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.

MRP14 (S100A9) is the major calciumbinding protein of neutrophils and monocytes. Targeted gene disruption reveals an essential role of this S100 protein for transendothelial migration of phagocytes. The underlying molecular mechanism comprises major alterations of cytoskeletal metabolism. MRP14, in complex with its binding partner MRP8 (S100A8), promotes polymerization of microtubules. MRP14 is specifically phosphorylated by p38 mitogen- IntroductionAlthough the initial steps of leukocyte adhesion to endothelial cells during inflammatory reactions have been well characterized in recent years, mechanisms of transmigration remain far less well understood. 1,2 During transendothelial migration leukocytes extensively remodel their cytoskeletal structures in an orchestrated interplay of intracellular signaling pathways involving activation of specific protein kinases and transient elevation of intracellular calcium concentrations. [3][4][5] Recent reports have focused on the actin filament system and its regulation by the small guanosine triphosphate (GTP)-binding proteins RhoA, Cdc42, and Rac1. Less is known about regulation of the other 2 major cytoskeletal components, intermediate filaments and microtubules (MTs). [6][7][8][9] Phagocytes are characterized by a highly dynamic turnover of MTs during transmigration, but the specific proteins that regulate these events have not yet been identified. 10,11 Reorganization of MTs is controlled by modulation of intracellular calcium levels and specific protein phosphorylation. 3,5,12,13 Elevation of intracellular calcium concentrations induces conformational changes of calciumbinding proteins allowing interaction with distinct intracellular targets. The major calcium-binding molecules expressed in neutrophils and monocytes are myeloid-related protein 8 (MRP8 [S100A8]) and MRP14 (S100A9), 2 members of the S100 protein family. 14,15 S100 proteins exhibit functions during various cellular processes such as cell cycle progression and modulation of cytoskeletal-membrane interactions. However, none of the numerous effects of S100 proteins observed in vitro has so far been convincingly confirmed in vivo. 16 Targeted disruption of the MRP8 gene resulted in a lethal phenotype not allowing further functional analysis. 17 On the other hand, MRP14 Ϫ/Ϫ mice are viable but, at the first glance, do not exhibit an obvious phenotype. 18,19 Calciuminduced complexes of MRP8 and MRP14 colocalize with intermediate filaments and MTs on activation of isolated monocytes. [20][21][22][23] Indirect evidence suggests that interaction of MRP8/MRP14 complexes with these cytoskeletal components is modulated by phosphorylation of MRP14 (phospho-MRP14) at Thr113, 23,24 but neither the specific targets within the MT system nor the molecular mechanisms of MRP8/MRP14 action have been identified.In the present study, we demonstrate that the MRP8/MRP14 complex promotes polymerization of MTs via direct interaction with tubulin. MRP14 acts as a regulatory subunit in the MRP8/ MRP14 complex and integrates input...

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.

IntroductionMyeloid-related protein 8 (MRP8) and MRP14, both S100 proteins, are the major calcium-binding proteins expressed in phagocytes during specific stages of differentiation. 1,2 They form stable complexes and are present in circulating neutrophils and monocytes, representing the first cells invading inflammatory lesions. 3 The protein complex is found in inflammatory fluids in distinct inflammatory conditions, including rheumatoid arthritis, allograft rejection, inflammatory bowel disease, and lung disease. [4][5][6][7][8][9] Prerequisite for its secretion is the contact of phagocytes with extracellular matrix proteins or inflamed endothelium, resulting in elevated intracellular calcium levels and activated protein kinase C. 10,11 MRP8/MRP14 is thereby released specifically at inflammatory sites and leads to increased serum levels in correlation with the degree of inflammation, indicating an extracellular role of these molecules in inflammatory processes. However, little is known about the extracellular functions of MRP8/MRP14. The protein complex is deposited on endothelia for which different mechanisms are proposed. MRP14 has been shown to bind specifically to human microvascular endothelial cells (HMECs) by way of heparan sulphate proteoglycans. 12 Another group 13 reported that MRP8/MRP14 binds to novel carboxylated N-glycans expressed on inflammatory activated endothelial cells (ECs). Blocking these N-glycans with specific antibodies inhibited leukocyte extravasation in a murine model. 13 The hypothesis of a prominent role of MRP8/MRP14 for leukocyte recruitment is further supported by the finding that MRP8/MRP14 increases the binding capacity of CD11b-CD18 on leukocytes to intracellular adhesion molecule-1 (ICAM-1) on endothelium. 14 A recently identified inflammatory disorder, with the hallmark of an extraordinarily high abundance of MRP8 and MRP14, finally underscores a direct pathogenetic role for these 2 molecules in inflammation in vivo. 15 Thus, multiple findings indicate important interactions between MRP8/MRP14 and ECs, whereas the functional consequences and the underlying molecular mechanisms are completely unknown.In our study, oligonucleotide microarray analysis of HMECs demonstrated that MRP8/MRP14 directly induces a distinct inflammatory, thrombogenic response in microvascular ECs. The inflammatory response is characterized by the induction of proinflammatory chemokines and adhesion molecules and by increased vascular permeability. Patients, materials, and methods Purification of MRP8 and MRP14MRP8 and MRP14 were purified from human granulocytes as described previously. 16 The purity of the protein was greater than 98%, as verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MALDI-MS) ( Figure 1A), as described elsewhere. 17 MRP8/MRP14-containing stock solutions (1.5 mg/mL) were essentially free of endotoxin, as tested by a limulus lysate assay (E-Toxate Reagent Kit, sensitive to 0.05-0.1 endotoxin U/mL; Sigma, Deisenhofen, Germany). ...