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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

To examine the role of chemokines, S100A8, and S100A9 in neutrophil accumulation induced by the causative agent of gout, monosodium urate monohydrate (MSU) crystals.

Methods

MSU crystal–induced neutrophil migration was studied in the murine air-pouch model. Release of chemokines, S100A8, S100A9, and S100A8/A9 in response to MSU crystals was quantified by enzyme-linked immunosorbent assays. Recruited cells were counted following acetic blue staining, and the subpopulations were characterized by Wright-Giemsa staining of cytospins.

Results

MSU crystals induced the accumulation of neutrophils following injection in the air pouch, which correlated with the release of the chemokines CXCL1, CXCL2, CCL2, and CCL3. However, none of these was found to play an important role in neutrophil migration induced by MSU crystals by passive immunization with antibodies directed against each chemokine. S100A8, S100A9, and S100A8/A9 were also found at high levels in the pouch exudates following injection of MSU crystals. In addition, injection of S100A8, S100A9, or S100A8/A9 led to the accumulation of neutrophils in the murine air pouch, demonstrating their proinflammatory activities in vivo. Passive immunization with anti-S100A8 and anti-S100A9 led to a total inhibition of the accumulation of neutrophils. Finally, S100A8/A9 was found at high concentrations in the synovial fluid of patients with gout.

Conclusion

S100A8 and S100A8/A9 are essential to neutrophil migration induced by MSU crystals. These results suggest that they might be involved in the pathogenesis of gout.

The acute inflammation of gouty arthritis is caused by the crystallization of sodium urate in the joint. Interaction between monosodium urate monohydrate (MSU) crystals and monocytes, platelets, synoviocytes, macrophages, and neutrophils within the articulation initiates an inflammatory response that involves the secretion of proinflammatory agents and chemotactic factors from these different cell types (1, 2). Some of these mediators induce the accumulation of neutrophils, which leads to enhancement of the inflammatory response (3–6). MSU crystal–stimulated neutrophils will also release oxygen radicals (7), cytokines such as interleukin-1 (IL-1) (8), and proteolytic enzymes (9), which leads to destruction of the articulation.

Neutrophil transendothelial migration is a critical stage in the development of this acute inflammatory reaction. To infiltrate the articulation, neutrophils must migrate from the blood through the endothelium and the synovial tissue (10). This migration occurs through a multistep process. First, interactions between selectins and glycans mediate neutrophil rolling along the endothelium. Neutrophils are then activated, leading to changes in β2 integrin to an active conformation. This change of conformation is thought to be induced by chemotactic factors, such as platelet-activating factor (PAF) or IL-8 expressed by endothelial cells. Activation of integrins causes neutrophils to adhere strongly to the endothelium, initiating extravasation. Once in the tissue, the cells follow concentration gradients of chemoattractants such as C5a, leukotriene B4 (LTB4), and IL-8.

Factors involved in neutrophil migration in the pathogenesis of gout remain largely unknown. While LTB4 is known to be produced by MSU crystal–activated neutrophils (11), inhibition of LTB4 synthesis does not reduce MSU crystal–induced neutrophil recruitment in the subcutaneous air-pouch model in rats (12). However, inhibition of PAF was shown to partially diminish MSU crystal–induced arthritis in rabbit articulations (13). Recently, IL-8 was suggested to be the major CXC chemokine involved in neutrophil migration in response to MSU crystals (5, 14, 15). Inactivation of IL-8 with specific blocking antibodies led to a significant reduction in neutrophil migration in rabbit articulations (16). However, this reduction was only observed 12 hours after MSU crystal injection, with no effect at earlier time points. This suggests that IL-8 is not responsible for the initiation of the inflammatory response induced by MSU crystals. Paradoxically, early neutrophil migration in response to MSU crystals is impaired in mice that are deficient in the murine IL-8 homolog CXCR2 (17). Since CXCR2 does not solely bind IL-8, the previous data suggest that other chemokines or inflammatory mediators could be involved at the beginning of the inflammatory response.

Recent studies suggest that the myeloid-related proteins (MRP) play an important role in neutrophil migration to inflammatory sites. These proteins constitute a subfamily of S100 proteins, of which 3 have been characterized: S100A8, S100A9, and S100A12 (18–20). These small proteins of 10–14 kd are constitutively expressed at high levels in the cytosol of neutrophils, monocytes, and activated macrophages (20–22). In the presence of calcium, S100A8 and S100A9 associate noncovalently to form the heterodimer S100A8/A9 (23–25). High concentrations of MRPs occur in serum of patients with diseases associated with increased neutrophil activity. Elevated levels of S100A8/A9 (>1 μg/ml) are observed at the inflamed site and in the serum of patients with various infections and inflammatory diseases, including rheumatoid arthritis and juvenile rheumatoid arthritis (23, 26–28). Local secretion of the proteins has also been detected in periodontal infections and during experimental murine abscesses (29, 30).

Several proinflammatory activities have been identified for MRPs. In vitro studies from our laboratory and others recently demonstrated that S100A8, S100A9, and S100A8/A9 are potent chemotactic factors for neutrophils, with an activity ranging from 10−12 to 10−10M (31–33). S100A8, S100A9, and S100A8/A9 were shown to also induce neutrophil adhesion to fibrinogen in a Mac-1–dependent manner (31, 34). In addition, intraperitoneal injection of murine S100A8 in mice stimulated the accumulation of activated neutrophils and macrophages within 4 hours (35). Finally, Eue et al showed that S100A9 and S100A8/A9 enhance monocyte adhesion to, and migration through, endothelial cells via interactions between Mac-1 and intercellular adhesion molecule 1 (36).

In this study, we hypothesized that chemotactic factors other than IL-8 could be involved in the early neutrophil migration associated with the inflammatory response to MSU crystals in the air-pouch model of acute urate-induced arthritis. The CXC chemokines studied were CXCL1 (KC, murine growth-related oncogene α [GROα]), CXCL2 (murine macrophage inflammatory protein 2 [MIP-2], murine GROβ/γ), and the CC chemokines CCL2 (JE, monocyte chemoattractant protein 1 [MCP-1]) and CCL3 (macrophage inflammatory protein 1α [MIP-1α]). The proteins S100A8 and S100A9 were also investigated in the course of the experiments. We made use of highly specific anti-murine chemokine antibodies against CXCL1, CXCL2, CCL2, and CCL3, as well as anti-murine S100A8 and S100A9. The results of these studies unequivocally prove that, of the mediators examined, only S100A8 and S100A9 are involved in neutrophil migration into the air pouch in response to MSU.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Recombinant proteins, polyclonal antibodies, and MSU crystals.

An expression vector containing the murine S100A8 complementary DNA (cDNA) was a generous gift from Professor H. J. Schluësener, University of Tübingen, Germany (37). Murine S100A9 cDNA was synthesized by reverse transcriptase–polymerase chain reaction from neutrophil RNA isolated using TRIzol reagent, according to the manufacturer's instructions (Gibco BRL, Grand Island, NY). The cDNA was then cloned into the pET28 expression vector (Novagen, Madison, WI) and transformed in Escherichia coli HMS174. Recombinant protein was expressed and purified as previously described (38). Contamination by endotoxins was <1 pg/μg of S100A8 or S100A9 protein, as measured by the Limulus amebocyte assay (Sigma, St. Louis, MO). Polyclonal antisera against human and murine recombinant S100A8 and S100A9 were generated after 4 repeated injections into NZW rabbits or CD1 rats at 4- or 2-week intervals, respectively. Antisera titers were determined using direct enzyme-linked immunosorbent assay (ELISA) and immunoblotting. IgG from antisera were purified by protein A affinity chromatography (Pierce, Rockford, IL). Absence of cross-reactivity of the purified IgG with other murine MRPs, S100 proteins, or proteins within the air-pouch exudates was confirmed by immunoprecipitation assays and Western blot analyses. Rabbit polyclonal antibodies directed against chemokines were prepared as previously described (39, 40). MSU crystals were prepared as previously described (41).

Air-pouch experiments.

Ten- to 12-week-old CD1 or BALB/c mice were obtained from Charles River Canada (Montreal, Quebec, Canada) or the Central Animal House at the University of Adelaide (Adelaide, South Australia, Australia). Air pouches were raised on the dorsum by subcutaneous injection of 3 ml of sterile air on days 0 and 3, as previously described (39, 40). On day 7, a suboptimal dose of MSU crystals (1.5 mg) suspended in a volume of 1 ml of endotoxin-free phosphate buffered saline (PBS; Sigma) was injected into the air pouches. Alternatively, 1 ml of murine S100A8 or S100A9 at concentrations ranging from 0.01 μg/ml to 10 μg/ml was injected into the air pouches. At specific times, the mice were killed by asphyxiation using CO2, the air pouches were washed once with 1 ml PBS–5 mM EDTA and then twice with 2 ml PBS–5 mM EDTA, and the exudates were centrifuged at 500g for 5 minutes at room temperature. Cells were counted with a hemocytometer following acetic blue staining. Characterization of leukocyte subpopulations was performed by Wright-Giemsa staining of cytospins (VWR, Mississauga, Ontario, Canada). In separate experiments, mice were injected intraperitoneally 16 hours prior to injection of MSU crystals into the air pouch with 200 μg of purified rabbit anti-murine CXCL1, CXCL2, CCL2, or CCL3 IgG (39, 40) or 2 mg of purified IgG from rabbit antisera against murine S100A8 and S100A9 to inhibit their activities. Injection of MSUM in both strains of mice led to a similar inflammatory reaction.

ELISAs.

The CXCL2, CCL3, and CCL2 ELISAs were performed as previously described (39, 40). For detection of CXCL1, Costar high-binding 96-well plates (Corning, Corning, NY) were coated overnight at 4°C with 100 μl of 1 μg/ml of goat anti-CXCL1 (R&D Systems, Minneapolis, MN). Plates were blocked with PBS/5% bovine serum albumin (BSA) for 1 hour at room temperature. After washing with PBS/0.5% Tween 20, standards and samples diluted in PBS/2% BSA (100 μl) were added and incubated for 2 hours at room temperature. After 3 washes, the plates were incubated with goat biotinylated anti-mouse CXCL1 antibody (0.05 μg/ml) diluted in PBS/2% BSA for 1 hour at room temperature. Following incubation, the plates were washed 3 times and incubated with 100 μl/well of poly-HRP-40–streptavidin (0.1 μg/ml; Research Diagnostics, Flanders, NJ) diluted in PBS/2% BSA/0.05% Tween 20 for 45 minutes at room temperature. After 3 washings, detection was carried out by adding 100 μl of tetramethylbenzidine solution (TMB-S) according to the manufacturer's instructions (Research Diagnostics), and the optical density (OD) was read at 500 nm. The concentration of CXCL1 and the other murine chemokines was derived from a standard curve generated by a known concentration of recombinant CXCL1 and other chemokines. The detection limits were <35 pg/ml for each of the chemokines tested.

The detection of human and murine S100A8, S100A9, and S100A8/A9 was achieved by coating 96-well plates with 100 μl/well of human S100A8/A9-specific monoclonal antibody 5.5 (generous gift of Nancy Hogg, ICRF, London, UK), or purified rabbit IgG against murine S100A8 or murine S100A9 (for the detection of murine S100A9 and S100A8/A9), diluted to a concentration of 1 μg/ml in 0.1M carbonate buffer pH 9.6. After overnight incubation, the plates were washed with PBS/0.1% Tween 20 and blocked with PBS/0.1% Tween 20/2% BSA for 30 minutes at room temperature. The samples and standards (100 μl) were added and incubated for 1 hour at room temperature. After 3 washes with PBS/0.1% Tween 20, the plates were incubated for 1 hour at room temperature with 100 μl/well of 1:10,000 dilution of antisera against human S100A9 (for the detection of human S100A8/A9) or with purified rat IgG against murine S100A9 or murine S100A8 (for the detection of murine S100A9, S100A8, and S100A8/A9). The plates were then washed 3 times and incubated with 100 μl/well of peroxidase-conjugated donkey anti-rabbit antibodies (1:7,500 dilution; Jackson ImmunoResearch, Mississauga, Ontario, Canada) or peroxidase-conjugated goat anti-rat antibodies (1:10,000 dilution; Jackson ImmunoResearch) in PBS/0.1% Tween 20/2% BSA for 1 hour at room temperature. After 3 washes, the presence of IgG was detected with 100 μl of TMB-S according to the manufacturer's instructions, and the OD was read at 500 nm. Concentrations of murine S100A8, S100A9, and S100A8/A9, and human S100A8/A9 were derived from a standard curve generated by known concentrations of recombinant murine S100A8, S100A9, and S100A8/A9, and human S100A8/A9. The detection limits were 4 ng/ml for murine S100A8 and S100A9, and 10 ng/ml and 100 pg/ml for murine and human S100A8/A9, respectively.

Patients.

Synovial fluids from 5 patients with osteoarthritis (OA) and 7 patients with gout were collected on heparin, centrifuged to remove cells, and frozen at −20°C until assayed. Plasma from heparinized blood of 6 of the same patients with gout and from 8 healthy donors was also collected and kept at −20°C until assayed.

Statistical analysis.

All statistical analyses were performed using InStat software (GraphPad Software, San Diego, CA). Statistical comparisons were made by analysis of variance for the number of leukocytes in air pouches and the secretion of chemokines and S100 proteins. Dunnett's multiple comparison test and Student's t-test were used to compare specific groups at a confidence interval of 95%.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Injection of MSU crystals into the murine air pouch induced the release of chemokines. We first evaluated the ability of MSU crystals to induce an inflammatory reaction in the murine air-pouch model. As shown in Figure 1, MSU crystals stimulated a significant accumulation of inflammatory cells when injected into the air pouch. Leukocyte recruitment was first detected 3 hours after injection and peaked at 9 hours, when a mean (±SEM) of 3.8 ± 0.6 × 106 leukocytes were recruited, before returning to control levels by 24 hours postinjection. More than 90% of the recruited leukocytes were neutrophils, with the rest being monocytes (data not shown).

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Figure 1. Accumulation of leukocytes in the air-pouch model induced by monosodium urate monohydrate (MSU) crystals. Air pouches were raised on the dorsum of 10- to 12-week-old mice. One milliliter of a suspension of MSU crystals (1.5 mg/ml) or phosphate buffered saline (PBS) was injected into the air pouches, and the mice were killed after increasing periods of time. The migrated leukocytes were harvested by washing the air pouch with PBS/EDTA (5 mM) and counted using a hemocytometer. A, Total leukocytes and B, number of neutrophils. Values are the mean ± SEM of neutrophils per pouch in at least 7 mice per group.

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To identify the chemotactic factors involved in this recruitment, we assayed the air-pouch exudates for CXCL1, CXCL2, CCL2, and CCL3 (Figure 2). CXCL1 (mean ± SEM 550 ± 145 pg) and CXCL2 (1,800 ± 170 pg) were detected in the air-pouch exudates in the first 3 hours following injection of MSU crystals. In contrast, concentrations lower than 100 pg of CXCL1 and CXCL2 were measured in PBS-injected animals. After 6 hours, the levels of both CXCL1 and CXCL2 diminished, and returned to control values within 9 hours postinjection (Figures 2A and B, respectively). The CC chemokines CCL2 and CCL3, which are chemotactic for monocytes but are also involved in neutrophil recruitment in vivo (40), followed a different kinetic pattern. CCL2 concentrations were maximal 2 hours (13,500 ± 3,950 pg) after injection, but remained stable for the next 6 hours (Figure 2C). Similarly, production of CCL3 was slower, with maximal secretion at 8 hours postinjection (1,290 ± 170 pg) (Figure 2D). The release of CXCL2 and CXCL1 therefore preceded neutrophil recruitment to the air pouch, while the release of CCL2 and CCL3 correlated directly with neutrophil recruitment to the pouch.

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Figure 2. Chemokine production in the air pouches of mice injected with MSU crystals. Air pouches were raised on the dorsum of 10- to 12-week-old mice. One milliliter of a suspension of MSU crystals (1.5 mg/ml) or PBS was injected into the air pouches, and the mice were killed after increasing periods of time. The air-pouch exudates were harvested by washing with 1 ml of PBS/EDTA (5 mM). A, CXCL1, B, CXCL2, C, CCL2, and D, CCL3 levels were then measured by enzyme-linked immunosorbent assays. Values are the mean ± SEM of at least 7 mice per group. See Figure 1 for other definitions.

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These results suggested that chemokines could be involved in MSU crystal–induced neutrophil recruitment. We therefore evaluated their role by inhibiting their activities using specific blocking antibodies. Inhibition of CXCL1, CXCL2, CCL2, or CCL3 activity by peritoneal injection of the specific antibodies prior to MSU crystal injection failed to prevent neutrophil recruitment to the pouch (Figure 3). The same purified antibodies, using similar conditions, had previously been demonstrated to inhibit the accumulation of neutrophils in the air pouch in response to tumor necrosis factor α (TNFα) and the Staphylococcus aureus superantigen (SEA) (39, 40). These results imply that the activity of each chemokine alone is not essential for neutrophil recruitment to the air pouch.

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Figure 3. Effects of blocking antibodies against the chemokines on neutrophil accumulation in air pouches in the presence of MSU crystals. Air pouches were raised on the dorsum of 10- to 12-week-old mice. Sixteen hours before the experiment, mice were injected intraperitoneally with 200 μg of purified IgG from rabbits immunized against CXCL1, CXCL2, CCL2, CCL3, or control IgG from an unimmunized rabbit. One milliliter of a suspension of MSU crystals (1.5 mg/ml) or PBS was then injected in the air pouches, and the mice were killed after 6 hours. The migrated leukocytes were harvested by washing the air pouch with PBS/EDTA (5 mM) and counted using a hemocytometer. Values are the mean ± SEM of at least 7 mice per group. See Figure 1 for other definitions.

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Release of MRPs into the air pouch in response to MSU crystal injection.

We recently demonstrated that S100A8 and S100A9 are highly potent chemotactic factors for neutrophils (31). Knowing that high levels of MRPs are present in several inflammatory processes (23, 27, 28), we quantified the presence of MRPs in the air-pouch exudates following MSU crystal injection. Low concentrations of S100A8, S100A9, and S100A8/A9 were detected in air-pouch exudates of control mice. Injection of MSU crystals led to the release of a mean (±SEM) of 7,700 ± 1,500 ng/ml of S100A8/A9. This release was detected as early as 3 hours postinjection and was maximal between 6 and 12 hours following injection of MSU crystals (Figure 4A). S100A8 and S100A9 homodimers were also present but at lower concentrations (905 ± 105 and 3,580 ± 545 ng/ml, respectively) (Figures 4B and C). The presence of MRPs in the pouch also correlated with neutrophil recruitment. These results suggest that MRPs could play a role in neutrophil recruitment in response to MSU crystals.

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Figure 4. Release of S100A8, S100A9, and S100A8/A9 in the air pouches of mice injected with MSU crystals. Air pouches were raised on the dorsum of 10- to 12-week-old mice. One milliliter of a suspension of MSU crystals (1.5 mg/ml) or PBS was injected into the air pouches, and the mice were killed after increasing periods of time. The air-pouch exudates were harvested by washing with 1 ml of PBS/EDTA (5 mM). A, S100A8/A9, B, S100A8, and C, S100A9 levels were then measured by enzyme-linked immunosorbent assays. Values are the mean ± SEM of at least 7 mice per group. See Figure 1 for definitions.

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Role of S100A8 and S100A9 in neutrophil recruitment induced by MSU crystals.

To determine a role for MRPs in MSU-induced leukocyte recruitment, 10 μg of recombinant murine S100A8 or S100A9 was injected into the air pouch. Injection of either murine S100A8 or S100A9 led to a significant accumulation of neutrophils in the air pouch (Figure 5A). Neutrophil recruitment occurred within 3 hours postinjection and was maximal (mean ± SEM 3.6 ± 0.4 and 4.7 ± 0.5 × 106 neutrophils/pouch for S100A8 and S100A9, respectively) at 6 hours postinjection, after which time it decreased rapidly. The number of neutrophils in the air pouch reached 2.4 × 106 and 0.9 × 106 neutrophils/pouch at 9 hours and 12 hours postinjection, respectively, and returned to control levels within 24 hours (Figure 5A). More than 95% of the migrated leukocytes were neutrophils, with the remainder being predominantly monocytes. As shown in Figure 5B, S100A8, S100A9, and S100A8/A9 induced leukocyte recruitment to the air pouch in a dose-dependent manner. Significant neutrophil recruitment occurred at injected doses of 1 μg (P < 0.01) and was maximal at 10 μg, the highest dose injected. Those doses are similar to the levels detected in the air pouches following injection of MSU crystals (Figures 4A–C).

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Figure 5. Effects of S100A8, S100A9, and S100A8/A9 on neutrophil accumulation in vivo. Air pouches were raised on the dorsum of 10- to 12-week-old mice. A, One milliliter of S100A8 (10 μg/ml), S100A9 (10 μg/ml), or PBS was injected into the air pouches, and the mice were killed after increasing periods of time. B, Increasing concentrations of S100A8, S100A9, and S100A8/A9 were injected into the air pouch. The migrated leukocytes were harvested by washing the air pouch with PBS/EDTA (5 mM) and counted using a hemocytometer. Values are the mean ± SEM of at least 7 mice per group. See Figure 1 for definitions.

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The role of S100A8 and S100A9 in neutrophil migration induced by MSU crystals was next investigated by inhibiting their activities using purified specific IgG neutralizing antibodies. Because of the difference between the concentrations of chemokines and S100A8, S100A9, and S100A8/A9 proteins in the air-pouch exudates, higher concentrations of blocking IgG were used to efficiently block S-100 protein–induced neutrophil migration. In preliminary experiments, peritoneal injection of anti-S100A8 and anti-S100A9 IgG specifically inhibited the recruitment induced into the air pouch by the injection of S100A8 and S100A9, respectively (data not shown). Injection of purified IgG from preimmunized rabbits prior to MSU crystal injection in the air pouch did not reduce neutrophil recruitment (Figure 6). Similarly, injection of anti-S100A9 alone did not significantly diminish neutrophil recruitment compared with control (PBS) (P > 0.05 by Dunnett's multiple comparison test). However, injection of anti-S100A8 significantly reduced neutrophil recruitment by 54% (P < 0.05 by Dunnett's multiple comparison test). Moreover, injection of both anti-S100A8 and anti-S100A9 inhibited by 96% the neutrophil recruitment induced by MSU crystals to the air pouch (P < 0.01). Since these antibodies bind to both homodimers and the S100A8/A9 heterodimer, injection of both antibodies could have inactivated not only S100A8 and S100A9, but also S100A8/A9 activity.

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Figure 6. Effects of anti-S100A8 and anti-S100A9 antibodies on neutrophil accumulation in air pouch in the presence of MSU crystals. Air pouches were raised on the dorsum of 10- to 12-week-old mice. Sixteen hours prior to the experiment, mice were injected intraperitoneally with 2 mg of purified IgG from rabbits immunized against S100A8 or S100A9 or control IgG from an unimmunized rabbit. One milliliter of a suspension of MSU crystals (1.5 mg/ml) or PBS was then injected into the air pouches, and the mice were killed after 6 hours. The migrated leukocytes were harvested by washing the air pouch with PBS/EDTA (5 mM) and counted using a hemocytometer. Values are the mean ± SEM of at least 4–7 mice per group. ∗ = P < 0.05 and ∗∗ = P < 0.01 versus MSU alone, by Dunnett's multiple comparison test. See Figure 1 for definitions.

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Measurements of S100A8/A9 in synovial fluids and plasma of patients with gouty arthritis.

To verify whether S100A8 and S100A9 were present in gout, we quantified S100A8/A9 by specific ELISA in synovial fluids and plasma of patients with gout. S100A8/A9 was almost absent from synovial fluids of OA patients (mean ± SEM 13.5 ± 3.7 ng/ml), a joint disease with little or no synovial inflammation (Figure 7A). In contrast, 40.3 ± 8.3 μg/ml of S100A8/A9 was measured in synovial fluids of patients with gout. S100A8/A9 was also detected in the plasma of the same patients, where it reached 470 ± 120 ng/ml, a concentration 50 times higher than concentrations measured in healthy donors (12.8 ± 3.4 ng/ml) (Figure 7B). These concentrations, which are higher than those detected in the murine air pouch following MSU crystal injection, are consistent with a possible role for S100A8 and S100A9 in the pathogenesis of gout.

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Figure 7. Measurement of S100A8/A9 in synovial fluids and plasma of patients with gout. S100A8/A9 was measured by enzyme-linked immunosorbent assay in A, synovial fluids of patients with gout or osteoarthritis and B, plasma of healthy donors and patients with gout. Each data point represents an individual sample; bars show the mean.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Although several molecules have been suspected in the past to play a role in the neutrophil migratory process in response to MSU deposition, the identity of the specific chemotactic factors involved has not yet been fully elucidated. In this study, we investigated the role of the chemokines CXCL1 and CXCL2, known to be chemotactic for neutrophils in vitro and in vivo, and CCL3 and CCL2, which have been shown to play an important role in the recruitment of neutrophils in vivo (39). Although all these chemokines were detected in air-pouch exudates of mice injected with MSU crystals, prior treatment of mice with specific neutralizing antibodies failed to prevent crystal-induced neutrophil migration into the air pouch. This prompted us to investigate the involvement of other chemotactic factors in neutrophil migration induced by MSU crystals. The proinflammatory proteins S100A8 and S100A9, which were also present in the air-pouch exudates, were found to induce neutrophil migration to the air pouch with a kinetic profile similar to that induced by MSU crystals. In addition, inactivation of both S100A8 and S100A9 totally inhibited neutrophil accumulation in response to MSU crystals, clearly demonstrating their involvement in this response. Since these proteins are also present at high concentrations in synovial fluids of patients with gout, we suggest that they play an essential role in gout pathogenesis.

A large number of inflammatory mediators are released by monocytes, macrophages, synovial cells, and neutrophils upon MSU crystal stimulation. For example, C5a, LTB4, PAF, and IL-8 are some of the neutrophil chemotactic factors that are present in the affected articulation and are suspected to be involved in the pathogenesis of gout (5, 13–15, 42, 43). Of all these chemotactic factors, only IL-8 has been demonstrated to be partially involved in neutrophil accumulation induced by MSU crystals. Inhibition of IL-8 activity by passive immunization reduced neutrophil accumulation by 50% 12 hours after injection of MSU crystals in rabbit articulations (16). IL-8 release in MSU crystal–induced rabbit arthritis occurs in a biphasic mode, with significant secretion detected after 2 and 12 hours (44). In spite of this, IL-8 is not involved in the early accumulation of neutrophils, since no inhibition was detected before 12 hours postinjection in the same model of MSU crystal–induced rabbit arthritis (16).

Since no murine equivalent of IL-8 has been identified, we investigated the role of the closely related CXC chemokines CXCL2 and CXCL1, as well as the CC chemokines CCL2 and CCL3, in neutrophil migration, concentrating our investigation on the first 6 hours post–MSU crystal injection. All of these chemokines had previously been shown to play a role in neutrophil migration in the air-pouch model in response to both TNFα and SEA (39, 40). The air-pouch model was selected since it has been used extensively in the past as a model of the synovial environment to study the mechanism of inflammation induced by different proinflammatory factors, including MSU (45–51). Designed to replicate the synovial lining, this model allows for the identification and study of emigrated leukocytes and molecules produced during an inflammatory response, although several features of the synovial environment are missing in air-pouch fibroblasts (52, 53). The kinetic profile of the production of these chemokines in the air pouch following MSU crystal injection suggests they could be involved in neutrophil migration in response to MSU crystals. However, individual inactivation with specific blocking antibodies failed to reduce neutrophil accumulation to the pouch, an observation consistent with the results of previous studies, indicating a dominant role for complement, rather than CXCL2 and CCL3, in neutrophil recruitment into the air pouch in response to phagocytic agents such as bacteria (54). Overall, the results of the present study suggest that CXCL2, CXCL1, CCL2, and CCL3 are not individually essential for neutrophil recruitment in response to MSU crystals. However, while the data strongly suggest that chemokines are not critically involved, there could be differences in the neutralizing ability of the anti-chemokine and anti-S100 protein antibodies.

Our results are in contrast with studies by others showing that early neutrophil migration in response to MSU crystals was impaired in mice deficient in the murine homolog of the IL-8 receptor CXCR2 (17). This could be partially explained by the fact that CXCR2 does not solely bind IL-8, suggesting that other chemokines could be involved. Moreover, the CXCR2 knockout mouse has an expansion of the neutrophil lineage, both in the bone marrow and in the spleen (55). A lack of neutrophil recruitment in response to IL-8 in this mutant strain may therefore be due, at least in part, to undefined developmental effects of the targeted deletion. Since chemokines show redundancy in their activity, it is also possible that the activity of each inactivated chemokine can be overcome by others present in the pouch (56–58). However, individual inactivation of CXCL1, CXCL2, CCL2, or CCL3 using the same antibodies reduced neutrophil accumulation following TNFα and SEA injection (39, 40). Nevertheless, the contribution of these chemokines to neutrophil migration differed according to the stimuli because inactivation of CXCL2 and CCL3, either alone or in combination, failed to inhibit leukocyte accumulation in response to bacteria in the air-pouch model (54). In the latter study, components of the complement pathway appeared to override the effect of CXCL2 and CCL3 in the recruitment of neutrophils (54). This strongly suggests that chemokine redundancy alone cannot explain the lack of inhibition by specific antibodies, suggesting that other factor(s) are also involved in MSU-induced inflammation.

Results presented in this study show that S100A8 and S100A9 could be these factors. S100A8, S100A9, and S100A8/A9 were detected at very high concentrations in the exudates of mice injected with MSU crystals and in the synovial fluid of patients with gout. The release was rapid, reaching 10−8M before 3 hours and ∼10−6M within 6 hours postinjection. The release of S100A8 and S100A9 also correlated temporally with neutrophil recruitment in the air-pouch exudates. The source of MRPs in the air-pouch following MSU crystal injection is not known. The correlation between the release of MRPs and neutrophil recruitment, and the fact that 30% of the neutrophil cytosolic proteins are MRPs, suggest that neutrophils are a major source of MRPs in the air pouch following MSU crystal injection. The release of MRPs by neutrophils, monocytes, and macrophages has previously been demonstrated (28, 59, 60); however, the possibility that the release of MRPs is a consequence of cellular lysis cannot be ruled out. Additional studies will be necessary to examine this possibility.

Inactivation of S100A9 by passive immunization did not reduce neutrophil recruitment. However, inactivation of S100A8 reduced neutrophil recruitment by 50%. These data indicate that S100A8 plays a more important role in MSU crystal–induced neutrophil recruitment than does S100A9. However, passive immunization with anti-S100A8 and anti-S100A9 prior to injection of MSU crystals led to a total inhibition of neutrophil recruitment to the air pouch, suggesting that both S100A8 and S100A9 play essential roles in the recruitment of neutrophils. The lack of an effect of anti-S100A9 antibodies alone, combined with our observation that anti-S100A8 antibodies, either alone or in combination with anti-S100A9 antibodies, significantly inhibited neutrophil recruitment in response to MSU, suggests that S100A8 and S100A8/A9 mediate this response.

Further studies will be required to determine whether either species or only the heterodimer is responsible for the observed response. Certainly, the heterocomplex S100A8/A9 is the major form found in the air pouch following MSU crystal injection and in synovial fluids of patients with gout. S100A8/A9 is also chemotactic for neutrophils in vitro and induces neutrophil accumulation in vivo (31). Together with this study, these results suggest that S100A8 and S100A9 could direct neutrophil chemotaxis at early time points, before inducing their retention at the inflammatory site by stimulating their adhesion. Further in vivo studies will be necessary to confirm this hypothesis.

In conclusion, we demonstrate that S100A8 and S100A9, probably in the form of an S100A8 monomer or homodimer or an S100A8/A9 heterodimer, play an essential role in neutrophil migration in response to MSU crystals in the air-pouch model in vivo. Since these proteins are found at high concentrations in the synovial fluids of patients with gout, we suggest that they might induce and/or sustain the massive accumulation of neutrophils to the affected articulation associated with gout.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors wish to thank Pascal Rouleau for technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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