M. Makhlouf

Alterations in macrophage G proteins are associated with endotoxin tolerance.

Previous studies have suggested that endotoxin tolerance induces macrophage desensitization to endotoxin through altered guanine nucleotide regulatory (G) protein function. In the present study the binding characteristics of the nonhydrolyzable GTP analogue GTP gamma [35S] to macrophage membranes from endotoxin tolerant and control rats were determined. Membranes were prepared from peritoneal macrophages harvested from rats 72 h after two sequential daily doses of vehicle or Salmonella enteritidis endotoxin (100 micrograms/kg on day 1 and 500 micrograms/kg on day 2). GTP gamma [35S] bound to a single class of sites that were saturable and displaceable in control and endotoxin tolerant macrophage membranes. The maximum specific binding of GTP gamma [35S] was significantly (P < 0.01) decreased in membranes from tolerant rats compared to control (Bmax = 39 +/- 7 pmol/mg protein in control vs. 11 +/- 2 pmol/mg protein in endotoxin tolerant; n = 5). There were no significant differences in the Kd values. To determine whether the reduced GTP gamma S binding was due to decreases in G proteins, macrophage membrane G protein content was determined by western blotting with specific antisera to Gi1,2 alpha, Gi3 alpha, Gs alpha, and the beta subunit of G. Scanning densitometric analysis demonstrated differential decreases in tolerant macrophage membrane G proteins. Gi3 alpha was reduced the most to 48 +/- 8% of controls (n = 3), and this reduction was significant compared to those of other G proteins. Gi1,2 alpha and G beta were reduced to 73 +/- 5% (n = 3) and 65 +/- 4% (n = 3) of control values, respectively. Gs alpha(L) and Gs alpha(H) were reduced to 61 +/- 5% (n = 3) and 68 +/- 3% (n = 3) of control, respectively. These results demonstrate that endotoxin tolerant macrophages exhibit decreased membrane GTP binding capacity and differential reductions in the content of specific G proteins. The cellular mechanisms leading to such alterations in G proteins and their functional significance in the acquisition of endotoxin tolerance merit further investigation.

Altered macrophage function in tumor necrosis factor α- and endotoxin-induced tolerance

Pretreatment of rats with a sublethal dose of human recombinant tumor necrosis factor-α (hrTNFα, 10 μg/kg i.p.) or Salmonella enteritidis LPS (100 μg/kg, i.p.) prevented death when a lethal dose of S. enteritidis lipopolysaccharide (LPS, 15 mg/kg i.p.) was administered 24 h later. The resistance to the lethal effect of LPS was associated with similar alterations of the functional phenotype of peritoneal macrophages from both groups. In ex vivo studies, peritoneal macrophages were harvested 24 h after vehicle (control), hrTNFα or LPS injection and stimulated in vitro with LPS. In macrophages collected from control rats, LPS stimulated arachidonic acid (AA) metabolism, as assessed by 6-keto-prostaglandin F1α (6-keto-PGF1α) levels, nitric oxide (NO) production, as assessed by nitrite, and interleukin 6 (IL-6) production. In macrophages from hrTNFα-pretreated or LPS pretreated rats, basal and LPS-stimulated 6-keto-PGF1α production were significantly reduced compared to controls, while nitrite production was increased (P < 0.001). LPS induced IL-6 synthesis was not affected in macrophages from hrTNFα-pretreated rats but was significantly reduced in stimulated macrophages from LPS treated rats. Furthermore, the macrophage membrane content of guanine nucleotide binding regulatory (G) protein subunits was determined. Macrophages collected from hrTNFα-pretreated rats exhibited a marked reduction of the membrane content of the Giα3 subunit compared to control macrophages, whereas the Giα1,2 and Gβ subunits were not significantly affected. The decrease in Giα3 in hrTNFα treated rats is similar to that previously observed in macrophages from LPS tolerant rats. The results demonstrate that hrTNFα induces cross tolerance to the lethal effect of LPS, and that tolerance induced by TNF or LPS is associated with differential changes in peritoneal macrophage mediator production. These changes may, in part, be a consequence of altered signal transduction via specific G proteins.

Increased prostacyclin and PGE2 stimulated cAMP production by macrophages from endotoxin-tolerant rats

Sublethal administration of lipopolysaccharide (LPS) renders rats tolerant to multiple lethal stimuli. Tolerant macrophages exhibit differential alterations in LPS-stimulated cytokine and inflammatory mediator release. Increased cAMP levels stimulated by PGE2 or prostacyclin (PGI2) result in differential effects on LPS-induced cytokine release and protect against the pathophysiological changes of endotoxemia. In the present studies, we sought to determine whether PGE2- and PGI2-stimulated cAMP levels are altered in tolerant macrophages. Incubation of macrophages with cicaprost or 11-deoxy-PGE1 in the presence of phosphodiesterase inhibitors resulted in significantly higher (2.5- to 6.5-fold) cAMP concentrations in tolerant macrophages compared with control. In contrast, isoproterenol-stimulated cAMP levels were not significantly different between control and tolerant cells. Also, incubation of tolerant macrophages with LPS did not result in significantly elevated cAMP levels. Prostacyclin (IP) receptor mRNA levels were significantly increased in tolerant cells compared with controls, whereas [3H]PGE2binding and PGE2 EP4 receptor mRNA levels were not significantly changed. These studies suggest that LPS tolerance induces selective alterations in eicosanoid regulation of cAMP formation.

Tolerance to LPS decreases macrophage G protein content

The effects of tolerizing doses of LPS on mRNA and protein levels of three different G protein subunits were investigated to understand the mechanism(s) responsible for the reduction in Gialpha protein content in LPS tolerance. Tolerance was induced in rats using Salmonella enteritidis LPS (intraperitoneal route) with a single dose of 100 microg/kg. Peritoneal macrophages were harvested 6 and 24 h later. In some studies, a second dose of LPS 500 microg/kg was given on the following day, and peritoneal macrophages were harvested 5 days after the first injection. Macrophage RNA or a crude membrane fraction was prepared from macrophages, and the mRNA level or the protein content for Gialpha3, Gialpha2, and Gsalpha was analyzed using Northern or Western blots, respectively. Compared with the control levels, the message for Gialpha3 was reduced (p < .025) at 6 and 24 h and 5 day time periods after LPS treatment. The Gialpha2 mRNA was increased relative to the control levels (p < .05) at 6 h and 5 days after LPS treatment, respectively, and Gsalpha message was not significantly changed. The half-life of Gialpha3 mRNA was not significantly different in control versus tolerant macrophages. The Gialpha3 mRNA and membrane protein were not significantly changed by incubation with LPS for intervals up to 6 h in vitro. Macrophage membrane Gialpha3 and Gialpha1 and 2 protein content from tolerant rats were reduced compared with the controls at 6 and 24 h, respectively (p < .05). These studies are consistent with our previous observations of selective changes in macrophage Gialpha protein content in LPS tolerance and raise the possibility that this may affect signal transduction events in these cells.

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

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 endotoxins 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.