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Published online before print December 30, 2005
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* Departments of Rheumatology and Inflammation Research and
Virology, University of Göteborg, Sweden
1 Correspondence: Department of Rheumatology and Inflammation Research, Guldhedsgatan 10A, S-413 46 Göteborg, Sweden. E-mail: fariba.zare{at}rheuma.gu.se
ABSTRACT
Uric acid, the naturally occurring degradation product of purine metabolism, is a danger signal, driving maturation of dendritic cells. It is well known that uric acid crystals display potent proinflammatory properties—the cause of gout—whereas the biological properties of soluble uric acid are less well documented. We have demonstrated previously that nucleic acids of endogenous and exogenous origin display proinflammatory properties. The aim of the present study was to assess the impact of soluble uric acid on in vivo inflammatory responses. Mice were administered with uric acid suspension in saline or saline alone prior to induction of neutrophil-mediated inflammation, delayed-type hypersensitivity, histamin-induced edema (measure of vasodilation capacity), as well as double-stranded (ds)RNA-triggered arthritis. Frequency and severity of arthritis were decreased significantly in mice exposed to dsRNA and simultaneously treated with uric acid as compared with saline-treated controls. Also, granulocyte-mediated inflammatory response and vasodilation capacity were reduced significantly in mice treated with uric acid as compared with their control group. The data suggest that down-regulation of inflammation was mediated by skewing the inflammatory response from the peripheral sites to the peritoneal cavity and down-regulating vasodilatatory capacity and thereby affecting leukocyte migration. In contrast, the T cell-mediated delayed-type hypersensitivity reaction was not affected significantly in mice exposed to uric acid. These findings demonstrate that uric acid displays a potent, distant anti-inflammatory effect in vivo. This property seems to be mediated by down-regulation of neutrophil influx to the site of inflammatory insult.
Key Words: inflammation • neutrophils
INTRODUCTION
The immune system is activated, not only by microbes but also by altered endogenous cells and their products. It has been reported that dying cells in their cytoplasm contain copious amounts of uric acid [1 ], which in a crystalline form, is one of the self-molecules that acts as an endogenous danger signal for the immune system [1 ]. In addition, uric acid, a degradation product from nucleotides, plays a critical role in a number of biochemical processes, including, for example, DNA and RNA synthesis and energy transfer [1 , 2 ]. Uric acid drives the maturation of dendritic cells (DCs), leading to an increase in antigen presentation and expression of costimulatory molecules such as CD80 and CD86 [3 ]. The mature DCs can then effectively prime the T cell response [3 ]. DCs become activated, express Toll-like receptors, and interact with exogenous (bacteria, viruses, parasites, fungi) and endogenous (uric acid crystals, gp 96, and hsp 70 from necrotic cells, apoptotic bodies) products. DCs become activated further in response to such signals, often resulting in immune activation and inflammation.
As the injured cells rapidly degrade their DNA and RNA, the liberated purines will be converted into uric acid, leading to its accumulation [2 ]. The production of uric acid does not require protein synthesis, which enables dying cells to produce more danger signal and explain why this increase is not blocked by protein synthesis inhibitors [2 ]. Only crystalline but not soluble uric acid is capable of activating DCs. Soluble uric acid, a physiological by-product of nucleic acid metabolism, is normally present at high concentrations in the blood and in the intercellular fluid [4 ]. As the physiological concentration of uric acid is near-saturating, crystallization occurs easily, even when a small excess is released during cellular damage caused by, e.g., infection, tumor growth, or autoimmune disease. As a result, crystalline uric acid might be enriched in human joints and kidneys, giving rise to gout and interstitial nephritis, respectively.
In the present study, we investigated the impact of soluble rather than crystalline uric acid on the in vivo inflammatory responses. We found that soluble uric acid, acting by down-regulation of neutrophil influx to the site of inflammatory focus, gives rise to an anti-inflammatory effect in vivo, thereby balancing the impact of the crystalline form of the same molecule.
MATERIALS AND METHODS
Mice
Naval Medical Research Institute (NMRI) mice were purchased from B&K Universal AB (Stockholm, Sweden). Female mice, 6–8 weeks of age, were used in all the experiments. All mice were housed in the animal facility of the Department of Rheumatology and Inflammation Research, University of Göteborg (Sweden). Mice were kept under standard conditions of temperature and light and were fed laboratory chow and water ad libitum. The Ethical Committee of the University of Göteborg approved the study, and the requirements of the National Board for Laboratory Animals were followed.
Uric acid and injection
Uric acid obtained from Sigma-Aldrich (Stockholm, Sweden) was used as a suspension in saline. Mice received 500 mg/kg body weight (BW) uric acid in a 100-µl saline suspension per injection [intraperitoneally (i.p.)] or saline alone as a control in four daily doses. The above dosage was previously shown to suppress murine experimental allergic encephalomyelitis [5
]. A single dose of uric acid transiently raises its serum levels in mice to levels approaching those seen in human sera [5
]. However, uric acid will be metabolized rapidly to allantoin in mice by urate oxidase, an enzyme that is absent in humans [6
].
Preparation and injection of double-stranded (ds)RNA with JetPEITM
dsRNA in the form of polyinosinic-polycytidylic acid (polyIC) was purchased from Sigma-Aldrich. dsRNA was administered intra-articularily, complexed in vitro to JetPEI (Invitrogen, Carlsbad, CA) in a total volume of 20 µl. Preparation of dsRNA and JetPEI was done according to the manufacturer’s instructions for in vivo transfection. The ratio of negatively charged phosphates in dsRNA and positively charged nitrogen residues in JetPEI may affect the transfection efficacy. An initial test to determine the optimal ratio between dsRNA and JetPEI to induce arthritis revealed that 5 µg dsRNA complex to JetPEI at a N/P ratio of seven was optimal [7
]. The N/P ratio is a measure of the ionic balance of the complexes and refers to the number of nitrogen residues of in vivo JetPEI per DNA phosphate.
Histopathological examination
Histopathologic examination of joints was performed after routine fixation, decalcification, and paraffin embedding. Sections were cut and stained with hematoxylin and eosin. All the slides were coded and evaluated blindly. Specimens were evaluated with regard to synovial hyperthrophy, pannus formation, and cartilage and subchondral bone destruction [8
]. The extent of synovitis was judged on an arbitrary scale from 0 to 3. No signs of inflammation represented Grade 0; Grade 1 was characterized by mild inflammation with hyperplasia of the synovial lining layer; Grades 2 and 3 represented increasing degrees of inflammation characterized by influx of inflammatory cells scattered throughout the synovial tissue.
T lymphocyte-mediated delayed-type hypersensitivity (DTH)
The mice were sensitized by epicutaneous application of 150 µl absolute ethanol:acetone (2:1) solution containing 3% oxazolone (OXA; Sigma-Aldrich) on the abdomen. Five days after the sensitization, the right ear was challenged on both sides by topical application of 30 µl 1% OXA dissolved in olive oil. Ear thickness of both ears was measured 24 h after the sensitization using an Oditest spring caliper (Kröplin, Hessen, Germany). The intensities of the DTH reactions were expressed as swelling, i.e., thickness of the right ear subtracted from thickness of the left ear (cmx10–3) [9
].
Determination of OXA-specific antibodies
The serum levels of specific antibodies against OXA were measured by an enzyme-linked immunosorbent assay (ELISA). Sera from immunized mice were obtained 1 or 6 days after the booster immunization with OXA. Microplates were coated overnight with 0.003% dog serum albumin-OXA in phosphate-buffered saline (PBS) and blocked with 0.5% bovine serum albumin (BSA) in PBS. Sera were diluted in BSA-PBS, whereas biotinylated antibodies and ExtrAvidin-peroxidase (Sigma-Aldrich) were diluted in physiological NaCl. The plates were incubated with sera, washed again, and incubated stepwise with biotinylated goat anti-mouse immunoglobulin G (IgG) antibody, diluted 1:1000 (Southern Biotechnology Associates, Inc., Birmingham, AL), ExtrAvidin peroxidase (1 µg/ml, Sigma-Aldirch), and buffer, including H2O2 and chromogen substrate (phosphatase substrate, Sigma-Aldrich). The absorbance at 405 nm was measured by spectrophotometer (Molecular Devices, Sunnyvale, CA).
Olive oil-induced inflammation
Olive oil-induced skin inflammation is granulocyte-mediated but T cell- and monocyte-independent [9
]. Olive oil (30 µl, Apoteksbolaget, Göteborg, Sweden) was injected intradermally (i.d.) in a hind footdorsum of mice under Ketalar/Dormitor anesthesia. Footpads were measured before and 24 h after injection using an Oditest spring caliper (Kröplin). The footpad swelling was expressed as footpad thickness after injection minus that before the injection (mm) [10
].
Histamin-induced edema formation
Histamin-induced edema is a measure of vascular permeability. Edema was induced by subcutaneous (s.c.) injection of 20 µl histamin solution (0.1 mg/ml in physiological saline, Apoteksbolaget) in a hind footpad. Thirty minutes after the injection, the edema formation was quantified, using an Oditest caliper (Kröplin), as the increase in footpad thickness in mm. Footpad swelling as a result of the injection of vehicle was subtracted from the values [10
].
Analysis of cytokine and chemokine levels
Serum interleukin (IL)-6 levels were measured by a bioassay method, which measures the biological activity of IL-6 and displays a high sensitivity and specificity. The cell line B9, dependent on IL-6 for growth, was used to determine the serum IL-6 levels. B9 cells were seeded into microtiter plates (Nunc, Roskilde, Denmark) at a concentration of 2.5 x 104 cell/ml in Iscove’s complete medium, and dilutions of the serum samples were added to the wells. After 72 h incubation, 3H thymidine was added, and 6 h later, the cells were harvested. The results were compared with a recombinant IL-6 standard. B9 cells were shown previously [10
] not to react with several recombinant cytokines, including IL-1
, IL-1ß, IL-12, IL-13, IL-5, granulocyte macrophage-colony stimulating factor, tumor necrosis factor , and interferon-
. There was only weak reactivity with IL-4.
Peritoneal fluid levels of chemokines were analyzed using macrophage-inflammatory protein-1 (MIP-1) and monocyte chemoattractant protein-1 (MCP-1) ELISA kits from R&D Systems (London, UK). The assays were performed as recommended by the manufacturer. The values below the detection limit were considered as zero.
Collection of peritoneal cells
NMRI mice received 500 mg/kg BW uric acid in a 100-µl saline suspension per injection (i.p.) or saline alone as a control as four daily doses for 4 days. After killing the mice, 1 ml sterile PBS was injected i.p. followed by aspiration. The total number of white blood cells (WBC) was calculated using an automated cell counter (Sysmex, Kobe, Japan). To assess the influence of uric acid on the total numbers of circulating polymorphonuclear (PMNC) and mononuclear (MNC) leukocytes, we performed differential counts of the leukocytes. Peritoneal smears were stained by the May-Grünwald-Giemsa technique, and the frequences of PMNC and MNC were estimated.
Migration of leukocytes to uric acid in vitro
Mouse peritoneal WBC were analyzed for the their ability to migrate toward a uric acid gradient using the ChemoTx® system with a pore size of 3 µm (Neuro Probe Inc., Gaithersburg, MD). Briefly 1 x 105 cells resuspended in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca2+ (1 mM), and Mg2+ (1.5 mM) in a final volume of 30 µl were added to the upper well. To the lower chamber, 30 µl uric acid (470 µg/ml or 47 µg/ml) was added. As a positive control, we used 5 x 10 –8 M hexapeptide WKYMVM [11
] and as a negative control, Krebs-Ringer glucose buffer (KRG) containing 0.1% BSA. All substances used were diluted in KRG containing 0.1% BSA. To assess potential of uric acid as a chemokinetic stimulus, upper and lower chambers were incubated with uric acid (470 µg/ml). All migration experiments were performed in triplicates for each stimulus. The cells were allowed to migrate for 90 min in a cell culture chamber (37°C, 5% CO2), whereafter, the upper leftover was removed followed by a centrifugation at 350 g at 4°C of the net. After addition of trypan blue, the migrated cells were counted using a microscope, and the mean of three wells was calculated.
Statistical analysis
Statistical comparisons were made by using the 
2 with Yates’ corrections, Mann Whitney, and Wilcoxon Signed Rank. All values are reported as the mean ± SEM. P values less than 0.05 were considered significant.
RESULTS
Effect of uric acid administration on dsRNA-induced arthritis
Mice were intra-articularly injected with polyIC as described previously [12
] with addition of JetPEI as the only modification. Some mice were treated i.p. with uric acid injections, whereas others, with saline alone. Administration of uric acid started 24 h prior to induction of arthritis and was continued (four daily doses) until the mice were killed 3 days later. dsRNA-triggered arthritis was suppressed significantly (P<0.001) in uric acid-treated NMRI mice as compared with saline-treated controls (Fig. 1A
1B
1C
1D
). The BW of all mice was measured before the treatment and at the time of sacrifice. Mice treated with uric acid showed some weight loss (41±0.7 g down to 36±0.7 g; P<0.0001) as compared with their control group [39±0.8 g vs. 40±0.9 g; not significant (N.S)].
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Sensitization and challenge with OXA resulted in significantly lower anti-OXA IgG ELISA antibody titers in mice that received uric acid compared with mice that received saline 1 day after the challenge (Fig. 3A ). In contrast, there were no differences regarding anti-OXA IgG antibody responses between the groups 6 days after the challenge with OXA (Fig. 3A) .
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Effect of uric acid administration on i.p. leukocyte accumulation
NMRI mice were treated for 4 days with i.p. injections of 500 mg/kg BW of uric acid in saline or saline alone as a control. After termination, the mice were killed, peritoneal cells were collected, and the total number of leukocytes was counted. Treatment with uric acid resulted in significantly greater accumulation of i.p. leukocytes than treatment with saline alone (Fig. 4A
). It is notable that mice injected with uric acid displayed a considerably higher proportion of PMNC in the peritoneum than mice injected with saline (Fig. 4B)
. Thus, uric acid seems to attract PMNC selectively.
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(1194±785 and 26±11 pg/ml, respectively) as compared with saline-injected control mice (445±265 and 15±12 pg/ml, respectively). To exclude chemokinesis, uric acid at the concentration of 470 µg/ml was included in lower and upper chambers. In that case, the migratory activity of PMNC did not differ significantly from KRG controls (Fig. 5)
.
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In our previous studies, we found that viral dsRNA [13 ] and bacterial DNA [14 ] give rise to arthritis following intra-articular exposure in healthy mice. The reason to study the proinflammatory properties of soluble uric acid in our present study is its status as the end-product of purine metabolism. We reasoned that following exposure to extrinsic nucleic acids with subsequent immune activation, the degradation product might be able to fine-tune responsiveness by down-regulation of inflammatory responses. In this respect, it has been shown recently that crystalline uric acid is a principal endogenous danger signal released from injured cells being able to alert immune cells of microbial attack [2 ]. In contrast, the role of soluble uric acid remains elusive.
In the present study, we used a relatively high concentration of uric acid. The reason for that is the known property of rodents to further degrade uric acid to its metabolite allantoin by action of urate oxidase. Thus, at the time of read-out of our in vivo experiments (12 h–4 days), the amount of uric acid in the circulation should be minimal if detectable at all, as previously demonstrated [5 ]. Using these premises, we demonstrate that systemic administration of soluble uric acid gives rise to a significant suppression of severity and frequency of dsRNA-triggered arthritis. This action seems to be mediated by deviation of PMNC responses together with down-regulation of vascular permeability.
The way that uric acid exerts these properties is not fully understood. However, it has been known for a relatively long time that uric acid acts as an antioxidant [15 ]. Such a mode of action down-regulates a number of potently proinflammatory molecules including nitric oxide, superoxide, and peroxynitrite [16 ]. Expression of peroxynitrite also contributes to some other proinflammatory processes including enhanced expression of intercellular adhesion molecule-1 and P-selectin by human endothelial cells [17 ], down-regulation of L-selectin expression, and finally, up-regulation of CD11/CD18 expression [18 ]. Also, these properties are readily inhibited by uric acid, the peroxynitrite scanavenger.
We also show that treatment with soluble uric acid has a significant, suppressive effect on in vivo granulocyte-mediated, olive oil-induced inflammation. This finding indicates that soluble uric acid, even here, displays anti-inflammatory properties, selectively directed to neutrophils and by-passing T cells/macrophages. Indeed, olive oil-triggered inflammation is as described previously, T cell/macrophages-independent [10 ].
Furthermore, DTH reaction, which is T cell/macrophages-dependent, was not affected by administration of uric acid. The latter finding shows that the action of uric acid displays a certain degree of immunomodulatory specificity. Finally, our data demonstrate that uric acid treatment reduced the vascular permeability triggered by histamin injection. This finding suggests that uric acid might act on endothelial tight junctions. Such a mode of action might be of great importance in immune reaction by controlling cell and plasma efflux to the site of inflammatory insult.
Taken together, the above data demonstrate that soluble uric acid displays an anti-inflammatory effect on distant acute, inflammatory responses. Despite its anti-inflammatory properties, several studies have demonstrated that soluble uric acid can generate free radicals [19 20 ] and has the ability to increase MCP-1 expression [23]. In agreement with these studies, we found that soluble uric acid induces accumulation of PMNC in the peritoneum. This property is supported by our in vitro result, which shows that soluble uric acid exerts chemotactic but not chemokinetic activity. Finally, treatment with uric acid leads to systemic production of IL-6, a well-known, proinflammatory cytokine. These findings indicate that soluble uric acid triggers local inflammation in the peritoneal cavity by enhancement of the invasion of inflammatory cells such as PMNC and thereby down-regulates influx of these cells to the distant site of inflammatory focus, preventing development of the various forms of distant inflammation.
ACKNOWLEDGEMENTS
This study was supported by grants from the Medical Society of Gothenburg, the Swedish Association against Rheumatism, the King Gustaf V:s Foundation, the Swedish Medical Research Council, the Inflammation Network, the Infection and Vaccinology Network, the Nanna Swartz’ Foundation, AME Wolff Foundation, Rune and Ulla Amlövs Trust, European Union grants, and the University of Göteborg. We thank Inge-Marie Jonsson, Margareta Verdrengh, and Berit Ericsson for excellent technical assistance.
Received August 1, 2005; revised October 14, 2005; accepted November 1, 2005.
REFERENCES
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