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.

A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We show here that hyperalgesia-inducing treatment with morphine causes downregulation of the K+-Cl− cotransporter KCC2, impairing Cl− homeostasis in spinal lamina l neurons. Restoring Eanion reversed the morphine-induced hyperalgesia without affecting tolerance. The hyperalgesia was also reversed by ablating spinal microglia. Morphine hyperalgesia, but not tolerance, required μ opioid receptor-dependent expression of P2X4 receptors (P2X4Rs) in microglia and μ-independent gating of the release of brain-derived neurotrophic factor (BDNF) by P2X4Rs. Blocking BDNF-TrkB signalling preserved Cl− homeostasis and reversed the hyperalgesia. Gene-targeted mice in which BDNF was deleted from microglia did not develop hyperalgesia to morphine. Yet, neither morphine antinociception nor tolerance was affected in these animals. Our findings dissociate morphine-induced hyperalgesia from tolerance and unveil the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target to prevent hyperalgesia without affecting morphine analgesia.

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.

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 enzymelinked 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.