Stimulation of Arachidonic Acid Metabolism by Adherence of Alveolar Macrophages to a Plastic Substrate: Modulation by Fetal Bovine Serum

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Abstract
In previous studies on arachidonic acid (AA) metabolism by pulmonary macrophages in vitro, we observed that the presence of serum in the culture medium influenced the profile of AA metabolites released. To further characterize this phenomenon, rat alveolar macrophages were placed in plastic tissue culture dishes and allowed to adhere in the presence or absence of 7.5% fetal bovine serum (FBS) for 1 h. Adherent cells were then maintained in medium (equilibration) with or without FBS for 3.5 h before stimulation with the calcium ionophore A23187. The release of thromboxane B2 (TXB2) (the stable metabolite of TXA2) and leukotriene B4 (LTB4) during culture was measured by radioimmunoassay and reverse-phase high pressure liquid chromatography, respectively, at the end of each culture step. Cell adhesion to the plastic substrate in FBS-free medium induced an intense stimulation of AA metabolism, with the release of both TXB2 and LTB4. Adhesion and the accompanying TXB2 release appear to be mediated by trypsin-sensitive components since trypsin-pretreated macrophages showed a dramatic reduction in both adherence and TXB2 synthesis. The presence of FBS during the attachment phase of culture reduced both adhesion and release of TXB2 and LTB4 by more than 50%. On the other hand, addition of FBS to cells that had completed adhesion in serum-free medium stimulated release of both metabolites. When challenged with calcium ionophore after 4.5 h of culture, macrophages that had adhered in FBS-free medium released a much smaller amount of TXB2 than did macrophages that had been cultured in the presence of FBS. If cells were equilibrated in medium containing FBS after serum-free adherence, they released an intermediate amount of both TXB2 and LTB4 upon stimulation with A23187. Cells that adhered to plastic in the presence or absence of FBS exhibited a dramatic shift toward the lipoxygenase pathway compared with the metabolic profile of cells treated with A3187 in suspension. We conclude that macrophage adhesion to plastic induces an intense initial stimulation of AA metabolism, with the activation of both the cyclooxygenase and lipoxygenase pathways. Consequently, this attachment leads to a significant modification of the profile of the AA metabolites secreted in response to further stimuli, which induce a predominant release of lipoxygenase metabolites. The presence of fetal bovine serum modulates the activation induced by initial adherence to plastic. The overall serum effect (inhibition or stimulation) is dependent upon the timing of its introduction to the culture system.