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Infectious Diseases Research Center, Laval University Hospital Center, Sainte-Foy, Quebec, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 integrins to an active conformation. This conformational change thought to be induced by lipid mediators such as platelet-activating factor or chemokines like IL-8, cause neutrophils to adhere strongly to the endothelium and extravasate. Once in the tissue, they follow concentration gradients of chemoattractants (such as C5a, leukotriene B4, and IL-8, or bacterial N-formylated peptides) toward the inflammatory site. Recent studies suggest that the myeloid-related proteins (MRPs)3 are involved in neutrophil migration. The MRPs S100A8 and S100A9 (10.6 and 13.5 kDa, respectively (2)) are calcium-binding proteins that belong to the S100 protein family (3, 4); they are expressed almost exclusively by cells of myeloid lineage. The MRPs are constitutively expressed in the cytosol of neutrophils where they comprise up to 30% of the cytosolic proteins (5). Monocytes and differentiated macrophages in inflamed tissues also express MRPs (4, 5, 6, 7, 8, 9). S100A8 and S100A9 form noncovalent homodimers and a heterodimer (S100A8/A9) in a calcium-dependent manner (10, 11, 12).
Local secretion of MRPs has been detected in chronic periodontal infections (13, 14) as well as in the serum of patients suffering from infections and inflammatory pathologies. High serum concentrations of MRPs have been found in advanced HIV infections, adult and juvenile rheumatoid arthritis, chronic bronchitis, cystic fibrosis, systemic lupus erythematosus, and granulomatous conditions, such as tuberculosis and sarcoidosis (10, 15, 16, 17, 18, 19, 20). S100A8/A9 is the main form found in the extracellular milieu and its in vitro release by neutrophils, activated monocytes, and macrophages has also been demonstrated (20, 21, 22, 23, 24, 25).
Extracellular S100A8, S100A9, and S100A8/A9 mediate regulatory and biological functions, including antiproliferative, antitumoral, antimicrobial, and antinociceptive activities (26, 27, 28, 29, 30, 31). Like other members of the S100 family (32, 33, 34), human MRPs and their murine homologs possess proinflammatory activities. However, there are disparities in the activities ascribed to these proteins pending a clear understanding of their activities in neutrophil migration to inflammatory sites.
Newton and Hogg (35) recently demonstrated that human S100A9 stimulates neutrophil adhesion to fibrinogen by activating the 2 integrin Mac-1 (CD11b/CD18). The promotion of this adhesion was negatively regulated by the formation of the heterocomplex with S100A8 implying that S100A8 as well as S100A8/A9 do not stimulate neutrophil adhesion (35). Eue et al. (36) also reported that S100A8 do not enhanced monocyte adhesion to endothelial cells. However, in contrast to the latter study, they demonstrated that not only S100A9 but also S100A8/A9 enhanced monocyte adhesion to endothelial cells via Mac-1/ICAM-1 interactions. These results suggest that S100A8 does not negatively regulate S100A9 activity by forming S100A8/A9.
Studies conducted using murine MRPs demonstrated that murine S100A8, called CP-10, is a potent chemotactic factor (activity at 10-13–10-11 M) for neutrophils (37), and induces sustained leukocyte recruitment in vivo (38, 39). Moreover, like human S100A9, murine S100A8 does not stimulate calcium flux, shedding of L-selectin, or up-regulation of the 2 integrin Mac-1 on neutrophils (35, 37, 40). In contrast to its murine homolog, human S100A8 is supposedly not chemotactic for neutrophils, but has been reported to be chemotactic for periodontal ligament cells (35, 41).
Therefore, the exact activities of S100A8 and S100A8/A9, particularly their effects on neutrophil migration, remain unknown. Harrison et al. (42) showed that murine S100A8 chemotactic activity can be efficiently inhibited by oxidation of the protein, demonstrating a certain susceptibility of S100A8 to inactivation. S100A9 activity appeared to be differently affected by oxidation (42). Therefore, the discrepancies in MRP activity could be due to differences in protein activity arising from interlaboratory variation in purification protocols or methodologies.
In this study, a different and gentle approach was used for the production and purification of the human S100A8, S100A9, and S100A8/A9 proteins. Recombinant proteins were generated and their proinflammatory properties toward neutrophils were characterized in vitro. S100A8, S100A9, and S100A8/A9 proved to be chemotactic for neutrophils in vitro and also stimulated shedding of L-selectin and up-regulation and activation of Mac-1. They also stimulated neutrophil adhesion to fibrinogen in a Mac-1-dependent manner. Neutrophil adhesion was also associated with an increase in intracellular calcium levels, but not with actin polymerization. Finally, injection of the human S100A8, S100A9, or S100A8/A9 into a murine air pouch led to a transient accumulation of neutrophils, therefore confirming their activity in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The fluorescent dyes fura 2-AM and calcein-AM were purchased from Calbiochem (San Diego, CA). ALEXA 488-conjugated goat anti-mouse specific Abs and SYPRO orange protein gel stain were purchased from Molecular Probes (Eugene, OR). LPS purified from Escherichia coli serotype O26 B6, fMLP, and fibrinogen were purchased from Sigma-Aldrich (St. Louis, MO). Anti-CD11b-, CD18-, and CD32-specific hybridomas OKM1, IB4, and IV.3, respectively, were obtained from the American Type Culture Collection (Manassas, VA) and were produced as purified ascites. Anti-CD51 (
v3) was obtained from Chemicon International (Temecula, CA). Digitonin was purchased from Calbiochem. FITC-phalloidin was purchased from Sigma-Aldrich. Anti-CD62L was purchased from BD PharMingen (San Diego, CA) and mAb 24 was a generous gift from Dr. N. Hogg (Imperial Cancer Research Fund, London, U.K.).
Recombinant proteins
Cloning, expression, and purification of human S100A8 and S100A9 was described previously (43). Briefly, human S100A8 and S100A9 (full-length) cDNA were cloned into the pET28 expression vector (Novagen, Madison, WI). Recombinant protein expression was induced with 1 mM isopropyl -D-thiogalactoside in E. coli HMS174 (Boehringer Mannheim, Mannheim, Germany) for 16 h at 16°C. After incubation, the bacteria were centrifuged at 5000 x g for 10 min and the pellet was resuspended in PBS/NaCl (0.5 M)/imidazole (1 mM) and lysed by sonication. The lysate was then centrifuged at 55,000 x g for 30 min at 4°C, and the supernatant was collected. Recombinant His-Tag S100A8 and S100A9 were purified using a nickel column. S100A8 or S100A9 bound to the column were freed from their His-Tag by incubation with 10 U of biotinylated thrombin (Novagen) for 20 h at room temperature. Recombinant S100A8 and S100A9 were eluted with PBS. The digestion and elution processes were repeated once to cleave the remaining undigested recombinant proteins, and streptavidin-agarose (Novagen) was added to remove the contaminating thrombin. Finally, the protein solutions were passed through a polymyxin B-agarose column (Pierce, Rockford, IL) to remove endotoxins. Control for bacterial contamination of the recombinant proteins consisted of untransformed HMS 174, stimulated, lysed, and processed for purification like the recombinant proteins. Experiments using this control were conducted using the same dilutions as for MRPs. Contamination by endotoxins were <1 pg/µg S100A8 or S100A9 protein as measured by the Limulus amebocyte assay (Sigma-Aldrich). The proteins were kept at -80°C for up to 2 mo until used.
Production of mAbs
Female BALB/c mice (4 wk old) were immunized by i.p. injections with 30 µg of purified recombinant S100A8 or S100A9 in 50 µl of endotoxin-free PBS (Sigma-Aldrich) mixed in an equal volume of CFA. Ab responses were enhanced by injections 14 days later with S100A8 or S100A9 using IFA, and final boost was given on day 28 with proteins alone. On day 31, spleen cells from the immunized mice were fused with SP2 murine myeloma cells and cultured in hypoxanthine/amethopterin/thymidine selection medium. Culture supernatants of the hybridomas were screened by ELISA using plates coated with 1 µg/ml recombinant proteins in 0.1 M carbonate buffer (pH 9.6). Positive hybridoma cells were cloned by limiting dilution. The mAb clones KVC/3E2 and KVC/2B9 showed the most distinctive recognition of the recombinant proteins S100A8 and S100A9, respectively. Both clones were isotyped as IgG1 kappa. Specificity of the mAbs was confirmed by ELISA and Western blot analysis.
Neutrophil purification
Peripheral blood was collected in heparinized tubes from healthy adult volunteers. Neutrophils were isolated as previously described by Boyum (44) and resuspended in HBSS-H (HBSS supplemented with 10 mM HEPES (pH 7.4)) containing 1.3 mM Ca2+ and 0.8 mM Mg2+. The purity and cell viability of neutrophil preparations were consistently >98% as assessed by acetic blue staining and trypan blue exclusion, respectively.
Chemotaxis assays
Neutrophil chemotaxis assays were performed in modified Boyden chambers. Increasing concentrations of MRPs or 10-8 M fMLP diluted in HBSS-H containing 1.3 mM Ca2+ and 0.8 mM Mg2+ was added to the lower well of the chambers. Polycarbonate filters (8-µm pore; Neuroprobe, Gaithersburg, MD) were put in place and the upper chambers were secured. Neutrophils (5 x 104 cells in 200 µl) were added to the upper chambers and allowed to migrate through the membrane for 30 min at 37°C. The upper chambers were washed once with PBS, and the membrane was then fixed in methanol and stained with Wright-Giemsa. Cells that had migrated through the membrane were counted using a microscope in five high-power fields. The results are expressed as mean number of migrated cells per high-power field. Directional migration was confirmed by adding increasing concentrations of MRPs to the upper wells. In separate experiments, 10 µg/ml anti-S100A8 and/or anti-S100A9 were added to the lower chambers before addition of neutrophils to the upper chamber.
Flow cytometry analysis
Neutrophils (2 x 106 cells/ml) were stimulated with MRPs (2 x 10-6 M) at 37°C for 30 min. For the detection of CD62L, CD11b, and CD51 expression, neutrophils (4 x 105) were incubated with 1 µg of CD62L-, CD11b-, or CD51-specific Abs, respectively, on ice for 25 min. After two washes in PBS/0.2% BSA/0.1% azide, neutrophils were incubated with ALEXA 488-conjugated goat anti-mouse specific Ab for 25 min on ice, washed twice, and fixed in PBS containing 2% formaldehyde. CD18 activation was detected by adding 1 µg of mAb 24 to 5 x 105 neutrophils after 20-min stimulation with MRPs and incubating at 37°C for 10 min. Cells were washed twice with PBS/0.2% BSA/0.1% azide and incubated with ALEXA 488-conjugated goat anti-mouse specific Ab for 45 min at RT. After two washes, neutrophils were fixed in PBS containing 2% formaldehyde. Detection was done with a FACSCalibur (BD Biosciences, Mountain View, CA) flow cytometer.
Adhesion
Neutrophils were resuspended at 5 x 106 cells/ml in HBSS-H containing 1.3 mM Ca2+ and 0.8 mM Mg2+ and labeled with 5 µM intracellular fluorescent dye calcein-AM for 30 min at 37°C. After washing, labeled cells were resuspended in HBSS-H containing 1.3 mM Ca2+ and 0.8 mM Mg2+ at 5 x 106 cells/ml, and 50 µl was added to Costar 96-well plates, which had been coated overnight with 2 mg/ml fibrinogen (50 µl/well). These wells contained 50 µl of increasing concentrations (2x) of human S100A8, S100A9, S100A8/A9, equal volumes of mock protein purification solution, or 10-7 M fMLP. Neutrophils were allowed to adhere for 30 min at 37°C before being washed three times by immersion in cold PBS. The adherent cells were lysed by adding ddH2O, and the fluorescence was measured using a 96-well plate fluorescence reader. In some experiments, S100A8, S100A9, S100A8/A9, or 10-7 M fMLP were boiled or treated at room temperature with 10 or 100 µM NaOCl for 15 min and diluted to a final concentration of 20 µg/ml before stimulation. In other experiments, neutrophils were preincubated for 20 min with 20 µg/ml Abs directed against CD18, CD51, or CD32 (negative control) before being added to the wells.
Actin polymerization
Neutrophils were resuspended at a concentration of 2 x 106 cells/ml in HBSS-H containing 1.3 mM Ca2+ and 0.8 mM Mg2+. The reaction was initiated by mixing 5 x 104 cells (250 µl) with the MRP preparations at final concentrations of 1 x 10-6–5 x 10-13 M. After increasing periods of incubation at RT, the reaction was stopped by adding 50 µl of 3.7% formaldehyde and placed immediately on ice. The cells were permeabilized, and the F-actin was stained for 60 min on ice with a mixture of 0.05% (w/v) digitonin and 2 µM FITC-phalloidin. Cells were then pelleted and washed twice in HBSS-H and resuspended in HBSS-H. The fluorescence was measured by flow cytometry on a FACSCalibur (BD Biosciences).
Intracellular calcium measurements
Neutrophils (107 cells/ml) were loaded with fura 2-AM (1 µM; 30 min at 37°C), washed, and resuspended at 2 x 106 cells/ml in HBSS-H supplemented with 1.3 mM CaCl2. The fluorescence was measured in a spectrofluorometer (SLM 8000; SLM-Aminco, Urbana, IL) in presence of fMLP (10-7 M) or increasing concentrations of human S100A8, S100A9, or S100A8/A9 at excitation and emission wavelengths of 340 and 510 nm, respectively. The internal calcium concentrations were calculated as described by Tsien et al. (45).
Air pouch experiments
Ten- to 12-wk-old CD-1 and C3H/HeJ mice were obtained from Charles River (St. Columban, Quebec, Canada) and The Jackson Laboratory (Bar Harbor, ME), respectively. Air pouches were raised on the dorsum by s.c. injection of 3 ml of sterile air on days 0 and 3. On day 7, 1 ml of S100A8, S100A9, or an equimolar ratio of S100A8 and S100A9 dissolved in endotoxin-free PBS at concentrations ranging from 0.1 to 10 µg/ml or LPS (1 µg/ml) was injected into the air pouches. At specific time points, the mice were killed by CO2 asphyxiation, 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 500 x g for 5 min at room temperature. Cells were counted with a hematocytometer following acetic blue staining. Characterization of leukocyte subpopulations migrating into the pouch space was performed by Wright-Giemsa staining of cytospins.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, discrepancies exist between the activities of S100A8, S100A9, and S100A8/A9. These differences could be consecutive to improper folding or inactivation of the proteins during purification. To alleviate these problems, His-tagged recombinant proteins were generated and produced in E. coli using a pEt28 expression vector. Production yields were kept low by inducing protein synthesis at 16°C to maximize proper folding, and the purification process was performed at physiological pH, in absence of potentially denaturing reagents such as DTT and imidazole. In addition, the purification protocol did not include any unfolding-refolding steps. Proteins with only three extra N-terminal amino acids (Gly-Ser-His-Met- ...) were generated after thrombin cleavage of the polyhistidine tail.
Recombinant MRPs were first analyzed on SDS-PAGE gels and stained with SYPRO orange (sensitivity limit of 4–8 ng/band). As shown in Fig. 1A, S100A8 and S100A9 produced using this protocol migrated to their expected molecular masses (8 and 14 kDa, respectively) and were free of contamination from bacteria-derived proteins and thrombin. These results also confirmed the absence of thrombin-cleaved products of S100A8 or S100A9. Analysis of S100A8 and S100A9 on nonreducing gels revealed the presence of monomers and homodimers (Fig. 1B). As expected, S100A8 and S100A9 also formed the heterodimer S100A8/A9 when mixed together (Fig. 1B). Although we observed variability in the relative concentrations of monomers, homodimers, and heterodimers, these concentrations were similar to the ones observed by other laboratories for native and recombinant human or murine proteins (11, 42, 46). The presence of monomers might result from the dissociation of homodimers and heterodimers caused by SDS in the nonreducing gels.
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Recombinant MRP activities were first evaluated in vitro in chemotaxis assays using modified Boyden chambers. S100A8, S100A9, and S100A8/A9 proved to be chemotactic for neutrophils at concentrations varying from 10-12–10-10 M (Fig. 2, A–C). Neutrophil migration was inhibited by adding mAbs against S100A8 and/or S100A9 to the bottom chambers (Fig. 2, D–F), demonstrating that the migration detected was consecutive to activation of neutrophils by the recombinant S100 proteins. To confirm that cell migration was the result of induced directional migration, increasing concentrations of MRPs were added to the upper chambers. As shown in Tables I, II and III, addition of the same proteins to the upper chambers significantly reduced neutrophil migration through the membranes, confirming that S100A8, S100A9, and S100A8/A9 induced neutrophil chemotaxis. A small increased migration of neutrophils has also been observed when 1 x 10-11 M MRPs were added to the upper chambers, suggesting that they also stimulated random migration of neutrophils.
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S100A9 and S100A8/A9 had previously been shown to stimulate neutrophil adhesion (35, 36). To verify whether our recombinant MRPs also stimulated adhesion, neutrophils were incubated with S100A8, S100A9, and S100A8/A9, and the level of expression of the adhesion molecules CD62L (L-selectin), CD11b (Mac-1), and CD51 (v3) on the cell surface was examined. In addition, the state of activation of Mac-1 was evaluated using the activation reporter mAb 24. As shown in Fig. 3, MRPs induced shedding of L-selectin (CD62L) from the neutrophil surface. In contrast, expression of CD11b, but not CD51 was up-regulated following stimulation with MRPs. As Mac-1 is stored in specific and gelatinase granules, this suggests that MRPs induced neutrophil degranulation. Stimulation with MRPs also led to the change of 2 integrin conformation to an active form, as reported by the activation reporter mAb 24.
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The effect of MRPs on the expression of adhesion molecule on neutrophils suggested that they modify neutrophil adhesion. Thus, we examined neutrophil adhesion to fibrinogen following stimulation with MRPs. Stimulation of neutrophil adhesion by S100A8/A9 was comparable to that of fMLP while the same concentrations of S100A8 and S100A9 stimulated a weaker adhesion (Fig. 4). Maximal adhesion was observed between 10-7 and 10-6 M S100A8/A9 and at 10-6 M S100A8 or S100A9. This effect was inhibited by >80% when the proteins were heat inactivated (Fig. 5A). Boiling fMLP did not reduce its activity; thus our results were not confounded by the proteins being contaminated with formyl peptides. Moreover, stimulation of neutrophils with a mock protein preparation did not induce their adhesion (Fig. 5B), ruling out the possibility that the activities attributed to the MRPs were a consequence of bacterial or thrombin contamination.
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MRP-stimulated neutrophil adhesion to fibrinogen is mediated by Mac-1 but not V3
Mac-1 (CD11b/CD18) and V3 (CD51/CD61) are two integrins expressed by neutrophils known to bind fibrinogen (48, 49). To study the mechanism of MRP-induced neutrophil adhesion, their role in neutrophil adhesion to fibrinogen was evaluated. Specific Abs directed against CD18 blocked MRP-stimulated and unstimulated neutrophil adhesion to fibrinogen. In contrast, the anti-CD51 Ab did not diminish neutrophil adhesion. CD32 Ab, used as negative control, did not influence neutrophil adhesion either (Fig. 6). These results demonstrate that MRP-stimulated neutrophil adhesion to fibrinogen is mediated by the 2 integrin Mac-1.
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To evaluate the level of activation of neutrophils, the effect of MRPs on actin polymerization was next investigated. fMLP induced a rapid and transient neutrophil actin polymerization at a concentration of 10-7 M. However, S100A8, S100A9, and S100A8/A9 did not induce significant neutrophil actin polymerization (Fig. 7). This contrasts with previously reported observations indicating that murine neutrophils stimulated with murine S100A8 had increased actin polymerization (37). These results indicate that, unlike classical chemotactic factors, the human MRPs possess restricted activating activities toward neutrophils.
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Calcium mobilization is also frequently associated with neutrophil adhesion and chemotaxis. Because murine S100A8 does not induce calcium mobilization in neutrophils (37) and S100A12 induces calcium mobilization in monocytoid cells, but not in neutrophils (Ref.50 ; M. Talbot and P. A. Tessier, unpublished observations), we investigated the effect of MRPs on calcium mobilization. The effect of S100A8, S100A9, and S100A8/A9 on calcium mobilization in human neutrophils was next investigated. Human neutrophils had resting Ca2+ levels of 100 nM. Neutrophil stimulation with fMLP (10-8 M) led to a transient increase in Ca2+ (Fig. 8A). Stimulation of neutrophils with S100A8, S100A9, and S100A8/A9 also increased calcium levels, although not as high as fMLP (Fig. 8, B, C, and D). This mobilization was observed at concentrations of 10-6–10-7 M but not at lower concentrations of MRPs.
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To determine the biological activities of the MRPs, S100A8 and S100A9 were injected into the murine air pouch and cells migrating to the air pouch environment were quantified. Injection of both S100A8 and S100A9 led to the accumulation of leukocytes. From 90 to 95% of the migrating leukocytes were neutrophils (Fig. 9A). Neutrophils were recruited to the air pouch within 3 h postinjection. The accumulation was maximal between 6 and 12 h postinjection, dropping to control levels by 24 h (Fig. 9A). Monocytes were also recruited to the air pouch, with maximum migration observed at 12 h postinjection (Fig. 9B). No significant migration was detected in PBS- or mock protein preparation-injected mice (data not shown), suggesting that these activities are not caused by contamination of the proteins with bacteria-derived Ni-binding proteins or thrombin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To date, the proinflammatory activities of the MRPs remained elusive. Human S100A9 has been reported to stimulate neutrophil adhesion to fibrinogen, but its activity was negatively regulated by forming the heterocomplex S100A8/A9 with S100A8 (35). Another study recently demonstrated that S100A9, S100A8/A9, but not S100A8, enhanced monocyte adhesion to endothelial cells, indicating that S100A8 does not negatively regulate S100A9 activity toward monocytes by forming S100A8/A9 (36). In addition, S100A8/A9 was also associated with monocyte recruitment by facilitating their migration through the endothelium (36). In these reports, S100A8 had no activity toward neutrophils and monocytes. The only activities attributed to human S100A8 were its capacity to activate HIV replication in human monocytic infected cells (52) and its chemotactic activity toward periodontal ligament cells (41). In addition, we recently demonstrated that S100A8 stimulates HIV-1 transcription and production in infected T lymphocytes through NF-
B activation (43). Therefore, it was unclear whether S100A8 exerts proinflammatory activities by itself. In this study, we demonstrate that human S100A8 possess proinflammatory activities toward neutrophils. We show that recombinant S100A9, S100A8/A9, and also S100A8 stimulate human neutrophil chemotaxis, adhesion, and other proinflammatory activities, proving that they all exert activities toward neutrophils.
Murine S100A8 and, to a lower level, S100A9 can be efficiently oxidized by OCl- anions and copper, sometimes resulting in the inactivation of S100A8 chemotactic activity (42, 48). In this study, we present similar results with human S100A8 and demonstrate that S100A8 but not S100A9 or S100A8/A9 can be inactivated by oxidation with NaOCl. These results demonstrate that human S100A8 possesses a similar susceptibility toward inactivation by oxidation as does its murine counterpart. This result suggests that the absence of S100A8 activity described by others might be due to oxidation and consequent deactivation of the protein during purification. In addition, the activities for S100A9 and S100A8/A9 reported by others could be explained by their relative resistance to inactivation by oxidation. Heat denaturation, which consists of unfolding and refolding of the proteins, also inactivates the proteins. The presence of denaturing agents such as DTT or imidazole during the purification process could therefore result in their inactivation. Thus, we propose that the discrepancies in MRPs activities could have resulted from interlaboratory variance in methodology, in particular protein oxidation or denaturation during the purification process or during the manipulation of the proteins, which could be responsible for the inactivation of the MRPs.
In the present study, S100A8, S100A9, and S100A8/A9 proved to be potent chemoattractants for neutrophils at concentrations of 10-12–10-10 M. Their chemotactic activities are similar to those of another MRP, S100A12, which is chemotactic for neutrophils at a concentration of 10-9 M (50). Murine S100A8 is also chemotactic for neutrophils but induces chemotaxis at lower concentrations (10-13–10-11 M) (37). The chemotactic activity of the S100 proteins is not restricted to neutrophils. Murine S100A8 is also chemotactic for mononuclear cells but not lymphocytes (38, 39, 53). In addition, S100A12 is chemotactic for monocytes and THP-1 cells (50), while another S100 protein, S100A7, is chemotactic for neutrophils and CD4+ T lymphocytes (34). Finally, S100L (S100A2) is chemotactic toward eosinophils (33). The chemotactic activity shared by several S100 proteins suggests that this family of proteins might represent a new group of chemotactic factors.
MRPs proved not only to stimulate chemotaxis of neutrophils but also to induce neutrophil adhesion and calcium mobilization. However, not all MRPs have the same activating potential toward neutrophils. Although all MRPs induced neutrophil chemotaxis at relatively similar concentrations, S100A8/A9 stimulated neutrophil adhesion at lower concentration than did S100A9 or S100A8, the latter being a less potent inducer. In addition, the activities of the MRPs varied according to their concentration. At lower concentrations, MRPs were chemotactic for neutrophils, while they stimulated neutrophil adhesion at higher concentrations. This suggests that MRPs could attract neutrophils at low concentrations, while higher concentrations could help retain them at the inflammatory site. Surprisingly, neutrophil migration stimulated by MRPs occurred without increases in polymerized actin content. However, actin cytoskeleton reorganization might occur following stimulation with S100 proteins while the total amount of polymerized actin remained unchanged.
MRPs have been described in several infections and immune diseases including tuberculosis, bronchitis, and arthritis (10, 15, 16, 17, 18, 19, 20), suggesting a role for these proteins in the inflammatory process. Unpublished observations from our laboratory demonstrate that neutrophil recruitment induced by monosodium urate crystals, the etiologic agent of gout, is inhibited when both S100A8 and S100A9 are inactivated by passive immunization of mice with specific anti-S100A8 and anti-S100A9 Abs (C. Ryckman, K. Vandal, and P.A. Tessier, manuscript in preparation). These results confirm the importance of S100A8, S100A9, and S100A8/A9 in neutrophil migration to inflammatory sites in vivo. Stimulation of neutrophil chemotaxis and adhesion by S100A8, S100A9, and S100A8/A9, corroborates MRP’s implication in neutrophil localization, adhesion, and transendothelial migration to inflammatory sites, shedding new light on the role of MRPs in neutrophil migration during inflammation.
The current paradigm of leukocyte migration suggests that, following selectin-induced rolling, neutrophils are activated by chemokines, resulting in a change of conformation of 2 integrins to their active conformation. This results in neutrophil strong adhesion to the endothelium and allows the transendothelial migration to occur. Leukocytes are then guided to the inflammatory site by a gradient of chemotactic factors. Chemokines have been suggested to act as activators of both neutrophil adhesion to endothelium and chemotaxis in the tissue. The results presented in this study suggest that MRPs could play the same role. S100A8 and S100A9 have been detected on the endothelium in inflamed tissues (54), and S100A9 has been shown to bind specifically to 2-macroglobulin on human endothelial cells (55), suggesting that they could induce neutrophil strong adhesion to the endothelium. In addition, the presence of S100A8/A9 has been described at sites of inflammation, suggesting that it could direct neutrophil migration in the tissue as well. Therefore, the MRPs could exert relatively the same activities as chemokines. Further studies will be necessary to confirm these hypotheses.
In conclusion, the present study demonstrates that S100A8, S100A9, and S100A8/A9 are potent inducers of neutrophil chemotaxis and adhesion. Together with other S100 proteins such as S100A12, S100A7 and S100A2, they could represent a new class of chemotactic factors contributing to neutrophil migration to inflammatory sites.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Philippe A. Tessier, Room RC 709, Infectious Diseases Research Center, Laval University Hospital Center, 2705 Laurier Boulevard, Sainte-Foy, Quebec, Canada G1V 4G2. E-mail address: Philippe.Tessier{at}crchul.ulaval.ca
3 Abbreviation used in this paper: MRP, myeloid-related protein.
Received for publication April 26, 2002. Accepted for publication January 3, 2003.
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References |
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induction of the proinflammatory cytokine interleukin-6 in neurons. J. Neurochem. 74:143.[Medline]
2 integrin Mac-1 on neutrophils. J. Immunol. 160:1427.v3 requires tyrosine 178 in the v subunit. Blood 97:175.This article has been cited by other articles:
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  J. Sharma, Q. Li, B. B. Mishra, M. J. Georges, and J. M. Teale Vaccination with an Attenuated Strain of Francisella novicida Prevents T-Cell Depletion and Protects Mice Infected with the Wild-Type Strain from Severe Sepsis Infect. Immun., October 1, 2009; 77(10): 4314 - 4326. [Abstract] [Full Text] [PDF]
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L. Reyes, S. Alvarez, A. Allam, M. Reinhard, and M. B. Brown Complicated Urinary Tract Infection Is Associated with Uroepithelial Expression of Proinflammatory Protein S100A8 Infect. Immun., October 1, 2009; 77(10): 4265 - 4274. [Abstract] [Full Text] [PDF]
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 S. Y. Lim, M. J. Raftery, J. Goyette, K. Hsu, and C. L. Geczy Oxidative modifications of S100 proteins: functional regulation by redox J. Leukoc. Biol., September 1, 2009; 86(3): 577 - 587. [Abstract] [Full Text] [PDF]
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J. Sharma, Q. Li, B. B. Mishra, C. Pena, and J. M. Teale Lethal pulmonary infection with Francisella novicida is associated with severe sepsis J. Leukoc. Biol., September 1, 2009; 86(3): 491 - 504. [Abstract] [Full Text] [PDF]
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 K. Croce, H. Gao, Y. Wang, T. Mooroka, M. Sakuma, C. Shi, G. K. Sukhova, R. R.S. Packard, N. Hogg, P. Libby, et al. Myeloid-Related Protein-8/14 Is Critical for the Biological Response to Vascular Injury Circulation, August 4, 2009; 120(5): 427 - 436. [Abstract] [Full Text] [PDF]
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 P L E M van Lent, L Grevers, A B Blom, A Sloetjes, J S Mort, T Vogl, W Nacken, W B van den Berg, and J Roth Myeloid-related proteins S100A8/S100A9 regulate joint inflammation and cartilage destruction during antigen-induced arthritis Ann Rheum Dis, December 1, 2008; 67(12): 1750 - 1758. [Abstract] [Full Text] [PDF]
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 G. Bode, A. Luken, C. Kerkhoff, J. Roth, S. Ludwig, and W. Nacken Interaction between S100A8/A9 and Annexin A6 Is Involved in the Calcium-induced Cell Surface Exposition of S100A8/A9 J. Biol. Chem., November 14, 2008; 283(46): 31776 - 31784. [Abstract] [Full Text] [PDF]
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 P. Sinha, C. Okoro, D. Foell, H. H. Freeze, S. Ostrand-Rosenberg, and G. Srikrishna Proinflammatory S100 Proteins Regulate the Accumulation of Myeloid-Derived Suppressor Cells J. Immunol., October 1, 2008; 181(7): 4666 - 4675. [Abstract] [Full Text] [PDF]
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P. Pouliot, I. Plante, M.-A. Raquil, P. A. Tessier, and M. Olivier Myeloid-Related Proteins Rapidly Modulate Macrophage Nitric Oxide Production during Innate Immune Response J. Immunol., September 1, 2008; 181(5): 3595 - 3601. [Abstract] [Full Text] [PDF]
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 K. F. Chung and I. M. Adcock Multifaceted mechanisms in COPD: inflammation, immunity, and tissue repair and destruction Eur. Respir. J., June 1, 2008; 31(6): 1334 - 1356. [Abstract] [Full Text] [PDF]
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 L. J. Quinton, M. R. Jones, B. E. Robson, B. T. Simms, J. A. Whitsett, and J. P. Mizgerd Alveolar Epithelial STAT3, IL-6 Family Cytokines, and Host Defense during Escherichia coli Pneumonia Am. J. Respir. Cell Mol. Biol., June 1, 2008; 38(6): 699 - 706. [Abstract] [Full Text] [PDF]
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 G. Z. Feuerstein Cardiac RAGE in Sepsis: Call TOLL Free for Anti-RAGE Circ. Res., May 23, 2008; 102(10): 1153 - 1154. [Full Text] [PDF]
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J. H. Boyd, B. Kan, H. Roberts, Y. Wang, and K. R. Walley S100A8 and S100A9 Mediate Endotoxin-Induced Cardiomyocyte Dysfunction via the Receptor for Advanced Glycation End Products Circ. Res., May 23, 2008; 102(10): 1239 - 1246. [Abstract] [Full Text] [PDF]
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M.-A. Raquil, N. Anceriz, P. Rouleau, and P. A. Tessier Blockade of Antimicrobial Proteins S100A8 and S100A9 Inhibits Phagocyte Migration to the Alveoli in Streptococcal Pneumonia J. Immunol., March 1, 2008; 180(5): 3366 - 3374. [Abstract] [Full Text] [PDF]
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 M. Sakaguchi, H. Sonegawa, H. Murata, M. Kitazoe, J.-i. Futami, K. Kataoka, H. Yamada, and N.-h. Huh S100A11, an Dual Mediator for Growth Regulation of Human Keratinocytes Mol. Biol. Cell, January 1, 2008; 19(1): 78 - 85. [Abstract] [Full Text] [PDF]
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 B. J. McMorran, S. A. Ouvry Patat, J. B. Carlin, K. Grimwood, A. Jones, D. S. Armstrong, J. C. Galati, P. J. Cooper, C. A. Byrnes, P. W. Francis, et al. Novel Neutrophil-Derived Proteins in Bronchoalveolar Lavage Fluid Indicate an Exaggerated Inflammatory Response in Pediatric Cystic Fibrosis Patients Clin. Chem., October 1, 2007; 53(10): 1782 - 1791. [Abstract] [Full Text] [PDF]
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 D. Stygar, B. Masironi, H. Eriksson, and L. Sahlin Studies on estrogen receptor (ER) {alpha} and {beta} responses on gene regulation in peripheral blood leukocytes in vivo using selective ER agonists J. Endocrinol., July 1, 2007; 194(1): 101 - 119. [Abstract] [Full Text] [PDF]
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K. Boniface, C. Diveu, F. Morel, N. Pedretti, J. Froger, E. Ravon, M. Garcia, E. Venereau, L. Preisser, E. Guignouard, et al. Oncostatin M Secreted by Skin Infiltrating T Lymphocytes Is a Potent Keratinocyte Activator Involved in Skin Inflammation J. Immunol., April 1, 2007; 178(7): 4615 - 4622. [Abstract] [Full Text] [PDF]
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H. Y. Sroussi, J. Berline, and J. M. Palefsky Oxidation of methionine 63 and 83 regulates the effect of S100A9 on the migration of neutrophils in vitro J. Leukoc. Biol., March 1, 2007; 81(3): 818 - 824. [Abstract] [Full Text] [PDF]
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D. Foell, H. Wittkowski, T. Vogl, and J. Roth S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules J. Leukoc. Biol., January 1, 2007; 81(1): 28 - 37. [Abstract] [Full Text] [PDF]
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K. J. Greenlee, D. B. Corry, D. A. Engler, R. K. Matsunami, P. Tessier, R. G. Cook, Z. Werb, and F. Kheradmand Proteomic Identification of In Vivo Substrates for Matrix Metalloproteinases 2 and 9 Reveals a Mechanism for Resolution of Inflammation J. Immunol., November 15, 2006; 177(10): 7312 - 7321. [Abstract] [Full Text] [PDF]
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 H.Y. Sroussi, J. Berline, P. Dazin, P. Green, and J.M. Palefsky S100A8 Triggers Oxidation-sensitive Repulsion of Neutrophils Journal of Dental Research, September 1, 2006; 85(9): 829 - 833. [Abstract] [Full Text] [PDF]
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R. R. Montgomery, K. Schreck, X. Wang, and S. E. Malawista Human Neutrophil Calprotectin Reduces the Susceptibility of Borrelia burgdorferi to Penicillin Infect. Immun., April 1, 2006; 74(4): 2468 - 2472. [Abstract] [Full Text] [PDF]
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 J. D. Graham, M. L. Yager, H. D. Hill, K. Byth, G. M. O'Neill, and C. L. Clarke Altered Progesterone Receptor Isoform Expression Remodels Progestin Responsiveness of Breast Cancer Cells Mol. Endocrinol., November 1, 2005; 19(11): 2713 - 2735. [Abstract] [Full Text] [PDF]
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M. Srivastava, S. Jung, J. Wilhelm, L. Fink, F. Buhling, T. Welte, R. M. Bohle, W. Seeger, J. Lohmeyer, and U. A. Maus The Inflammatory versus Constitutive Trafficking of Mononuclear Phagocytes into the Alveolar Space of Mice Is Associated with Drastic Changes in Their Gene Expression Profiles J. Immunol., August 1, 2005; 175(3): 1884 - 1893. [Abstract] [Full Text] [PDF]
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J. Abe, T. Jibiki, S. Noma, T. Nakajima, H. Saito, and M. Terai Gene Expression Profiling of the Effect of High-Dose Intravenous Ig in Patients with Kawasaki Disease J. Immunol., May 1, 2005; 174(9): 5837 - 5845. [Abstract] [Full Text] [PDF]
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 A. Astolfi, L. Landuzzi, G. Nicoletti, C. De Giovanni, S. Croci, A. Palladini, S. Ferrini, M. Iezzi, P. Musiani, F. Cavallo, et al. Gene Expression Analysis of Immune-Mediated Arrest of Tumorigenesis in a Transgenic Mouse Model of HER-2/neu-Positive Basal-Like Mammary Carcinoma Am. J. Pathol., April 1, 2005; 166(4): 1205 - 1216. [Abstract] [Full Text] [PDF]
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K. Boniface, F.-X. Bernard, M. Garcia, A. L. Gurney, J.-C. Lecron, and F. Morel IL-22 Inhibits Epidermal Differentiation and Induces Proinflammatory Gene Expression and Migration of Human Keratinocytes J. Immunol., March 15, 2005; 174(6): 3695 - 3702. [Abstract] [Full Text] [PDF]
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K. Hsu, R. J. Passey, Y. Endoh, F. Rahimi, P. Youssef, T. Yen, and C. L. Geczy Regulation of S100A8 by Glucocorticoids J. Immunol., February 15, 2005; 174(4): 2318 - 2326. [Abstract] [Full Text] [PDF]
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C. Cataisson, A. J. Pearson, S. Torgerson, S. A. Nedospasov, and S. H. Yuspa Protein Kinase C{alpha}-Mediated Chemotaxis of Neutrophils Requires NF-{kappa}B Activity but Is Independent of TNF{alpha} Signaling in Mouse Skin In Vivo J. Immunol., February 1, 2005; 174(3): 1686 - 1692. [Abstract] [Full Text] [PDF]
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M. Jaramillo, M. Godbout, P. H. Naccache, and M. Olivier Signaling Events Involved in Macrophage Chemokine Expression in Response to Monosodium Urate Crystals J. Biol. Chem., December 10, 2004; 279(50): 52797 - 52805. [Abstract] [Full Text] [PDF]
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C. Ryckman, C. Gilbert, R. de Medicis, A. Lussier, K. Vandal, and P. A. Tessier Monosodium urate monohydrate crystals induce the release of the proinflammatory protein S100A8/A9 from neutrophils J. Leukoc. Biol., August 1, 2004; 76(2): 433 - 440. [Abstract] [Full Text] [PDF]
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P. B. Sugerman, S. B. Faber, L. M. Willis, A. Petrovic, G. F. Murphy, J. Pappo, D. Silberstein, and M. R. M. van den Brink Kinetics of Gene Expression in Murine Cutaneous Graft-versus-Host Disease Am. J. Pathol., June 1, 2004; 164(6): 2189 - 2202. [Abstract] [Full Text] [PDF]
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M. Jaramillo, I. Plante, N. Ouellet, K. Vandal, P. A. Tessier, and M. Olivier Hemozoin-Inducible Proinflammatory Events In Vivo: Potential Role in Malaria Infection J. Immunol., March 1, 2004; 172(5): 3101 - 3110. [Abstract] [Full Text] [PDF]
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J. Roth, T. Vogl, C. Sunderkotter, and C. Sorg Chemotactic activity of S100A8 and S100A9. J. Immunol., December 1, 2003; 171(11): 5651 - 5651. [Full Text] [PDF]
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K. Vandal, P. Rouleau, A. Boivin, C. Ryckman, M. Talbot, and P. A. Tessier Blockade of S100A8 and S100A9 Suppresses Neutrophil Migration in Response to Lipopolysaccharide J. Immunol., September 1, 2003; 171(5): 2602 - 2609. [Abstract] [Full Text] [PDF]
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