependent on signaling through a P2R. Although the various inhibitors we used have different capacities to block different P2Rs (19), in the absence of detailed dose-response experiments with each inhibitor, it is premature to speculate on which receptor(s) is relevant to the effects we have observed. We have recently obtained preliminary data showing that Abs to the P2X7R can inhibit some responses to ATP, but additional work needs to be performed to establish the importance of this receptor. It also remains possible that ATPS may signal through non-P2Rs. Of course, the experiments examining the effects of ATPS on MHC, CD86, CD80, and cytokine expression were performed on XS106 cells, rather than fresh LCs; therefore, the results must be interpreted cautiously.
These observations have several important implications. First, ATP is most likely an endogenous regulator of LC function within the skin. Damaged cells are known to release ATP into the extracellular environment (10), and we hypothesize that the release of ATP from keratinocytes after injury or irritation of the skin leads to ATP-induced up-regulation of LC function as a homeostatic protective mechanism. This observation may also explain why contact allergens are invariably also contact irritants; irritation may be necessary for up-regulation of APC function and subsequent induction of immunity to the allergen (20). In this regard, purinergic agonists may represent a class of danger signals that alert the immune system to dying cells in a manner analogous to that recently proposed for uric acid (21). Interestingly, it appears that the stimulatory effect of P2 agonists is maximal in the presence of additional stimulatory signals. In our experiments, LPS and GM-CSF provided these signals. Physiologically, in the setting of infection, such a signal(s) could be derived from a pathogen. The results seen with our CHS and DTH experiments demonstrate that immunity induced with an epicutaneously applied hapten or our intradermally administered vaccine is significantly boosted without an additional signal being supplied, or that the immunogen itself supplies a second signal or induces the release of such a signal endogenously. Second, and most importantly, the immunostimulatory properties of ATP agonists may be exploited for adjuvant activity to enhance the efficacy of vaccines. As stated above, there is great interest in the development of new adjuvants for use with vaccines. All currently available adjuvants have limitations, and the development of novel, more effective, and safer adjuvants could have practical benefits for increasing the efficacy and utility of both prophylactic and therapeutic vaccines. Although additional work remains to be performed, understanding the molecular and cellular mechanisms by which purinergic agonists exert their immunostimulatory effects and the range of immune responses that may be enhanced is of considerable importance.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported in part by National Institutes of Health Grant AI59226 (to R.D.G. and J.A.W.), a grant from the Abercrombie Foundation, a grant from the Edith C. Blum Foundation, contributions from the Ann L. and Herbert J. Siegel Philanthropic Fund, a gift from the Jacob L. and Lillian Holtzmann Foundation, contributions from Mr. and Mrs. Blair O’Connor, and a gift from Call on an Angel.
2 Address correspondence and reprint requests to Dr. Richard D. Granstein, Department of Dermatology, Weill Medical College of Cornell University, 1300 York Avenue, Room F-342, New York, NY 10021. E-mail address: rdgranst{at}med.cornell.edu
3 Abbreviations used in this paper: LC, Langerhans cells; CHS, contact hypersensitivity; CM, complete medium; DNFB, dinitrofluorobenzine; DTH, delayed-type hypersensitivity; EC, epidermal cell; KLH, keyhole limpet hemocyanin; PPNDS, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate); TAA, tumor-associated Ag.
Received for publication October 19, 2004. Accepted for publication April 12, 2005.
References
Singh, M., D. T. O’Hagan. 2002. Recent advances in vaccine adjuvants. Pharm. Res. 19: 715-728.
Hunter, R. L.. 2002. Overview of vaccine adjuvants: present and future. Vaccine 20: S7-S12.
Alving, C. R.. 2002. Design and selection of vaccine adjuvants: animal models and human trials. Vaccine 20: S56-S64.
Streilen, J. W., P. R. Bergstresser. 1984. LC: APC of the epidermis. Immunobiology 186: 285-300.
Valladeau, J., O. Ravel, C. Dezutter-Dambuyant, K. Moore, M. Kleijmeer, Y. Liu, V. Duvert-Frances, C. Vincent, D. Schmitt, J. Davoust, et al 2000. Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules. Immunity 12: 71-81.
Nestle, F. O., X. G. Zheng, C. B. Thompson, L. A. Turka, B. J. Nickoloff. 1993. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets. J. Immunol. 151: 6535-6545.(Richard D. Granstein2,*, )