Expression and function of the endothelial protein C receptor in human neutrophils.
Activation of protein C by thrombin bound to thrombomodulin is enhanced by endothelial protein C receptor. This pathway may inhibit inflammation. We investigated effects of protein C and activated protein C on neutrophils as well as whether an endothelial protein C receptor is involved in mediating protein C effects. Neutrophils were from venous blood of healthy donors. Cell migration, respiratory burst, phagocytic activity, and apoptosis were studied by micropore filter assays and fluorometry. Receptor expression was investigated by reverse transcriptase-polymerase chain reaction (PCR) for mRNA, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of immunoprecipitated receptor protein, and fluorescence-activated cell-sorter scanner (FACS) analysis using the anti-endothelial protein C receptor antibody RCR-252. Neither protein C nor activated protein C induced migration, yet both of them inhibited neutrophil chemotaxis triggered by interleukin-8, formyl-Met-Leu-Phe, antithrombin, or C5a. A protein C activation-blocking antibody against endothelial protein C receptor diminished inhibitory effects of protein C or activated protein C on migration. No effect of either protein C preparation was seen in neutrophil's respiratory burst, bacterial phagocytosis, or apoptosis assays. Endothelial protein C receptor immunoreactivity was confirmed on neutrophils by FACS. De novo synthesis is suggested by endothelial protein C receptor mRNA expression as demonstrated by reverse transcriptase PCR and immunoprecipitation SDS-PAGE analyses. Data suggest that an endothelial protein C receptor is expressed by human neutrophils whose active site ligation with either protein C or activated protein C arrests directed cell migration. Inhibitory effects of these components of the protein C pathway on neutrophil function may play a role in the protein C-based treatment of severe sepsis.
Sturn
Daniel H
DH
Department of Internal Medicine, University of Innsbruck, Austria.
Kaneider
Nicole C
NC
Feistritzer
Clemens
C
Djanani
Angela
A
Fukudome
Kenji
K
Wiedermann
Christian J
CJ
D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001779:Blood Coagulation Factors / Q000096:biosynthesis / Q000502:physiology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000502:physiology; D015936:Complement C5a / Q000494:pharmacology; D005434:Flow Cytometry; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D016209:Interleukin-8 / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011233:Precipitin Tests; D011486:Protein C / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000096:biosynthesis / Q000097:blood / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D016897:Respiratory Burst / Q000187:drug effects / Q000502:physiology
D000894:Anti-Inflammatory Agents, Non-Steroidal; D000906:Antibodies; D000991:Antithrombins; D001779:Blood Coagulation Factors; D006629:Hirudins; D016209:Interleukin-8; D011486:Protein C; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C091963:activated protein C receptor; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a