Monosodium urate monohydrate crystals induce the release of the proinflammatory protein S100A8/A9 from neutrophils.

The neutrophil cytoplasmic protein S100A8/A9 (along with S100A8 and S100A9) is chemotactic and stimulates neutrophil adhesion by activating the beta2-integrin CD11b/CD18. It is also essential to neutrophil migration in vivo in response to monosodium urate monohydrate (MSUM) crystals, the principal etiologic agent of gout. S100A8/A9 is present in the synovial fluid of patients with gout and arthritis and is secreted by activated monocytes; however, its mechanism of release by neutrophils remains unknown. The aim of this study was to identify the mechanism of stimulation of the release of S100A8/A9 by MSUM-activated neutrophils. Here, we show that S100A8/A9 is released by neutrophils stimulated with MSUM crystals and that this release could be enhanced by preincubating neutrophils with granulocyte macrophage-colony stimulating factor. Antibodies directed against CD11b and CD16 blocked the release induced by MSUM crystals, suggesting that Fc receptor for immunoglobulin G (FcgammaR)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.

Role of S100A8 and S100A9 in neutrophil recruitment in response to monosodium urate monohydrate crystals in the air-pouch model of acute gouty arthritis.

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.

Blockade of S100A8 and S100A9 suppresses neutrophil migration in response to lipopolysaccharide.

Recently, proinflammatory activities had been described for S100A8 and S100A9, two proteins found at inflammatory sites and within the neutrophil cytoplasm. In this study, we investigated the role of these proteins in neutrophil migration in vivo in response to LPS. LPS was injected into the murine air pouch, which led to the release of S100A8, S100A9, and S100A8/A9 in the pouch exudates that preceded accumulation of neutrophils. Passive immunization against S100A8 and S100A9 led to a 52% inhibition of neutrophil migration in response to LPS at 3 h postinjection. Injection of LPS was also associated with an increase in peripheral blood neutrophils and the presence in serum of S100A9 and S100A8/A9. Intravenous injection of S100A8, S100A9, or S100A8/A9 augmented the number of circulating neutrophils and diminished the number of neutrophils in the bone marrow, demonstrating that S100A8 and S100A9 induced the mobilization of neutrophils from the bone marrow to the blood. Finally, passive immunization with anti-S100A9 inhibited the neutrophilia associated with LPS injection in the air pouch. These results suggest that S100A8 and S100A9 play a role in the inflammatory response to LPS by inducing the release of neutrophils from the bone marrow and directing their migration to the inflammatory site.

The calcium-binding protein S100A12 induces neutrophil adhesion, migration, and release from bone marrow in mouse at concentrations similar to those found in human inflammatory arthritis.

We investigated the proinflammatory activities of S100A12 in the context of synovial inflammation. S100A12 levels were increased in the synovial fluids and plasma of patients with gout, rheumatoid arthritis, psoriatic arthritis, and undetectable in osteoarthritis, a noninflammatory disorder. S100A12 proved to induce neutrophil adhesion to fibrinogen via Mac-1 at concentrations similar to those found in the synovial fluids. Similar concentrations induced the recruitment of large numbers of neutrophils and monocytes in the murine air pouch model. To characterize the effect of increased S100A12 plasma levels, mice were injected intravenously with S100A12. This led to the mobilization of neutrophils from the bone marrow to the peripheral blood. These results suggest that S100A12 stimulates the accumulation of neutrophil by inducing their release from the bone marrow, as well as by activating their adhesion and migration toward inflammatory sites.

Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion.

S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that S100A8, S100A9, and S100A8/A9 caused neutrophil chemotaxis at concentrations of 10(-12)-10(-9) M. S100A8, S100A9, and S100A8/A9 stimulated shedding of L-selectin, up-regulated and activated Mac-1, and induced neutrophil adhesion to fibrinogen in vitro. Neutralization with Ab showed that this adhesion was mediated by Mac-1. Neutrophil adhesion was also associated with an increase in intracellular calcium levels. However, neutrophil activation by S100A8, S100A9, and S100A8/A9 did not induce actin polymerization. Finally, injection of S100A8, S100A9, or S100A8/A9 into a murine air pouch model led to rapid, transient accumulation of neutrophils confirming their activities in vivo. These studies 1) show that S100A8, S100A9, and S100A8/A9 are potent stimulators of neutrophils and 2) strongly suggest that these proteins are involved in neutrophil migration to inflammatory sites.

HIV-1 transcription and virus production are both accentuated by the proinflammatory myeloid-related proteins in human CD4+ T lymphocytes.

S100A8, S100A9, and S100A12, collectively known as myeloid-related proteins (MRPs), are highly expressed by the myeloid cell lineage and are found in the extracellular milieu during infections and inflammatory conditions. Recent data showed high levels of MRPs in the serum of HIV type 1 (HIV-1)-infected patients which correlated with disease progression and low CD4(+) counts. Therefore, we set out to investigate the effect of MRPs on HIV-1 replication. We observed a 4- to 5-fold induction of virus production in J1.1, a human T lymphoid cell line latently infected with HIV-1, following treatment with MRPs. Using luciferase-based reporter gene assays, we demonstrated that MRPs induce a dose- and time-dependent activation of the HIV-1 long terminal repeat promoter region that could be blocked by specific anti-MRP polyclonal Abs and by physical denaturation of these proteins. The MRP-mediated induction was acting through the HIV-1 enhancer sequence and was dependent upon NF-kappaB activity. These latter results were also confirmed by EMSA experiments conducted in Jurkat cells and freshly isolated PBMCs. In conclusion, we demonstrate that MRPs induce HIV-1 transcriptional activity and viral replication in infected CD4(+) T-lymphocytes at concentrations similar to those found in the serum of HIV-1-infected patients.