Anke Strey

Myeloid-related proteins 8 and 14 are specifically secreted during interaction of phagocytes and activated endothelium and are useful markers for monitoring disease activity in pauciarticular-onset juvenile rheumatoid arthritis.

OBJECTIVE To analyze which physiologic stimuli induce secretion of myeloid-related protein 8 (MRP8) and MRP14, two S100 proteins expressed in neutrophils and monocytes, and to determine whether serum concentrations of these proteins are reliable parameters for monitoring inflammatory activity in pauciarticular juvenile rheumatoid arthritis (JRA). METHODS Secretion of MRP8 and MRP14 was analyzed using a coculture system of endothelial cells and monocytes. Concentrations of MRP8/MRP14 in the serum and synovial fluid of JRA patients or culture medium were determined by enzyme-linked immunosorbent assay. The expression of MRP8 and MRP14 by leukocytes in synovial tissue or fluid was investigated using immunohistochemistry. RESULTS MRP8 and MRP14 were specifically released during interaction of activated monocytes with tumor necrosis factor-stimulated endothelial cells. Secretion was mediated via an increase in intracellular calcium levels in monocytes. In contrast, contact with resting endothelium inhibited protein kinase C-induced secretion of the proteins by monocytes. In JRA patients, MRP8 and MRP14 were strongly expressed in infiltrating neutrophils and monocytes within the inflamed joints and could be found in significantly higher concentrations in synovial fluid (mean 42,800 ng/ml) compared with serum (2,060 ng/ml). Concentrations of MRP8/MRP14 in serum correlated well with those in synovial fluid (r = 0.78) and showed a strong correlation with disease activity (r = 0.62). After intraarticular triamcinolone therapy, the serum concentrations of MRP8/MRP14 decreased significantly in therapy responders, whereas no differences were found in patients who showed no clinical benefit. CONCLUSION MRP8 and MRP14 are specifically released during the interaction of monocytes with inflammatory activated endothelium, probably at sites of local inflammation. Their serum concentrations represent a useful marker for monitoring local inflammation in JRA.

Loss of S100A9 (MRP14) Results in Reduced Interleukin-8-Induced CD11b Surface Expression, a Polarized Microfilament System, and Diminished Responsiveness to Chemoattractants In Vitro

ABSTRACT The S100A9 (MRP14) protein is abundantly expressed in myeloid cells and has been associated with various inflammatory diseases. The S100A9-deficient mice described here were viable, fertile, and generally of healthy appearance. The myelopoietic potential of the S100A9-null bone marrow was normal. S100A8, the heterodimerization partner of S100A9 was not detectable in peripheral blood cells, suggesting that even a deficiency in both S100A8 and S100A9 proteins was compatible with viable and mature neutrophils. Surprisingly, the invasion of S100A9-deficient leukocytes into the peritoneum and into the skin in vivo was indistinguishable from that in wild-type mice. However, stimulation of S100A9-deficient neutrophils with interleukin-8 in vitro failed to provoke an up-regulation of CD11b. Migration upon a chemotactic stimulus through an endothelial monolayer was markedly diminished in S100A9-deficient neutrophils. Attenuated chemokinesis of the S100A9-deficient neutrophils was observed by using a three-dimensional collagen matrix migration assay. The altered migratory behavior was associated with a microfilament system that was highly polarized in unstimulated S100A9-deficient neutrophils. Our data suggest that loss of the calcium-binding S100A9 protein reduces the responsiveness of the neutrophils upon chemoattractant stimuli at least in vitro. Alternative pathways for neutrophil emigration may be responsible for the lack of any effect in the two in vivo models we have investigated so far.

Agonists of proteinase-activated receptor-2 modulate human neutrophil cytokine secretion, expression of cell adhesion molecules, and migration within 3-D collagen lattices

Proteinase‐activated receptor‐2 (PAR2) belongs to a novel subfamily of G‐protein‐coupled receptors with seven‐transmembrane domains. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and allergic or bacterial proteases. This receptor is expressed by various cells and seems to be crucially involved during inflammation and the immune response. As previously reported, human neutrophils express functional PAR2. However, the precise physiological role of PAR2 on human neutrophils and its implication in human diseases remain unclear. We demonstrate that PAR2 agonist‐stimulated human neutrophils show significantly enhanced migration in 3‐D collagen lattices. PAR2 agonist stimulation also induced down‐regulation of L‐selectin display and up‐regulation of membrane‐activated complex‐1 very late antigen‐4 integrin expression on the neutrophil cell surface. Moreover, PAR2 stimulation results in an increased secretion of the cytokines interleukin (IL)‐1β, IL‐8, and IL‐6 by human neutrophils. These data indicate that PAR2 plays an important role in human neutrophil activation and may affect key neutrophil functions by regulating cell motility in the extracellular matrix, selectin shedding, and up‐regulation of integrin expression and by stimulating the secretion of inflammatory mediators. Thus, PAR2 may represent a potential therapeutic target for the treatment of diseases involving activated neutrophils.

Wnt signaling regulates transendothelial migration of monocytes.

The Wnt‐signaling pathway plays a critical role in directing cell fate during embryogenesis. Several lines of evidence also suggest a role in inflammatory processes. Here, we analyzed whether Wnt signaling plays a role in leukocyte inflammatory responses. Monocytes from healthy donors expressed different Frizzled receptors, which are ligands for the Wnt molecules. Activation of the Wnt/β‐catenin pathway by LiCl or Wnt3a increased β‐catenin protein levels in monocytes but not in granulocytes. It is interesting that the activation of Wnt/β‐catenin signaling via Wnt3a in monocytes resulted in a decrease in migration through an endothelial layer (human dermal microvascular endothelial cell‐1). Further experiments revealed that the decrease in transendothelial migration was associated with specific monocyte adherence to endothelial cells after Wnt exposure. The specificity was verified by a lack of Wnt3a‐induced adhesion to fibronectin, laminin, or collagen compared with endothelial interaction. Analysis of the distribution of β‐catenin revealed a Wnt3a‐induced increase of β‐catenin in the cytoplasm. Wnt3a exposure did not result in any activation of the classical Wnt‐target gene c‐myc or a Wnt‐target gene involved in cell adhesion (Connexin43). Our study implicates for the first time a role of canonical Wnt signaling in inflammatory processes in monocytes.

Endothelial Rho signaling is required for monocyte transendothelial migration.

Bacterial toxins affecting Rho activity in microvascular endothelial cells were employed to elucidate whether endothelial Rho participates in regulating the migration of monocytes across monolayers of cultured endothelial cells. Inactivation of Rho by the Clostridium C3 exoenzyme resulted in an increased adhesion of peripheral blood monocytes to the endothelium and a decreased rate of transendothelial monocyte migration. Cytotoxic necrotizing factor 1‐mediated activation of endothelial Rho also reduced the rate of monocyte transmigration, but did not affect monocyte–endothelium adhesion. Thus, efficient leukocyte extravasation requires Rho signaling not only within the migrating leukocytes but also within the endothelial lining of the vessel wall.

Agonists of proteinase-activated receptor-2 affect transendothelial migration and apoptosis of human neutrophils

Abstract:  Skin is the first barrier preventing microorganism invasion in host. Wounds destroy this defense barrier and, without an appropriate care, may lead to sepsis. Neutrophil activation and immigration plays an important role at the inflammatory stage of wound healing. Neutrophils are known to express proteinase‐activated receptors (PARs), which can be activated by serine proteases, also by enzymes involved in wound healing. We previously reported that PAR2 agonists up‐regulate cell adhesion molecule expression and cytokine production by human neutrophils. Here, we demonstrate that PAR2 agonists (serine proteases as well as synthetic peptides) reduce transendothelial migration of neutrophils and prolong their life in vitro. Synthetic PAR2 agonist also enhanced protective interferon (IFN)γ‐induced FcγRI expression at neutrophil cell surface. Of note, IFNγ is a cytokine, which was used in clinical trials to reactivate human neutrophil functions during sepsis. Moreover, we observed a significant increase of PAR2 expression on cell surface of neutrophils from septic patients as compared with healthy volunteers. Together, our results indicate that PAR2 may be involved in the pathophysiology of neutrophil‐endothelial interactions during wound healing or later during sepsis in humans, potentially by affecting neutrophil apoptosis, transendothelial migration and Fcγ receptor‐mediated phagocytosis.