Monocyte Respiratory Burst Research Articles

Enhancement of human peripheral blood monocyte respiratory burst activity by aggregated C-reactive protein.

We had previously demonstrated that C-reactive protein (CRP), an acute phase reactant, when aggregated or coupled to a ligand, interacts with monocytes and certain human peripheral blood lymphocytes. The purpose of the present study, after further characterizing the binding interaction of CRP with human monocytes, was to focus on the biological response of monocytes to CRP binding. Flow cytometric analysis of human mononuclear leukocytes, following incubation with fluoresceinated heat-aggregated CRP (Agg-CRP), revealed that while greater than 70% of monocytes bound Agg-CRP, only 8% of lymphocytes demonstrated positive fluorescence. Furthermore, mean channel fluorescence values indicated that monocytes bound greater amounts of Agg-CRP per cell than did lymphocytes. Luminol-enhanced chemiluminescence (CL) was used as a measure of monocyte respiratory burst activity. Monocytes were stimulated only minimally by Agg-CRP alone; however, Agg-CRP substantially enhanced the CL response to heat-aggregated IgG. This Agg-CRP enhancing effect was selective for IgG-initiated monocyte activation, as no augmentation in CL was observed following cell stimulation with phorbol myristate acetate or serum-opsonized zymosan. These results demonstrate that aggregated CRP binds to the major proportion of human monocytes and selectively augments Fc receptor-mediated stimulation of monocyte oxidative metabolism.

Relation of Cytosolic Calcium to the Microbicidal Activation of Blood Monocytes by Recombinant Interferon

Oxygen metabolism and calcium ion (Ca++) handling in blood monocytes were greatly affected by treatment with recombinant gamma interferon (rIFN-gamma). Incubating the monocytes with rIFN-gamma resulted in increased basal production of superoxide anion, as well as a significantly enhanced respiratory burst in response to concanavalin A (Con A). A concomitant increase in microbicidal activity against Listeria monocytogenes was observed, and cell membrane permeability to Ca++ was enhanced by treatment with rIFN-gamma. Con A stimulation was also associated with acute rises in cytosolic-free calcium, and these calcium transients were greatly augmented in cells pretreated with rIFN-gamma. The enhanced respiratory burst and the increased calcium transients were completely neutralized by a monoclonal antibody to rIFN-gamma. Furthermore, enhancement of the respiratory burst and calcium transients occurred in a parallel dose-response range for rIFN-gamma. Blocking the intracellular release of calcium into cytosol by TMB-8 abrogated the capacity of rIFN-gamma to enhance the monocyte respiratory burst. Thus, not only was rIFN-gamma treatment of monocytes associated with altered calcium metabolism, but calcium from intracellular pools was essential to the expression of this activated state during Con A stimulation.

Redistribution of protein kinase C activity in human monocytes: correlation with activation of the respiratory burst.

Protein kinase C (PKC) was found to be present in purified human monocytes and lymphocytes isolated by countercurrent centrifugal elutriation. In unstimulated monocytes and lymphocytes, approximately 90% of the PKC activity was cytosolic when the cells were disrupted in the presence of EGTA. The role of this kinase in the stimulation of the respiratory burst in monocytes was investigated. Phorbol esters capable of triggering the release of O2- caused a loss of PKC activity from the cytosol and the appearance of the kinase activity in the particulate cell fraction. Kinase activity was partially extractable from the particulate fraction by 0.1% Triton X-100, whereupon it demonstrated calcium and lipid dependence. The EC50 for the phorbols in initiating the respiratory burst correlated well with their EC50 for stimulating the appearance of PKC activity in the particulate fraction (R = 0.998). Redistribution of PKC activity in monocytes by phorbol myristate acetate (PMA) was rapid and appeared to precede the release of O2-. PMA also shifted PKC activity from the cytosol to the extractable particulate fraction of lymphocytes. We conclude that redistribution of PKC activity by active phorbols or other cell stimulants could provide substrate specificity for phosphorylation reactions. By shifting PKC activity to the monocyte particulate fraction, active phorbols may initiate the phosphorylation of a substrate required for stimulation of the respiratory burst.