Activation mechanisms of adherent human neutrophils.
The mechanism by which unstimulated human neutrophils initiate a respiratory burst on adherence to a surface has been examined. When neutrophils adhere to a plastic surface, they immediately generate a sustained burst of superoxide (O2-). However, this respiratory burst is not initiated by adherence alone, since neutrophils attached to fibronectin fail to mount a response. Adhesion to plastic is calcium (Ca2+) independent, but O2- production requires Ca2(+)-containing buffer in the initiation phase, that is, during adhesion and the early phase of O2- production. The Ca2(+)-dependent step was shown to involve protein kinase C (PK-C) in that the O2- production, but not adherence, was blocked with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and PK-C was found to translocate from the cytosol to the membrane on adhesion. Furthermore, it may be inferred that this translocation results in the generation of a Ca2+ independent form of PK-C, PK-M, since leupeptin, which inhibits the generation of PK-M, also blocked O2- production. This finding was corroborated by showing that after 5 minutes in a Ca2(+)-containing buffer, enough time to initiate O2- production and PK-C translocation, Ca2+ is no longer required for sustained O2- release. These results, in aggregate, demonstrate that neutrophils are activated by adhesion to plastic to generate O2-, a PK-C-dependent process that appears to involve a Ca2(+)-independent form of the kinase, PK-M. Why adherent neutrophils generate a respiratory burst on plastic and not fibronectin surfaces probably reflects activation of distinct receptors, whose nature must still be defined. Another issue to address is the priming effect of adhesion, since cells adherent to plastic- or fibronectin-coated surfaces have an enhanced O2- response to formylmethionyl-leucine-phenylalanine (FMLP) compared with neutrophils stimulated in suspension. This may relate to increased Ca2+ mobilization, an important mediator of priming for FMLP responses. Thus, adhesion as a priming event does not necessarily initiate cell effector function, and the further elucidation of the plastic and fibronectin models suggests a means of characterizing the crucial event that control neutrophil activation.
William B. Castle Hematology Research Laboratory, Boston City Hospital, MA.
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Ginis
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Tauber
D002118:Calcium / Q000502:physiology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008213:Lymphocyte Activation / Q000502:physiology*; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism; D010969:Plastics / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology
D010969:Plastics; D011493:Protein Kinase C; D010100:Oxygen; D002118:Calcium