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). ...
