Published online before print April 23, 2004
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* Centres de Recherche en Infectiologie and
Rhumatologie et Immunologie, CRCHUL, Université Laval, Quebec, Canada; and
Unité des maladies rhumatismales, Centre Hospitalier Universitaire, Université de Sherbrooke, Quebec, Canada
1Correspondence: Centre de Recherche en Infectiologie, Room RC 709, CRCHUL, Université Laval, 2705, Laurier Blvd., Sainte-Foy, Quebec, Canada G1V 4G2. E-mail: Philippe.Tessier{at}crchul.ulaval.ca
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R)IIIB (CD16) and CD11b/CD18 were involved in the stimulation by MSUM crystals. Neutrophil preincubation with the Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine and the Syk tyrosine kinase inhibitor trans-3,3',4,5'-tetrahydrozystilbene significantly reduced the release of S100A8/A9, suggesting that the Src tyrosine kinase family and Syk were involved. In addition, wortmannin reduced neutrophil release of S100A8/A9, indicating a potential involvement of phosphatidylinolitol-3 kinase in this release. Preincubation of neutrophils with the tubulin depolymerization promoters nocodazole and vincristine reduced MSUM-induced release, suggesting a tubulin-associated pathway of release. These results indicate that S100A8/A9 is released by MSUM crystal-stimulated neutrophils following activation of CD11b, CD16, Src kinases, Syk, and tubulin polymerization.
Key Words: leukocytes • inflammation • cellular activation • calcium-binding proteins
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S100A8 and S100A9 are small, cytosolic proteins that exist as homodimers, but the presence of calcium seems to increase the formation of the heterocomplex S100A8/A9 [7 8 9 ], which is the major form found in the extracellular milieu. S100A8 and S100A9 are highly expressed in neutrophil and monocyte cytosols but can also be expressed by other cell types such as keratinocytes, epithelial, and endothelial cells [10 11 12 13 14 15 ]. S100A8, S100A9, and S100A8/A9 stimulate leukocyte chemotaxis, adhesion, and migration [16 17 18 19 ]. As these proteins are found on the endothelium near inflammatory sites, they are suspected to play a role in leukocyte migration to inflammatory sites [20 ].
Elevated serum levels of S100A8/A9 (more than 1 µg/ml) are detected in patients suffering from various infections and inflammatory pathologies such as cystic fibrosis, tuberculosis, and juvenile rheumatoid arthritis [7 , 21 , 22 ]. Local release of the proteins has also been detected in periodontal infections and in experimental murine abscesses [23 , 24 ]. The extracellular presence of S100A8/A9 suggests that S100A8 and S100A9 can be released actively or as a result of cell necrosis. Like interleukin (IL)-1 and fibroblast growth factor ß (FGFß), S100A8 and S100A9 are expressed and stored in the cytosol, indicating that they are secreted via a secretion pathway distinct from the endoplasmic reticulum (ER)/Golgi classical route. It has been shown that pokeweed mitogen-stimulated as well as Mycobacterium bovis-infected monocytes release S100A9 and S100A8/A9 but not S100A8 [21 , 25 ]. This secretion was inhibited by combined treatment with the T helper cell type 2 cytokines IL-10 and IL-4 [25 ]. Active secretion of S100A8 and S100A9 by monocytes following activation of protein kinase C has also been demonstrated [26 ]. Moreover, that release occurred through a tubulin-associated pathway [26 ]. Specific release of S100A8/A9 by stimulated neutrophils has also been reported by monitoring the disappearance of S100A8/A9 from neutrophil cytosol following stimulation with C5a and formyl-Met-Leu-Phe (fMLP) [27 ]. However, the signal-transduction pathway, leading to S100A8/A9 secretion by neutrophils and the mechanism of secretion, remains unknown.
We recently showed the involvement of S100A8, S100A9, and S100A8/A9 in neutrophil recruitment during MSUM crystal-induced inflammatory reaction in a murine air pouch model [6
]. The release of S100A8, S100A9, and S100A8/A9 correlated with neutrophil infiltration to the pouch, suggesting neutrophils as the major source of these proteins. We therefore set out to investigate and characterize the release of S100A8/A9 by MSUM crystal-stimulated neutrophils in vitro. In the present study, we show that S100A8/A9 is specifically released by MSUM crystal-activated neutrophils. S100A8/A9 release was stimulated through the receptors CD11b/CD18 [membrane activated complex-1 (Mac-1)] and CD16 [Fc receptor for immunoglobulin G (IgG; FcR)III], which have been previously identified as the two major receptors implicated in neutrophil stimulation by MSUM crystals [28
]. The release of S100A8/A9 occurred at least in part through a tubulin-associated mechanism and was dependent on Src kinases, Syk, and phosphatidylinositol-3 kinase (PI-3K), confirming that this release was an active phenomenon.
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RIII) was purchased from Accurate Chemical and Scientific Corp. (Westbury, NY). Mouse monoclonal anti-human CD11b (MEM-170) was purchased from EXBIO Praha, A.S. (Víde
ská, Czech Republic). Antilactate dehydrogenase (anti-LDH) and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MI).
Neutrophil purification
Peripheral blood was collected in heparinized tubes from healthy adult volunteers. Neutrophils were isolated as described previously by Boyum [31
] with some modification. Briefly, leukocytes were obtained after erythrocyte sedimentation in 2% Dextran T-500 followed by centrifugation on Ficoll-Paque cushions. Contaminating erythrocytes were removed by hypotonic (water) lysis for 15 s. Neutrophils were resuspended in Hanks’ balanced saline solution (HBSS) containing 1.3 mM Ca2+ and 10 mM HEPES, pH 7.4 (H-HBSS). The purity and cell viability of neutrophil preparations were consistently greater than 98%, as assessed by acetic blue staining and trypan blue exclusion, respectively. Cell preparation with nonspherical cell morphology (an indication of preactivation) or exhibiting any other activation-related phenotype was excluded from the study.
Purification of S100A8/A9
Neutrophils were lysed by sonication and centrifuged at 15,000 gfor 5 min, and the supernatant was collected. S100A8/A9 was purified by fast performance liquid chromatography using a UNOTMQ-12 anion exchange column from Bio-Rad (Hercules, CA). Buffer A contained 50 mM Tris/HCl, pH 8.5, 1 mM dithiothreitol, 1 mM EDTA, and 1 mM EGTA and Buffer B, made of Buffer A plus 1 M NaCl. The proteins were separated in a 0–20% gradient of Buffer B in Buffer A. Elution profiles were obtained by reading the absorption at 280 nm. Fractions containing the eluted S100A8/A9 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before being pooled and dialyzed overnight in phosphate-buffered saline (PBS) containing 1 mM CaCl2. S100A8/A9 purity was confirmed by SDS-PAGE. Less than 20% of the S100A8 and S100A9 was in homodimer forms. This preparation was used as a standard in S100A8/A9 enzyme-linked immunosorbent assay (ELISA).
Neutrophil activation
Neutrophils were resuspended in H-HBSS to a final concentration of 10 x 106 cells/ml and were incubated at 37°C with 0.1–3 mg/ml MSUM crystals for increasing periods. Stimulation was stopped by centrifugation at 17,000 g for 15 s, and the supernatants containing S100A8 and S100A9 were harvested for detection by ELISA. In separate experiments, neutrophils (10x106 cells/ml) were primed with 200 pM GM-CSF for 30 min at 37°C before being washed with H-HBSS and incubated with the stimuli or preincubated with 10 µM vincristine, 500 ng/ml nocodazole, and 5 µg/ml cytochalasin B at 37°C for 1 h; or increasing concentrations of wortmannin, PP2, or piceatannol for 20 min before the addition of the stimuli. Alternatively, neutrophils (15x106 cells/ml) were preincubated with 5 µg/ml anti-CD16 and/or 5 µg/ml anti-CD11b or 5 µg/ml isotype-control IgG and/or IgM for 2 min at 37°C before addition of the stimuli. Neutrophils were then stimulated with 1.5 mg/ml MSUM crystals for 1 h.
Immunoblot analysis
Cell lysates and supernatants from stimulated neutrophils were separated on 15% SDS-PAGE and transferred to Sequi-Blot polyvinylidene difluoride 0.2 µm membranes (Bio-Rad) by electroblotting. Nonspecific binding sites on the membranes were blocked by incubating the filters in Tris-buffered saline (TBS)–Tween 20 0.2% containing 5% powered skim milk for 1 h at room temperature. The membranes were then incubated for 1 h at room temperature with Rab1-8 (1/1000) polyclonal antibody or anti-LDH-specific antibody. All antibodies were diluted in TBS–Tween 20 0.2% containing 0.5% powdered milk. After four washes with TBS–Tween 20 0.2%, the membranes were incubated with peroxidase-conjugated donkey anti-rabbit IgG (1/10,000) or goat anti-mouse IgG (1/10,000) for 1 h at room temperature. Following incubation, the filters were washed four times in TBS–Tween 20 0.2%, and peroxidase activity was revealed by developing the membranes in Renaissance chemiluminescence reagent (NEN, Ville St. Laurent, Canada).
Detection of cell necrosis
Following neutrophil incubation with MSUM crystals or H-HBSS, propidium iodide was added to a final concentration of 50 ng/ml in H-HBSS or H-HBSS containing 0.5% Triton X-100 as positive control for the detection of lysed cells, and neutrophils were incubated for 5 min at room temperature. Neutrophils were then centrifuged at 17,000 g for 15 s and resuspended in H-HBSS.Cells were then deposited on microscope slides, and the fluorescence was visualized using an inverted confocal microscope (Olympus, Melville, NY). The presence of propidium iodide staining inside the nuclei indicated cellular lysis.
ELISA
Costar high-binding 96-well plates (Corning, NY) were coated with 100 µl/well S100A8/A9 conformational epitope-specific monoclonal antibody 5.5, diluted to a concentration of 1 µg/ml in 0.1 M carbonate buffer, pH 9.6, and left overnight at 4°C. Following incubation, the plates were washed with PBS/0.1% Tween-20 and blocked with PBS/0.1% Tween-20/2% bovine serum albumin (BSA; 100 µl/well) for 30 min at room temperature. The samples and standards (100 µl) were added and incubated for 40 min at room temperature. After three washes with PBS/0.1% Tween-20, the plates were incubated with 100 µl/well S100A9 polyclonal antibody (Rab1-14, diluted 1/10,000 in PBS/0.1% Tween-20/2% BSA) for 40 min at room temperature. Following incubation, the plates were washed three times and incubated with 100 µl/well peroxidase-conjugated donkey anti-rabbit IgG at a dilution of 1/7500 in PBS/0.1% Tween-20/2% BSA for 40 min at room temperature. After three washes, the presence of IgG was detected with 100 µl TMB-S, according to the manufacturer’s instructions; the reaction was stopped by adding 100 µl 0.18 M H2SO4, and the optical density was read at 500 nm.
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Figure 1. Release of S100A8/A9 by MSUM-activated human neutrophils. (A) Neutrophils (10x106 cells/ml) were incubated at 37°C for 1 h with increasing concentrations of MSUM (MSU) crystals. (B) Neutrophils (10x106 cells/ml) were stimulated with medium alone [H-HBSS (HBSS)] or 3 mg/ml MSUM (MSU) crystals for increasing periods of time. The reactions were stopped by centrifugation at 17,000 g for 15 s, and the supernatants were collected. Levels of S100A8/A9 in the supernatants were analyzed by ELISA. Results are mean ± SEM of three independent experiments performed on different donors.
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Figure 2. Release of LDH and S100A8 by MSUM crystal-stimulated neutrophils. Neutrophils were stimulated with MSUM (MSU) crystals (3 mg/ml) for 1 h at 37°C. The reaction was stopped by centrifugation at 17,000 g for 15 s, and the supernatant was collected and separated on SDS-PAGE. Total cell lysates (Cells) were obtained by lysing the neutrophils in loading buffer. LDH and S100A8 were detected by immunoblot. Results are from one experiment representative of two other experiments performed on neutrophils from different donors.
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Figure 3. Confocal images demonstrating the absence of cellular lysis when neutrophils are stimulated with MSUM crystals. Cells were incubated in medium alone (A and B) or stimulated with 1.5 mg/ml MSUM crystals (C and D) for 1 h at 37°C. After stimulation, propidium iodide was added to a final concentration of 50 ng/ml and incubated for 5 min at room temperature. Neutrophils were then centrifuged at 17,000 g for 15 s and resuspended at a concentration of 40 x 106 cells/ml in H-HBSS (A and C) or H-HBSS containing 0.5% Triton X-100 as positive control for the detection of lysed cells (B and D). Confocal images are from one experiment representative of two others performed on neutrophils from different donors.
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Figure 4. Priming of neutrophils with GM-CSF enhances S100A8/A9 release when neutrophils are stimulated with MSUM (MSU) crystals. Neutrophils (10x106 cells/ml) were pretreated with 200 pM GM-CSF or its control buffer [H-HBSS (HBSS)] for 30 min at 37°C. Cells were stimulated with MSUM crystals (1.5 mg/ml) for 1 h at 37°C. The reaction was stopped by centrifugation at 17,000 g for 15 s, and the supernatant was collected. Levels of S100A8/A9 in the supernatant were analyzed by ELISA. Results are the mean ± SEM of three experiments performed on different donors. *, P < 0.05, paired t-test.
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RIIIb (CD16) and Mac-1 (CD11b/CD18) [28
]. To determine if those two molecules were involved in the stimulation of S100A8/A9 release by neutrophils following activation with MSUM crystals, cells were stimulated with MSUM crystals with or without specific blocking antibodies. Antibodies directed against CD11b or CD16 blocked the release of S100A8/A9 induced by MSUM crystals by 48–58% (Fig. 5
). This inhibition climbed to 77% when the two antibodies were combined. As expected, isotype-matched antibodies had no effect on the release of S100A8/A9 by activated neutrophils, demonstrating the specificity of the effect detected. These results demonstrate the involvement of CD11b and CD16 in the stimulation of S100A8/A9 release by MSUM crystal-stimulated neutrophils.
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Figure 5. Stimulation of S100A8/A9 release from MSUM crystal-stimulated neutrophils occurs through CD16 and CD11b. Neutrophils were preincubated with 5 µg/ml anti-CD16 and/or 5 µg/ml anti-CD11b or 5 µg/ml isotype-matched antibody for 2 min at 37°C before addition of MSUM (MSU) crystals at a final concentration of 1.5 mg/ml. After 1 h incubation, the levels of S100A8/A9 in the supernatant were analyzed by ELISA. Results are the mean ± SEM of six experiments from different donors. *, P < 0.05, and **, P < 0.01, compared with control, Dunnett multiple comparison test.
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Figure 6. Inhibition of Src tyrosine kinase family, Syk tyrosine kinase, or PI-3K reduces S100A8/A9 release from MSUM (MSU) crystal-stimulated neutrophils. Neutrophils were preincubated with increasing concentrations of (A) the Src tyrosine kinase family inhibitor PP2, (B) the Syk tyrosine kinase inhibitor piceatannol, or (C) the PI-3K inhibitor wortmannin for 20 min at 37°C and stimulated with 1.5 mg/ml MSUM crystals for 1 h at 37°C. The reaction was stopped by centrifugation at 17,000 g for 15 s, and the supernatant was harvested. The levels of S100A8/A9 in the supernatant were analyzed by ELISA. Results are the mean ± SEM of five experiments from different donors. *, P < 0.05, and **, P < 0.01, compared with control, Dunnett multiple comparison test.
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We also evaluated the role of PI-3K in a MSUM, crystal-stimulated transduction pathway leading to S100A8/A9 release, as PI-3K has also been associated with neutrophil functions such as chemotaxis and phagocytosis. [36 37 38 39 ]. Preincu-bation with the PI-3K inhibitor wortmannin reduced the release of S100A8/A9 from neutrophils stimulated with MSUM crystals in a dose-dependent manner (Fig. 6C) . Maximum inhibition was observed in the presence of 200 nM wortmannin with a 74% reduction of S100A8/A9 release compared with neutrophils incubated with MSUM alone. Similar results were obtained using LY294002, another PI-3K inhibitor (data not shown). Together, these results also confirm that the release of S100A8/A9 by MSUM crystal-stimulated neutrophils is an active phenomenon.
Effect of tubulin inhibitors on S100A8/A9 release
Being cytosolic, S100A8/A9 must follow a secretion pathway different from the classical secretory vesicule pathway. The alternative secretion pathway used by several cytoplasmic proteins such as IL-1 and FGFß remains mostly unknown or not completely characterized. Moreover, it is unclear whether all proteins secreted via this alternative pathway follow a similar route of secretion. In the case of S100A8/A9, activated monocytes were shown to release S100A8/A9 via a microtubule polymerization mechanism [26
]. To investigate if the same mechanism was involved in S100A8/A9 release by neutrophils, cells were incubated with the microtubule polymerization inhibitors nocodazole and vincristine. S100A8/A9 release was inhibited by up to 42% or 38% when neutrophils were incubated in the presence of nocodazole or vincristine, respectively (Fig. 7
), suggesting that like monocytes, the release of S100A8/A9 by neutrophils follows a tubulin-dependent pathway. Cytochalasin B, an actin polymerization inhibitor, diminished MSUM-induced S100A8/A9 release by 57% (as compared with nonstimulated neutrophils incubated with cytochalasin B). This suggests that cell movement, phagocytosis, or both are involved in the release of S100A8/A9.
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Figure 7. Tubulin polymerization is involved in the release of S100A8/A9 by neutrophils stimulated with MSUM crystals. Neutrophils (10x106 cells/ml) were pretreated for 1 h at 37°C with H-HBSS (Control), nocodazole (500 ng/ml), vincristine (10 µM), or cytochalasin B (5 µg/ml) and were stimulated with MSUM crystals (1.5 mg/ml) for 1 h at 37°C. The reaction was stopped by centrifugation at 17,000 g for 15 s, and the supernatant was harvested. S100A8/A9 in the supernatant was analyzed by ELISA. Results are the mean ± SEM of four experiments from different donors. *, P < 0.05, and **, P < 0.01, compared with control, Dunnett multiple comparison test.
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Others [21 , 26 ] have reported secretion of S100A8/A9 from monocytes, but this secretion pathway remains mostly undefined. Although secretion of S100A8 and S100A9 by neutrophils has been suggested, the mechanism of their release is still unclear. A recent study suggested that S100A8/A9 is released following stimulation of neutrophils with fMLP or C5a [27 ]. Others have showed by immunohistochemistry that incubation of neutrophils with opsonized zymosan leads to the disappearance of S100A8 in neutrophil cytosol [40 ]. However, these studies relied on indirect methods to reveal the release of S100A8 and S100A8/A9 by neutrophils. Our results clearly show that neutrophils can specifically release S100A8/A9 in a dose- and time-dependent manner upon MSUM crystal stimulation. High concentrations of S100A8/A9 (10–5 M) were detected in MSUM-stimulated neutrophil supernatants. At these concentrations, S100A8/A9 stimulates neutrophil adhesion, and S100A8/A9 induces neutrophil chemotaxis at lower concentrations (10–10 M). High concentrations at the site of inflammation could thus contribute to the retention of neutrophils by enhancing their adhesion, and diffusion of S100 proteins from the inflammatory site could induce neutrophil chemotaxis.
Voganatsi et al. [41 ] associated the release of S100A8/A9 from Candida albicans-stimulated whole blood with the release of LDH, suggesting that S100A8/A9 release was a consequence of cellular lysis. However, it was unclear whether the same cells released LDH and S100A8/A9. In the present study, only traces of LDH were detected in the supernatant of neutrophils stimulated with MSUM crystals, confirming that the release of S100A8/A9 was not a consequence of cellular lysis. Furthermore, confocal microscopy showing the absence of propidium iodide staining in neutrophils provided clear evidence that the release of S100A8/A9 by MSUM-stimulated neutrophils was not the result of cellular lysis or temporary membrane disruption. Moreover, the inhibition of this release by specific inhibitors and the increase of this release when neutrophils are primed with GM-CSF provide further evidence for an active secretion. This result is in agreement with studies demonstrating that activation of neutrophils with MSUM crystals at concentrations as high as 3 mg/ml was not associated with cell necrosis as detected by trypan blue exclusion and LDH release [42 ].
Ruling out cell lysis as the cause of S100A8/A9 release, this prompted us to study its pathway of secretion. S100A8 and S100A9 do not possess a signal peptide to enable their secretion through the classic ER/Golgi/granule route used by enzymes such as gelatinase and cytokines, like IL-8, which would imply that they are secreted via an alternative pathway. It is plausible that as in phorbol 12-myristate 13-acetate-stimulated monocytes [26 ], the alternative secretory pathway used by S100A8/A9 in neutrophils involves tubulin polymerization. Indeed, vincristine and nocodazole, which promote microtubule depolymerization, impeded the release of S100A8/A9 following stimulation with MSUM crystals, which suggest that S100A8/A9 release is mediated by microtubule polymerization. Although the results suggest that S100A8/A9 release could be mediated in monocytes and neutrophils by a tubulin-dependent mechanism, tubulin polymerization inhibitors could also inhibit MSUM crystal-induced signal transduction [43 ]. Therefore, it is not possible to ascertain whether the effects of the tubulin polymerization inhibitors observed in the present study (that is, inhibition of S100A8/A9 release) are caused by inhibition within the secretory pathway or by inhibition of more upstream signaling events that promote secretion.
Despite numerous findings, the transduction signals leading to the activation of neutrophils by MSUM crystals remain mostly unknown. Nevertheless, tyrosine phosphorylation is known to play a critical role in MSUM crystal activation of numerous signaling pathways [44 , 45 ]. The Src family tyrosine kinase [32 , 46 ] as well as Syk [35 ] and PI-3K [47 , 48 ] are associated with MSUM crystal activation. Results presented here demonstrate the involvement of these molecules in the induction of S100A8/A9 release by MSUM crystal-stimulated neutrophils. It is unclear whether Src kinases, Syk, and PI-3K participate in a single or parallel signal-transduction pathways leading to S100A8/A9 release. Indeed, the lack of total inhibition by the specific inhibitors points toward parallel routes of transduction signal. However, the possibility that the inhibitors used did not completely inhibit their target kinases (possibly explaining the partial inhibition detected) cannot be ruled out. In addition, the order in which they are involved also remains unknown. Further studies are needed to answer these questions.
Neutrophil activation by MSUM crystals is thought to occur primarily through FcRIIIB (CD16) and Mac-1 (CD11b/CD18) [28
]. Here, we show that the same molecules are associated with the release of S100A8/A9 following activation with MSUM crystals. Occupation of CD11b and CD16 with specific antibodies inhibited the release of S100A8/A9 by 77%. Inhibitors of Src kinases, Syk, and PI-3K, which are also linked to CD11b and CD16 transduction pathways [49
50
51
52
53
54
55
56
], blocked neutrophil release of S100A8/A9 by approximately the same order of magnitude. These findings suggest that the release of S100A8/A9 by MSUM crystal-stimulated neutrophils could result from a direct activation of CD16 and CD11b. The lack of total inhibition also suggests the contribution of another as-yet unknown receptor using a different transduction pathway.
Several studies showed that the presence of neutrophils is necessary to induce an inflammatory reaction by MSUM crystals [57 , 58 ]. This implies that the proinflammatory factors released by neutrophils during MSUM stimulation are crucial for enhancing the inflammatory response. We previously demonstrated that S100A8 and S100A9 are essentials to neutrophil migration in response to MSUM crystals [6 ]. In this study, we demonstrate for the first time that neutrophils release the proinflammatory protein S100A8/A9. These results therefore further contribute to understanding the mechanisms leading to the inflammatory response associated with gout.
Received June 26, 2003; revised March 3, 2004; accepted March 17, 2004.
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RIIIB (CD16) and CD11b in response to inflammatory microcrystals FASEB J. 12,209-220 in inflammation Science 287,1049-1053 J. Biol. Chem. 275,23636-23641 RI activation of phospholipase C 1 and protein kinase C in dibutyryl cAMP-differentiated U937 cells is dependent solely on the tyrosine-kinase activated form of phosphatidylinositol-3-kinase Immunology 98,1-8[CrossRef][Medline] receptor IIA and Fc receptor IIIB signaling pathways in human neutrophils J. Immunol. 164,350-360 receptor signaling in macrophages and neutrophils Mol. Cell. Biol. 18,4209-4220 RIIa and Fc RIIIb, depends on the activity of different families of intracellular kinases Immunobiology 203,616-628[Medline]This article has been cited by other articles:
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