Endotoxin-induced desensitization of THP-1 cells is not associated with altered G protein binding or content

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Abstract
In rats endotoxin tolerance is characterized by decreased endotoxin-stimulated peritoneal macrophage arachidonic acid metabolism and decreased GTP binding protein function. The hypothesis that THP-1 cells can be altered in a similar manner by pretreatment with endotoxin was tested. These studies examined endotoxin's ability to stimulate eicosanoid and tumor necrosis factor α (TNFα) in control and desensitized THP 1 cells. Additionally, membrane GTPγ 35S binding and Western blot analyses with specific antisera to G i1,2α, Gi3a, Gαcommon, and the β subunit of G in control and endotoxin-desensitized THP-1 cells were assessed. Endotoxin (10 μg/ml) stimulated thromboxane (Tx) B2 production in THP-1 cells. Pretreatment with pertussis toxin (PT), resulted in significant inhibition of TxB2 production at concentrations not inhibited by equimolar concentrations of PT-B protomer. The latter observations suggest a role of PT-sensitive G protein in endotoxin activation of THP-1 cells. Pre-exposure to endotoxin (1 μg/ml) for 18 h desensitized THP-1 cells to endotoxin-stimulated TxB2 production and endotoxin-stimulated TNFα. To determine if endotoxin pretreatment affects G protein function, THP-1 cell membranes were isolated from endotoxin pretreated and control cells for equilibrium binding with GTPγ35S, a nonhydrolyzable analog of GTP. Neither the total number of binding sites (Bmax) nor the dissociation constant (Kd) for GTPγ35S in desensitized THP-1 cells were significantly different from those of control cells. PT-catalyzed ADP-ribosylation of G proteins in control and LPS-desensitized THP-1 cells demonstrated no difference in the quantity of G protein labelled versus desensitized cells. Immunoblots also showed no difference between control and desensitized cells in the membrane content of specific heterotrimeric G proteins. The data demonstrate that pre-exposure to endotoxin desensitizes the cells subsequent endotoxin stimulation of mediator production. However, unlike the in vivo rat model, this is not associated with a decrease in G protein binding or content.