Sarah Ashton

Effect of cross-tolerance between endotoxin and TNF-α or IL-1β on cellular signaling and mediator production

Abstract: Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory‐mediator production. This phenomenon also confers cross‐tolerance to other stimuli including tumor necrosis factor (TNF) α and interleukin (IL)‐1β. Post‐receptor convergence of signal transduction pathways might occur after LPS, IL‐1β, and TNF‐α stimulation. Therefore, it was hypothesized that down‐regulation of common signaling molecules induces cross‐tolerance among these stimuli. LPS tolerance and cross‐tolerance were examined in THP‐1 cells. Phosphorylation of MAP kinases and degradation of inhibitor κBα (IκBα) DNA binding of nuclear factor‐κB (NF‐κB), and mediator production were examined. In naive cells, LPS, TNF‐α, and IL‐1β induced IκBα degradation, kinase phosphorylation, and NF‐κB DNA binding. LPS stimulation induced production of TNF‐α or TxB2 and degradation of IRAK. However, neither TNF‐α nor IL‐1β induced IRAK degradation or stimulated TNF‐α or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS‐induced TxB2 and TNF‐α production. LPS pretreatment induced cross‐tolerance to TNF‐α or IL‐1β stimulation. Pretreatment with TNF‐α induced cross‐tolerance to LPS‐induced signaling events and TxB2 production. Although pretreatment with IL‐1β did not induce cross‐tolerance to LPS‐induced signaling events, it strongly inhibited LPS TNF‐α and TxB2 production. These data demonstrate that IL‐1β induces cross‐tolerance to LPS‐induced mediator production without suppressing LPS‐induced signaling to MAP kinases or NF‐κB activation.

Peroxisome proliferator-activated receptor-gamma agonists modulate macrophage activation by gram-negative and gram-positive bacterial stimuli.

&NA; — Bacterial products, such as lipopolysaccharide (LPS) or heat‐killed Escherichia coli (EC), and heat‐killed Staphylococcus aureus (SA) are potent activators of macrophages (MØ). When stimulated by these bacterial components, MØ produce inflammatory mediators, such as nitric oxide (NO) and thromboxane (Tx) B2. Bacterial mediator production is preceded by the activation of various signal transduction pathways. Agonists that activate the peroxisome proliferatoractivated receptor‐gamma (PPAR&ggr;) have been shown to block MØ mediator production by LPS and other stimuli. However, very little is known about the effects of PPAR&ggr; agonists on SA‐ or EC‐induced MØ activation. Therefore, we investigated whether the PPAR&ggr; agonists 15‐deoxy‐&Dgr;12,14 prostaglandin J2 (15‐PGJ2) and troglitazone block LPS‐, EC‐, or SA‐induced mediator production. Rat peritoneal MØ were stimulated with LPS, EC, or SA (10 μg/mL) with or without increasing concentrations (0.1 to 10 μM) of each PPAR&ggr; agonist and NO and TxB2 production were measured. 15‐PGJ2 decreased LPS‐, EC‐, and SA‐induced NO and TxB2 production. However, troglitazone only inhibited the production of TxB2 by each stimuli. In parallel studies, the effects of PPAR&ggr; agonists on signaling pathways were evaluated. Rat peritoneal MØ were pretreated for 1 h with 15‐PGJ2 or troglitazone (1 or 10 μM) and then stimulated for 40 min with LPS, EC, or SA (10 μg/mL). Western blot analysis demonstrated that 15‐PGJ2 significantly inhibited LPS‐, EC‐, and SA‐induced ERK ½ activation and blocked I&kgr;B&agr; degradation. Troglitazone had no significant effect on either signaling protein. The data demonstrate that although both 15‐PGJ2 and troglitazone are considered PPAR&ggr; agonists, they differentially affect mediator production and cell signaling events. PPAR&ggr;‐independent effects of 15‐PGJ2 may contribute to its more potent anti‐inflammatory effects compared with troglitazone.

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.

Implication of G i proteins and Src tyrosine kinases in endotoxin-induced signal transduction events and mediator production

Previous studies have suggested that heterotrimeric G proteins and tyrosine kinases may be involved in lipopolysaccharide (LPS) signaling events. Signal transduction pathways activated by LPS were examined in human promonocytic THP-1 cells. We hypothesized that Gi proteins and Src tyrosine kinase differentially affect mitogen-activated protein (MAP) kinases (MAPK) and nuclear factor kappa (NF- B) activation. Post-receptor coupling to G i proteins were examined using pertussis toxin (PTx), which inhibits G i receptor-coupling. The involvement of the Src family of tyrosine kinases was examined using the selective Src tyrosine kinase inhibitor pyrazolopyrimidine-2 (PP2). Pretreatment of THP-1 cells with PTx attenuated LPS-induced activation of c-Jun-N-terminal kinase (JNK) and p38 kinase, and production of tumor necrosis factor-alpha (TNF-) and thromboxane B2 (TxB2). Pretreatment with PP2 inhibited TNF- and TxB2 production, but had no effect on p38 kinase or JNK signaling. Therefore, the G i-coupled signaling pathways and Src tyrosine kinase-coupled signaling pathways are necessary for LPS-induced TNF- and TxB2 production, but differ in their effects on MAPK activation. Neither PTx nor PP2 inhibited LPS-induced activation of interleukin receptor activated kinase (IRAK) or inhibitedtranslocation of NF- B. However, PP2 inhibitedLPS-inducedNF-B transactivation of a luciferase reporter gene construct in a concentration-dependent manner. Thus, LPS induction of Src tyrosine kinases may be essential in downstream NF- B transactivation of genes following DNA binding. PTx had no effect on NF- B activation of the reporter construct. These data suggest upstream divergence in signaling through G i pathways leading to MAPK activation and other signaling events leading to I B degradation and NF- B DNA binding.

Beneficial Effect of a CXCR4 Agonist in Murine Models of Systemic Inflammation

The chemokine CXC receptor 4 (CXCR4) is activated by stromal cell-derived factor (SDF-1α). CXCR4 may be part of a lipopolysaccharide (LPS) sensing co-clustering complex that modulates TLR4 activation and evidence suggest that SDF-1α can activate anti-inflammatory signaling pathways and suppress inflammation. In the present study we examined the hypothesis that the SDF-1α peptide analog and CXCR4 agonist CTCE-0214 is anti-inflammatory in three distinct models of murine systemic inflammation. Our findings demonstrate that CTCE-0214 in vivo significantly suppressed plasma tumor necrosis factor alpha (TNF-α) increases in acute endotoxemia and following zymosan-induced multiple organ dysfunction syndrome (MODS). In both models, CTCE-0214 did not suppress plasma increases in the anti-inflammatory cytokine interleukin (IL)-10. CTCE-0214 improved survival without antibiotics in a model of severe sepsis induced by cecal ligation and puncture (CLP). CTCE-0214 also decreased plasma increases in IL-6 but not TNF-α and IL-10 in response to CLP-induced inflammation. We demonstrated in a moderately severe model of CLP (one puncture) that IL-6 levels at 24 h were similar to sham controls. However in severe CLP (two punctures) plasma IL-6 levels were markedly elevated. Plasma SDF-1α levels varied inversely with the plasma IL-6. In addition to the beneficial effect of CTCE-0214 in these models of systemic inflammation in vivo, we also demonstrated that the analog dose dependently suppressed LPS-induced IL-6 production in bone marrow-derived macrophages. CTCE-0214 therefore may be beneficial in controlling inflammation sepsis and systemic inflammatory syndromes.

Effect of vasoactive intestinal polypeptide (VIP) on the in vitro and in vivo motility of the rabbit reproductive tract

Isolated segments of rabbit ampulla and isthmus and strips of uterus and cervix were spontaneously mechanically active in vitro, and this activity was inhibited in a dose-dependent manner by 2 to 200 ng/ml vasoactive intestinal polypeptide (VIP). The oviductal segments were the most inhibited, the uterine strips the least inhibited. These tissues were stimulated to contract in a dose-dependent manner by epinephrine (EPI), with the uterus and cervix being the more responsive. VIP (200 ng/ml) produced only a slight noncompetitive antagonism of this stimulation. Recordings made with miniature force transducers showed the isthmus, uterus, and cervix also to be spontaneously active in vivo. This activity was inhibited by injections (1, 10, and 20 micrograms) and infusions (1.0 and 2.5 micrograms/min) of VIP. The isthmus was the most inhibited, the cervix the least inhibited. Possible physiologic implications of these pharmacologic effects are discussed.

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.

Effect of the LTD4 receptor antagonist LY203647 on endotoxic shock sequelae in the rat

This study investigated the effect of a novel LTD4 receptor antagonist LY203647 on Salmonella enteritidis endotoxin-induced shock sequelae in anesthetized rats. LY203647 (30 mg/kg i.v.) or vehicle was given 10 min prior to endotoxin (10 mg/kg i.v.) or its vehicle, and the hematocrit, mean arterial pressure and circulating leukocyte counts were determined. LY203647 significantly inhibited endotoxin-induced hemoconcentration up to 90 min post-endotoxin (46.7 +/- 1.3 vs. 51.9 +/- 2.4% at 30 min post-endotoxin, 45.9 +/- 1.1 vs. 53.1 +/- 1.4% at 90 min post-endotoxin, N = 8-9, P less than 0.05). The endotoxin-induced decreases in mean arterial pressure were also attenuated by LY203647, -29 +/- 5 vs. -56 +/- 9 mm Hg at 60 min post-endotoxin and -42 +/- 4 vs. -60 +/- 9 mm Hg at 90 min post-endotoxin (N = 9-10, P less than 0.05). LY203647 also attenuated endotoxin-induced decreases in leukocyte count in arterial blood. A study of differential counts in circulating leukocytes (N = 3) showed that endotoxin induced complete disappearance in circulating neutrophils. The circulating lymphocyte count was decreased by 30 +/- 10 and 41 +/- 1% at 15 and 30 min post-endotoxin, respectively. LY203647 inhibited endotoxin-induced lymphopenia (P less than 0.05) but failed to alter endotoxin-induced neutropenia. These data suggest that LTD4 may play an important role in mediating hemoconcentration, hypotensive and lymphocytopenic sequelae of endotoxin shock.

Vasoactive intestinal polypeptide stimulates nonovarian progesterone secretion in rabbits

Recent experiments conducted in this laboratory have shown that intravenous infusions of vasoactive intestinal polypeptide (VIP) induced significant increases in plasma progesterone (P) in female rabbits. The purpose of this study was to determine the organ source of this P and to clarify the mechanisms by which it is induced. Intravenous infusions of VIP (37.5, 75, and 150 pmol/kg per min for 60 min) produced acute dose-dependent increases in plasma P in intact estrous rabbits. In ovariectomized (OVX) animals, VIP infusion (75 pmol/kg per min) produced a P increase of the same magnitude. In animals both OVX and adrenalectomized (ADX), this VIP effect was eliminated. The only significant change noted in luteotropic hormone (LH) or follicle stimulating hormone (FSH) was a decrease in FSH immediately following VIP infusion (150 pmol/kg). VIP infusion significantly increased plasma cortisol in intact and OVX animals, but not in OVX/ADX animals. It is concluded that VIP primarily stimulates the adrenal component of P secretion in the rabbit, via mechanisms independent of LH or FSH.