Morphine hyperalgesia gated through microglia-mediated disruption of neuronal Cl⁻ homeostasis.

A major unresolved issue in treating pain is the paradoxical hyperalgesia produced by the gold-standard analgesic morphine and other opiates. We found that hyperalgesia-inducing treatment with morphine resulted in downregulation of the K(+)-Cl(-) co-transporter KCC2, impairing Cl(-) homeostasis in rat spinal lamina l neurons. Restoring the anion equilibrium potential 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 signaling preserved Cl(-) homeostasis and reversed the hyperalgesia. Gene-targeted mice in which Bdnf was deleted from microglia did not develop hyperalgesia to morphine. However, neither morphine antinociception nor tolerance was affected in these mice. Our findings dissociate morphine-induced hyperalgesia from tolerance and suggest the microglia-to-neuron P2X4-BDNF-KCC2 pathway as a therapeutic target for preventing hyperalgesia without affecting morphine analgesia.

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins.

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.

Toll-like receptor 2 represses nonpilus adhesin-induced signaling in acute infections with the Pseudomonas aeruginosa pilA mutant.

Expression of pili and associated proteins is an important means of host invasion by bacterial pathogens. Recent evidence has suggested that the binding of Pseudomonas aeruginosa through nonpilus adhesins may also be important in respiratory diseases, since adhesins bind mucins. Using wild-type C57BL/6 and TLR2KO mice, we compared the induction levels of the host response to P. aeruginosa that either expressed pili or lacked pilus expression due to a mutation in the structural gene pilA. In C57BL/6 mice, deletion of pili led to a decreased immune response, evidenced by a lower secretion of cytokines and a lack of neutrophil chemotaxis. By contrast, the P. aeruginosa pilA mutant induced a hyperresponsive phenotype in TLR2KO mice. TLR2KO mice showed an increased number of neutrophils in lavage fluid compared to the levels seen when either mouse strain was exposed to wild-type P. aeruginosa. Further analysis indicated that the increased neutrophil influx was associated with an increased expression of calgranulins, possibly through an induction of Toll-like receptor 4 (TLR4) expression. The hyperresponsive phenotype of TLR2KO mice exposed to the P. aeruginosa pilA mutant was associated with TLR4 induction and indicated that nonpilus adhesin-induced signaling was repressed by TLR2 function and, if not blocked by the host, could induce airway hyperresponsiveness.

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.

Hemozoin-inducible proinflammatory events in vivo: potential role in malaria infection.

During malaria infection, high levels of proinflammatory molecules (e.g., cytokines, chemokines) correlate with disease severity. Even if their role as activators of the host immune response has been studied, the direct contribution of hemozoin (HZ), a parasite metabolite, to such a strong induction is not fully understood. Previous in vitro studies demonstrated that both Plasmodium falciparum HZ and synthetic HZ (sHZ), beta-hematin, induce macrophage/monocyte chemokine and proinflammatory cytokine secretion. In the present study, we investigated the proinflammatory properties of sHZ in vivo. To this end, increasing doses of sHZ were injected either i.v. or into an air pouch generated on the dorsum of BALB/c mice over a 24-h period. Our results showed that sHZ is a strong modulator of leukocyte recruitment and more specifically of neutrophil and monocyte populations. In addition, evaluation of chemokine and cytokine mRNA and protein expression revealed that sHZ induces the expression of chemokines, macrophage-inflammatory protein (MIP)-1alpha/CCL3, MIP-1beta/CCL4, MIP-2/CXCL2, and monocyte chemoattractant protein-1/CCL2; chemokine receptors, CCR1, CCR2, CCR5, CXCR2, and CXCR4; cytokines, IL-1beta and IL-6; and myeloid-related proteins, S100A8, S100A9, and S100A8/A9, in the air pouch exudates. Of interest, chemokine and cytokine mRNA up-regulation were also detected in the liver of i.v. sHZ-injected mice. In conclusion, our study demonstrates that sHZ is a potent proinflammatory agent in vivo, which could contribute to the immunopathology related to malaria.

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.

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.

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.