Lawrence P. Fernando

SUPPRESSION OF COX-2 AND TNF-α mRNA IN ENDOTOXIN TOLERANCE: EFFECT OF CYCLOHEXIMIDE, ANTINOMYCIN D, AND OAKADAIC ACID

Lipopolysaccharide (LPS)-tolerant human promonocytic THP-1 cells produce decreased levels of inflammatory mediators such as eicosanoids and tumor necrosis factor alpha (TNFalpha) in response to LPS. We hypothesized that transcriptional repression by newly synthesized proteins may be a mechanism for the reduced cellular response to a secondary challenge with LPS. THP-1 cells were desensitized after a 3.5 h or 20 h pre-exposure to LPS (1 microg/mL) and subsequently challenged with LPS (10 microg/mL). In cells rendered tolerant by exposure to LPS for 20 h, LPS-induced expression of cyclooxygenase (Cox)-2 and TNFalpha mRNA was suppressed. Cycloheximide (10 microM) prevented the transcriptional down-regulation of Cox-2 mRNA and to a lesser extent, TNFalpha mRNA, in LPS-tolerant cells. Transcriptional arrest with actinomycin D stabilized steady-state expression of Cox-2 mRNA in naive and tolerant cells but destabilized TNFalpha mRNA expression in LPS-tolerant cells. The observation that in naive cells Cox-2 and TNFalpha mRNA levels subside at 3 to 4 h after LPS (10 microg/mL or 1 microg/mL) suggested that LPS tolerance may occur earlier. Therefore, in subsequent experiments, the effect of LPS pretreatment for only 3.5 h was examined. This abbreviated tolerance regimen diminished secondary LPS-induced Cox-2 mRNA expression but had a lesser effect on TNFalpha mRNA expression. However, cycloheximide augmented both Cox-2 and TNFalpha mRNA expression in this group. Also, the serine/threonine phosphatase inhibitor okadaic acid augmented Cox-2 and TNFalpha mRNA expression in the LPS-tolerant cells. Although LPS-induced TNFalpha production in LPS-tolerant cells was suppressed relative to the naive cells, okadaic acid induced comparable levels of TNFalpha in tolerant and naive cells. These findings support the concept that LPS tolerance is associated with induction of proteins that alter expression of certain genes. Expression of Cox-2 mRNA appears to be particularly sensitive to down-regulation and, to a lesser extent, TNFalpha mRNA. However, this seems to vary depending on the LPS pretreatment regimen. The ability of a phosphatase inhibitor to induce TNFalpha and expression of Cox-2 and TNFalpha mRNA in LPS tolerance suggests that there may be alterations in phosphorylation status of signaling pathways, transcriptional mechanisms, or post-transcriptional mRNA stability.

High molecular weight kininogen and factor XII binding to endothelial cells and astrocytes

We have quantitated the binding of high molecular weight kininogen (HK) to human microvascular endothelial cells of lung and dermal origin as well as to astrocytes and compared the results with those reported for human umbilical vein endothelial cells (HUVEC). We also reassessed parameters of binding to HUVEC employing cells in suspension as well as cells attached to the culture plate and report similar numbers of sites varying from 6.96x10(5) to 7.71x10(5) per cell. The present study shows that HK binds with high specificity and affinity to microvascular endothelial cells (Kd = 1.86 to 4.5 nM) compared to HUVEC (Kd = 10.35 nM) but with lower affinity to astrocytes (Kd = 23.73 nM). Human cytokeratin 1, urokinase plasminogen activator receptor and gC1qR were found to be HK binding proteins present at the surface of microvascular endothelial cells and astrocytes analogous to that seen in HUVEC, as assessed by inhibition of binding with antibody to each protein. Lung microvascular endothelial cells had approximately half the number of HK binding sites as HUVEC while dermal micro vascular endothelial cells and astrocytes had only 8-10% of the sites/cell. The affinity of binding to the microvascular endothelial cells was greater than HUVEC, the affinity of binding to astrocytes was considerably less, nevertheless binding to each cell type involves gC1qR, cytokeratin 1 and u-PAR to varying degrees. We also demonstrate, for the first time, that factor XII binds to all of these cell types in a saturable and Zn(+2) dependent manner. Given that factor XII accelerates the interactions among cell surfaces and proteins of the contact activation cascade to generate bradykinin, binding of factor XII (and the prekallikrein-HK complex) may serve as a mechanism by which these proteins are concentrated locally to facilitate their interactions.

Protective effect of tyrphostin AG-556 on shock induced by endotoxin or gram positive bacteria.

The effects of tyrphostin AG-556 (TYR), a tyrosine kinase inhibitor, were evaluated on shock induced by lipopolysaccharide (LPS) or group B streptococcus (GBS) in rats. Mortality and mean survival time were monitored. Plasma 6-keto prostaglandin F1alpha (6-keto PGF1alpha) was also measured at four hours after LPS injection. The effects of TYR on the production of 6-keto PGF1alpha thromboxane B2(TXB2) and nitrite (NO) from LPS or GBS stimulated in vitro peritoneal rat macrophage were also examined. Salmonella enteritidis LPS (12 mg/kg, i.v. ) (n=6) produced severe shock (100% mortality). Simultaneous treatment with TYR (n=6) significantly (p < 0.01) extended mean survival time and 33% of rats survived. Plasma 6-keto PGF1alpha concentrations were increased in LPS controls, whereas TYR (5 mg/kg) significantly (p < 0.05) decreased the production. Animals treated with GBS/D-galactosamine (n=9) also exhibited shock with 100% lethality and TYR again prolonged survival time (p < 0.05) with 55% of the animals surviving. To evaluate direct effects of TYR on mediator production induced by LPS or GBS, rat macrophages were stimulated with heat-killed GBS or LPS with or without TYR. Supernatants were collected at 24 h for determination of TXB2, 6-keto PGF1alpha and NO. All mediators measured were significantly increased (p < 0.05) with LPS or GBS. TYR inhibited (p < 0.05) the production of all mediators from macrophages induced by LPS or GBS. The decrease in eicosanoids was associated with a reduction of the content of cyclooxygenase-2 (COX-2) as determined by western blotting. Collectively, these results suggest that TYR ameliorates toxic shock induced by LPS or gram positive bacteria. This protection is associated with suppression of macrophage mediator production.

Assessment of the role of heparan sulfate in high molecular weight kininogen binding to human umbilical vein endothelial cells

Summary.  The assembly and activation of the kinin forming system components on human umbilical vein endothelial cells (HUVEC) have been studied in great detail. Proteins such as gC1qR, cytokeratin‐1 and u‐PAR have been identified to be responsible for Zn2+‐dependent binding of high molecular weight kininogen (HK) to HUVEC. Heparan sulfate has also been shown to have a major role in Zn2+‐dependent binding of HK to the endothelial cell line, Ea.hy 926. In this study, we have analyzed the possible contribution of heparan sulfate to high molecular weight kininogen binding to HUVEC using multiple approaches. The presence of heparan sulfate on HUVEC was analyzed by staining with an antibody specific for heparan sulfate. Incubation of the cells with bacterial heparinases removed the heparan sulfate from the cell surface to the level seen with a control antibody, however, the Zn2+‐dependent binding of HK was not affected. Further, blocking of heparan sulfate with a specific antibody to heparan sulfate even after digestion with heparinases did not reduce HK binding whereas antibodies to the proteins gC1qR and cytokeratin‐1 consistently reduced the binding of HK to the endothelial cells. The binding intensities of FITC‐labeled HK were similar in heparinase‐treated and ‐untreated HUVEC. The rate of kallikrein formation by the assembly of factor XII, HK and PK were similar in both heparinase‐treated and non‐treated HUVEC. All of these data indicate that heparan sulfate does not contribute significantly to HK binding to HUVEC.

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.

Suppressed thromboxane production in endotoxin-desensitized THP-1 cells is not a result of decreased prostaglandin H synthase activity.

ABSTRACT Pre-exposure of THP-1 cells to low concentrations of endotoxin (lipopolysaccharide, LPS) down-regulates thromboxane (Tx) A2, an arachidonic acid (AA) metabolite, production in response to a subsequent LPS stimulation. To further delineate the mechanisms of LPS-induced down-regulation of TxA2, we examined expression of prostaglandin H synthase (PGHS)-2 mRNA, changes in PGHS activity, and content of PGHS-1 and −2. Pre-exposure to LPS (1 ±g/mL for 18 h to desensitize cells) inhibits production of TxB2, the stable metabolite of TxA2, in response to secondary stimulation of LPS (10 ±g/mL), when compared with LPS-stimulated naive cells (p < .05, n = 5). LPS (10 ±g/mL) induced expression of PGHS-2 mRNA at 1 and 2 h in naive cells, but this expression was decreased in the LPS-desensitized cells. However, exogenous AA (16 ±M) or phorbol myristic acid (PMA), 3 ±M) stimulated greater TxB2 production in the LPS-desensitized cells than in the naive cells (p < .05). Protein content of PGHS-1 and −2 were examined by Western blot analysis, using antibodies specific for PGHS-1 and PGHS-2. Densitometric analysis demonstrated a significant increase in PGHS-2 induction in LPS-stimulated naive cells (405 ± 174%) over its respective basal group (p < .05, n = 5). PGHS-1 was constitutively present, but there was no significant difference in quantity between naive and LPS-desensitized basal or LPS-stimulated groups. Thus, despite the reduction in expression of PGHS-2 mRNA, these composite data demonstrate that down-regulation of PGHS activity (assessed with exogenous AA or PMA) cannot be responsible for the inhibition of AA metabolism observed in LPS desensitization.