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[Cancer Research 64, 5059-5062, August 1, 2004]
© 2004 American Association for Cancer Research

Advances in Brief

Uric Acid Promotes Tumor Immune Rejection

De-En Hu1, Alistair M. Moore1, Lindy L. Thomsen2 and Kevin M. Brindle1

1 Department of Biochemistry, University of Cambridge, Cambridge, and 2 Immunomodulation Section, Immunotherapeutics Department, GlaxoSmithKline, Stevenage, United Kingdom


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Uric acid released from dying cells has been shown recently to act as a danger signal for the immune system, stimulating dendritic cell maturation and enhancing T-cell responses to foreign antigens. Stimulation of dendritic cell maturation by uric acid has been proposed as a mechanism by which the immune system could generate responses against tumors. We show here that uric acid levels are elevated in tumors undergoing immune rejection and that the inhibition of uric acid production, by systemic administration of allopurinol, or the removal of uric acid, by administration of uricase, delayed tumor immune rejection, whereas subcutaneous administration of crystalline uric acid enhanced the rejection process.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Coinjection of dying cells with an antigen into animals is known to have an adjuvant effect, stimulating T-cell responses to the antigen (1) . Recently Shi et al. (2) purified this endogenous adjuvant activity from the cytosol of damaged cells and showed it to be uric acid, an end product of purine catabolism. This increases rapidly in concentration in injured cells as they degrade their RNA and DNA and explains previous observations that showed that the production of this endogenous adjuvant in injured cells did not require protein synthesis (1) . Injection of purified uric acid was shown to boost CTL responses in splenocytes isolated from mice, which had been primed with particulate antigens, by triggering increased dendritic cell expression of the costimulatory molecules CD86 and CD80. Allopurinol and uricase treatment, which substantially reduced plasma uric acid concentrations, was shown to markedly inhibit this T-cell priming. The concentrations of uric acid that stimulated dendritic cells corresponded to the point at which uric acid crystals were precipitated, and it was shown that preformed crystals were highly stimulatory, whereas soluble uric acid was not. Uric acid crystals are known to stimulate monocytes to produce inflammatory mediators (3) , and it seems likely that dendritic cells are stimulated in a similar way.

These data led Shi et al. (2) to propose a model for immune responses to tumors, in which dying tumor cells release uric acid and cellular antigens. The maturation of dendritic cells, which acquire these antigens and present them to the immune system, is promoted by uric acid; thus, stimulating the immune response to the tumor. We have investigated this model here by determining whether uric acid levels are increased in a tumor undergoing immune rejection and by examining the effects of increasing or decreasing uric acid levels on the rejection process.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Lines and Tumor Implantation.
The E.G7-OVA cell line was derived from the murine thymoma line EL-4 by transfection with a neomycin-selectable vector expressing full-length chicken ovalbumin (4) . Cells were cultured as a suspension in RPMI 1640 (Invitrogen Ltd., Paisley, United Kingdom) containing 10% heat-inactivated fetal calf serum (PAA Laboratories Ltd., Yeovil, Somerset, United Kingdom), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Clones expressing different levels of surface ovalbumin were selected as described previously (5) . Wild-type female C57BL/6 mice were purchased at 6–8 weeks of age from Charles River Ltd. (Thanet, United Kingdom) and tumor cells (5 x 106) were injected subcutaneously into their shaved flanks. Tumor size is reported as the product of the two largest perpendicular diameters (in square millimeters). Rasburicase, 1.5 mg/ml, a recombinant form of urate oxidase, was obtained from Sanofi-Synthelabo Inc. (New York, NY).

All of the experiments were conducted in compliance with a project license issued under the Animals (Scientific Procedures) Act 1986 and were designed with reference to the UK Coordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia. The work was approved by a local ethical review committee.

Tumor Histology.
Tumors were fixed in 10% formalin and embedded in paraffin. Five-micrometer-thick sections were cut and stained with H&E or Masson’s trichrome stain.

Induction and Measurement of Apoptosis in Vitro and in Vivo.
Cultured cells were induced to undergo apoptosis at a density of 106 cells/ml by the addition of 25 µM etoposide to the culture medium. To induce tumor apoptosis, we injected etoposide, 67 mg/kg, and cyclophosphamide, 100 mg/kg, into mice 24 hours prior to tumor excision. Cell apoptosis was scored by monitoring nuclear fragmentation, after cell staining with 50 µg/ml propidium iodide and 10 µg/ml acridine orange. Cells that possessed condensed or fragmented nuclei but intact plasma membranes were scored as apoptotic (6) . Tumor apoptosis was scored by counting the number of cells with condensed and fragmented nuclei in sections stained with Masson’s trichrome.

Measurement of Uric Acid Concentrations in Cell and Tumor Extracts.
Tumor tissue was mechanically homogenized [1 g of tissue in 2.5 ml of ice-cold buffer containing 250 mM Tri-HCl (pH 7.4), 10 mM EDTA, 10 mM EGTA, and 1 ml/50 ml protease inhibitor cocktail (Sigma)]; the homogenate was centrifuged at 16,000 x g, and the supernatants were passed through 0.45 µm Millex filters (Millipore Corporation, Bedford, MA). Cultured cells were harvested and were washed in ice-cold PBS, and then ~2 x 107 cells were resuspended in 2 ml of fresh extraction buffer. The buffer contained 50 mM Tris-HCl (pH 8.2), 2 mM DTT, 2 mM EDTA, and 1% Triton X-100. After a 10-second homogenization, the resulting extracts were kept on ice for 30 min and then were centrifuged for 15 minutes at 2,000 x g. The supernatants from cell and tumor homogenates were removed and were assayed for uric acid enzymatically (assay kit from ThermoTrace, Melbourne, Victoria, Australia).

Preparation of Uric Acid and Monosodium Urate Crystals.
Monosodium urate crystals were prepared by dissolving uric acid at a concentration of 5 mg/ml in 0.1 M sodium borate buffer, pH 8.5–9. The solution was then warmed to 55°C, and, after filtering, the supernatant was left to sit for more than 72 h, whereupon monosodium urate crystals formed.

Cell Growth Rates in Vitro.
These were determined either by cell counting under the microscope or by use of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT was added to cell suspensions at a final concentration of 0.5 mg/ml, and the mixture was incubated for 4 hours at 37°C. The cells were then harvested by centrifugation, the pellet was resuspended in DMSO, and the absorbance was measured at 540 nm.

Statistical Analysis.
Statistical analyses were performed using GraphPad Prism Software (Graphpad Software, Inc., San Diego, CA). ANOVA and t test were used to compare specific groups. Values shown are the means ± SE.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Induction of Tumor Apoptosis Results in Uric Acid Production.
Etoposide-induced apoptosis in EL-4 cells resulted in significant accumulation of uric acid, when compared with control cells (Fig. 1ACitation ; n = 9, P < 0.01). The amounts were comparable with those observed previously in damaged EL-4 cells (2) . Cells were harvested 18 h after etoposide addition, when fluorescence microscopy measurements indicated that 41 ± 2% of the cells were apoptotic and 19 ± 2% necrotic. Treatment of implanted EL-4 tumors with etoposide and cyclophosphamide also resulted in significant uric acid accumulation (n = 8), when compared with untreated tumors (n = 7, P < 0.01; Fig. 1BCitation ). Histological analysis of tumor sections indicated that ~38 ± 1% (30 sections from 3 tumors) of the cells showed signs of apoptosis (cell shrinkage and nuclear condensation).



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  		Fig. 1. Uric acid concentrations in EL-4 cell cultures (A) and in implanted EL-4 tumors (B) that had been induced to undergo apoptosis. Apoptosis was induced in EL-4 cells by treatment with 25 µM etoposide for 18 h. Apoptosis was induced in EL-4 tumors by i.v. injection of 67 mg/kg etoposide and 100 mg/kg cyclophosphamide into mice 13–15 days after tumor implantation and 24 h before tumor excision. Uric acid concentrations were determined enzymatically in tumor and cell homogenates (cells, n = 9; control tumors, n = 7; apoptotic tumors, n = 8).

 
Uric Acid Concentration Increases in Tumors Undergoing Immune Rejection.
EL-4 tumor cells can be rendered immunogenic by transfection with a vector expressing chicken ovalbumin (E.G7-OVA; 4 ). We showed previously that by cloning cells with different levels of surface ovalbumin expression we were able to titrate the immunogenicity of this tumor to produce a range of subcutaneous tumors that showed different frequencies of immune rejection (5 , 7) . Two lines were selected for the experiments described here. A line at nominal passage number 14, that produced tumors of which ~30% underwent spontaneous immune rejection and a line that expressed relatively high levels of surface ovalbumin and which produced tumors that always underwent rejection. Uric acid levels were increased significantly in nonregressing E.G7-OVA tumors (n = 12) when compared with EL-4 tumors (n = 12; P < 0.01) and these levels were increased further in regressing E.G7-OVA tumors (n = 9, P < 0.01; Fig. 2Citation ). Histological analysis of tumor sections indicated that ~43 ± 1% (n = 15) of the cells showed signs of apoptosis in regressing E.G7-OVA tumors, 19 ± 1% (n = 10) in nonregressing E.G7-OVA tumors and 6.6 ± 0.4% (n = 10) in EL-4 tumors.



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  		Fig. 2. Uric acid concentrations in tumors undergoing immune rejection. Tumors were implanted by subcutaneous injection of EL-4, or E.G7-OVA cells at nominal passage number 14, at t (time) = 0 days. Tumors were excised at between 12 and 14 days after tumor implantation and were homogenized; uric concentrations in the homogenates were determined enzymatically. Concentrations are expressed as micrograms per gram of tumor wet weight [n = 9, regressing E.G7-OVA tumors (E.G7R); n = 12 nonregressing E.G7-OVA tumors (EG7) and EL-4 tumors]. The regressing E.G7-OVA tumors were excised immediately after evidence of tumor shrinkage.

 
Inhibition of Tumor Immune Rejection by Allopurinol or Uricase.
The regression of E.G7-OVA tumors expressing relatively high levels of surface ovalbumin was delayed significantly at 12 days after tumor implantation, when compared with controls, by the administration of allopurinol in the drinking water (n = 12, P < 0.01, ANOVA; Fig. 3ACitation ). Regression was also delayed significantly at 10 days after tumor implantation by the administration of allopurinol i.p. (n = 12; P < 0.01, ANOVA; Fig. 3BCitation ) and at 12 days after tumor implantation by the administration of uricase, given i.v. (n = 12; P < 0.01, ANOVA; Fig. 3CCitation ). Allopurinol or uricase administration had no effect on the growth of nonimmunogenic EL-4 tumors (n = 5, P > 0.05, ANOVA; Fig. 3DCitation ); and, in vitro, 125 µg/ml allopurinol or 5 µg/ml uricase had no effect on the growth of either EL-4 or E.G7-OVA cells. The doubling times of untreated EL-4 and E.G7-OVA cells were 11.2 ± 0.12 hours and 11.7 ± 0.16 hours, respectively, whereas for allopurinol- or uricase-treated EL-4 cells, they were 12.1 ± 0.37 hours and 11.3 ± 0.12 hours, respectively; and for allopurinol- or uricase-treated E.G7-OVA cells, they were 12.5 ± 0.39 hours and 11.7 ± 0.25 hours, respectively (mean ± SE; n = 3). By the MTT assay, the growth rates of allopurinol- or uricase-treated EL-4 cells were 94.9 ± 2.6% and 99.7 ± 1.5%, respectively, of controls; and, for E.G7 cells, they were 94.7 ± 2.8% and 99.3 ± 3.0%, respectively, of controls (n = 5).



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  		Fig. 3. Inhibition of tumor immune rejection by allopurinol and uricase. Tumors were implanted by subcutaneous injection of E.G7-OVA cells that expressed relatively high levels of surface ovalbumin (A–C) or by injection of EL-4 cells (D), at t (time) = 0 days. The symbols represent the mean ± SE of the volumes reported as the product of the two largest perpendicular diameters; error bars, where not visible, lie within the symbols. A, {bullet}, animals with E.G7-OVA tumors, given allopurinol in their drinking water (0.256 g/liter) for 4 weeks prior to tumor implantation and throughout the period of tumor growth and subsequent regression (n = 12); {circ}, controls (n = 12). B, {bullet}, animals with E.G7-OVA tumors, given allopurinol (10 mg/kg dissolved in 0.1 M NaHCO3) i.p. daily for 7 days from the day after tumor implantation (n = 12). Controls ({circ}) received 0.1 M NaHCO3 alone (n = 12). C, {bullet}, animals with E.G7-OVA tumors, given 0.2 mg/kg uricase i.v. daily for 7 days from the day after tumor implantation (n = 12). {circ}, controls received PBS injections (n = 12). D, {bullet}, animals with EL-4 tumors, given allopurinol (10 mg/kg dissolved in 0.1 M NaHCO3) i.p. daily for 7 days from day 7 after tumor implantation (n = 5); {blacktriangleup}, animals with EL-4 tumors, given 0.2 mg/kg uricase i.v. daily for 7 days from day 7 after tumor implantation (n = 5). {circ}, controls received 0.1 M NaHCO3 alone (n = 5).

 
Stimulation of Tumor Immune Rejection by Subcutaneous Injection of Uric Acid Crystals.
Tumors that expressed lower levels of ovalbumin, and were thus less immunogenic and showed lower frequencies of spontaneous regression, were implanted by subcutaneous injection of E.G7-OVA cells at nominal passage 14. A mixture of soluble and crystalline uric acid (0.1 ml at a concentration of 1 mg/ml) was then injected 2 cm from the site of tumor implantation, daily for 3 days from the day after tumor implantation. Inoculation with uric acid crystals resulted in a significant increase in the rate of tumor rejection ({chi}2 = 4.34; P < 0.05), with 17 tumors of 30 undergoing spontaneous rejection (Fig. 4A)Citation , as compared with 9 of 30 of the controls (in which PBS was injected; Fig. 4BCitation ). Uric acid injection had no effect on the growth of nonimmunogenic EL-4 tumors (Fig. 4C)Citation .



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  		Fig. 4. Stimulation of tumor immune rejection by uric acid. Tumors were implanted by subcutaneous injection of E.G7-OVA cells at nominal passage number 14 (A and B) or by injection of EL-4 cells (C), at t (time) = 0 days. In A, a mixture of soluble and crystalline uric acid (0.1 ml at a concentration of 1 mg/ml) was injected subcutaneously 2 cm from the site of E.G7-OVA tumor implantation daily, for 3 days from the day after tumor implantation (n = 30); in B, saline was injected in place of uric acid (n = 30). A and B, {bullet}, tumor volumes reported as the product of the two largest perpendicular diameters. C, {bullet}, the mean ± SE of the volumes when a mixture of soluble and crystalline uric acid (0.1 ml at a concentration of 1 mg/ml) was injected subcutaneously 2 cm from the site of EL-4 tumor implantation daily, for 3 days from the day after tumor implantation (n = 10); {circ}, the mean ± SE of the volumes in control animals, in which saline was injected in place of uric acid (n = 10); error bars, where not visible, lie within the symbols.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
According to the "danger model," antigen-presenting cells are activated by danger or alarm signals released from injured cells to produce costimulatory molecules that promote T-cell responses to a copresented antigen (8 , 9) . Recently Shi et al. (2) identified uric acid as the principal endogenous danger signal released from injured cells. They showed that it stimulated dendritic cell maturation in vitro, increasing the expression of the costimulatory molecules, CD86 and CD80, and, when coinjected with a particulate antigen into mice, it enhanced the generation of a CD8+ T-cell response. Furthermore, the priming of an antigen-specific T-cell response, by coinjection of injured cells, was substantially reduced by treatment of the animals with allopurinol and uricase, which markedly decreased the plasma concentrations of uric acid. They suggested a "danger signal" model in which the release of uric acid by injured cells, produced as a result of DNA and RNA degradation, stimulates the maturation of dendritic cells, which also acquire and present antigens from the dying cells to T cells, thus enhancing the immune response. If, in the case of a tumor, the host is not tolerant of these antigens, then an immune response against the tumor will be generated.

We have shown here that tumors in which there is increased cell death, either as a result of treatment with a chemotherapeutic drug or as a consequence of immune rejection, show elevated levels of uric acid. This increase in uric acid production seemed to be involved in tumor immune rejection because treatment of mice bearing a highly immunogenic tumor, with allopurinol or uricase [which have been shown previously to lower plasma uric acid levels (2) ] significantly delayed tumor regression. Although allopurinol may have immunomodulatory effects that are unrelated to its effect on systemic levels of uric acid (10) , this is unlikely to be the case for uricase. Furthermore, Shi et al. (2) showed that the CTL response in splenocytes isolated from mice, immunized with activated dendritic cells that had been pulsed with a peptide corresponding to the antigenic epitope, was undiminished by treatment of the animals with allopurinol plus uricase. In further support of a role for uric acid in the immune rejection of this tumor, we have shown that subcutaneous inoculation of uric acid crystals, 2 cm from the site of tumor implantation, increases significantly the frequency of tumor immune rejection.

There is, however, a paradox in the role of uric acid as an enhancer of tumor immune rejection. We have shown previously that immune rejection of the E.G7-OVA tumor model involves the production of nitric oxide by the tumor cells. This is involved in their death, probably as a result of combination with superoxide to form peroxynitrite (5) , which is a potent cellular oxidant and mediator of cell death (11) . Uric acid is a natural peroxynitrite scavenger (12) and, therefore, might have been expected to protect the cells from immune cell-mediated death. Such a role is inconsistent with the observations that uricase and allopurinol treatment, which lowers plasma uric acid levels, delayed tumor immune rejection and that inoculation of uric acid crystals promoted rejection. Shi et al. (2) suggested that uric acid stimulated dendritic cell maturation only when crystalline and proposed that this chemical phase transition could be the key event that turns uric acid into a danger signal. Therefore, perhaps the role of uric acid as an antioxidant or as a stimulator of the immune response depends crucially on this transition, which will in turn depend on other factors in the tumor microenvironment and the surrounding tissue (2) .


   FOOTNOTES
 
Grant support: This work was supported by grants from the Medical Research Council and Cancer Research United Kingdom (CUK grant C197/A3514). A. Moore is in receipt of a CASE studentship from the Biotechnology and Biological Sciences Research Council.

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.

Requests for reprints: Kevin M. Brindle, Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom. Phone: 44-(0)-1223-333674; Fax: 44-(0)-223-766002; E-mail: kmb{at}mole.bio.cam.ac.uk

Received 5/ 6/04. Revised 6/ 9/04. Accepted 6/16/04.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Shi Y, Zheng WY, Rock KL. Cell injury releases endogenous adjuvants that stimulate cytotoxic T cell responses. Proc Natl Acad Sci USA, 97(26): 14590-5, 2000.
  2. Shi Y, Evans JE, Rock KL. Molecular identification of a danger signal that alerts the immune system to dying cells. Nature (Lond), 425(6957): 516-21, 2003.
  3. Landis RC, Yagnik DR, Florey O, et al Safe disposal of inflammatory monosodium urate monohydrate crystals by differentiated macrophages. Arthritis Rheum, 46(11): 3026-33, 2002.
  4. Moore MW, Carbone FR, Bevan MJ. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell, 54(6): 777-85, 1988.
  5. Hu D-E, Dyke SOM, Moore AM, Thomsen LL, Brindle KM. Tumor cell-derived nitric oxide is involved in the immune-rejection of an immunogenic murine lymphoma. Cancer Res, 64(1): 152-61, 2004.
  6. Anthony ML, Zhao M, Brindle KM. Inhibition of phosphatidylcholine biosynthesis following induction of apoptosis in HL-60 cells. J Biol Chem, 274(28): 19686-92, 1999.
  7. Hu D-E, Beauregard DA, Bearchell MC, Thomsen LL, Brindle KM. Early detection of tumour immune-rejection using magnetic resonance imaging. Br J Cancer, 88(7): 1135-42, 2003.
  8. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol, 12: 991-1045, 1994.[Medline]
  9. Matzinger P. The danger model: A renewed sense of self. Science (Wash DC), 296(5566): 301-5, 2002.
  10. Kato C, Sato K, Wakabayashi A, Eishi Y. The effects of allopurinol on immune function in normal BALB/c and SCID mice. Int J Immunopharmacol, 22(7): 547-56, 2000.
  11. Radi R, Cassina A, Hodara R, Quijano C, Castro L. Peroxynitrite reactions and formation in mitochondria. Free Radic Biol Med, 33(11): 1451-64, 2002.
  12. Hooper DC, Spitsin S, Kean RB, et al Uric acid, a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci USA, 95(2): 675-80, 1998.



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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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