Peter D. Zimmerman

Lipopolysaccharide, Tumor Necrosis Factor Alpha, or Interleukin-1β Triggers Reactivation of Latent Cytomegalovirus in Immunocompetent Mice

ABSTRACT We have previously shown that cytomegalovirus (CMV) can reactivate in lungs of nonimmunosuppressed patients during critical illness. Our recent work has shown that polymicrobial bacterial sepsis can trigger reactivation of latent murine CMV (MCMV). We hypothesize that MCMV reactivation following bacterial sepsis may be caused by inflammatory mediators. To test this hypothesis, BALB/c mice latently infected with Smith strain MCMV received sublethal intraperitoneal doses of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), or saline. Lung tissue homogenates were evaluated for viral reactivation 3 weeks after mediator injection. Because LPS is known to signal via Toll-like receptor 4 (TLR-4) in mice, further studies blocking this signaling mechanism were performed using monoclonal MTS510. Finally, mice were tested with intravenous TNF-α to determine whether this would cause reactivation. All mice receiving sublethal intraperitoneal doses of LPS, TNF-α, or IL-1β had pulmonary reactivation of latent MCMV 3 weeks following injection, and LPS caused MCMV reactivation with kinetics similar to those for sepsis. When TLR-4 signaling was blocked, exogenous LPS did not reactivate latent MCMV. Intravenous TNF-α administration at near-lethal doses did not reactivate MCMV. Exogenous intraperitoneal LPS, TNF-α, and IL-1β are all capable of reactivating CMV from latency in lungs of previously healthy mice. LPS reactivation of MCMV appears dependent on TLR-4 signaling. Interestingly, intravenous TNF-α did not trigger reactivation, suggesting possible mechanistic differences that are discussed. We conclude that inflammatory disease states besides sepsis may be capable of reactivating CMV from latency.

Interleukin-1β regulates the inducible cyclooxygenase in amnion-derived WISH cells

The purpose of this study was to determine the mechanism of enhanced prostaglandin synthesis in amnion-derived WISH cell cultures when stimulated by interleukin-1 beta (IL-1 beta). Confluent monolayer cultures of WISH cells were incubated with human recombinant IL-1 beta (0.001-10 ng/ml) for 0-24 hours, while control cells received medium alone. PGE2 production was measured by specific radioimmunoassay. IL-1 beta enhanced the production of PGE2 in a dose- and time-dependent manner with enhanced production detectable by 2 h following exposure. Immunoblot analysis using isoform-specific antibodies showed that the inducible cyclooxygenase enzyme, i.e., COX-2, was expressed by 2 h in IL-1 treated cells, while the constitutive COX-1 remained unaltered in its expression. Northern blot analysis demonstrated that COX-2 mRNA expression was not detected in untreated cells, but became evident after a 30-min exposure to IL-1 beta (10 ng/ml). COX-1 mRNA was detected under basal conditions and did not increase significantly following IL-1 beta treatment. The close parallel between the kinetics of COX-2 mRNA and protein expression and PGE2 accumulation in the medium, as well as the constitutive, unregulated nature of the COX-1 isoform, indicates that cytokine-driven PGE2 formation in WISH cells may be mediated by de novo expression of the novel COX-2 enzyme.

Interleukin-1 receptor antagonist blocks interleukin-1– induced expression of cyclooxygenase-2 in endometrium

OBJECTIVE Our purpose was to test the hypothesis that the interleukin-1 receptor antagonist can inhibit interleukin-1-induced prostaglandin production and de novo expression of the inducible cyclooxygenase-2 isoform in a human endometrial epithelial cell line. STUDY DESIGN A continuous line of human endometrial epithelial cells was established from a hysterectomy specimen from a nonmalignant uterus. Cells were maintained as a monolayer culture in medium 199 supplemented with 10% fetal bovine serum and 50 micrograms/ml gentamicin. Cultures were treated with cytokines (interleukin-1 alpha or interleukin-1 beta, interleukin-1 receptor antagonist, or tumor necrosis factor-alpha), and media were collected for analysis of prostaglandin E2 and prostaglandin F2 alpha) by radioimmunoassay, whereas cells were harvested for ribonucleic acid and protein extractions and subsequent Northern blot or Western blot analyses, respectively. RESULTS When endometrial cells were incubated with interleukin-1 alpha or interleukin-1 beta, each cytokine was shown to stimulate the production of prostaglandin E2 and prostaglandin F2 alpha in a time- and dose-dependent fashion, with interleukin-1 alpha being far more potent than interleukin-1 beta. Interleukin-1 receptor antagonist inhibited interleukin-1 alpha- and interleukin-1 beta-induced prostaglandin formation, with 50% inhibitory concentration values of 30 ng/ml for prostaglandin E2 and 90 ng/ml for prostaglandin F2 alpha. When Northern blots of interleukin-1 alpha-treated cells were probed with a complementary deoxyribonucleic acid fragment specific for either cyclooxygenase-1 or cyclooxygenase-2, rapid de novo induction of cyclooxygenase-2 messenger ribonucleic acid was observed; however, cyclooxygenase-1 expression was constant regardless of interleukin-1 alpha concentration or incubation time. Coincubation of cells with interleukin-1 alpha (10 ng/ml) and cycloheximide caused superinduction of cyclooxygenase-2 messenger ribonucleic acid but had no effect on the expression of cyclooxygenase-1 messenger ribonucleic acid. Actinomycin D completely abolished interleukin-1 alpha-induced cyclooxygenase-2 messenger ribonucleic acid expression, suggesting that the cytokine caused transcriptional activation of the cyclooxygenase-2 gene. Experiments were conducted to examine whether interleukin-1 receptor antagonist could suppress interleukin-1-induced cyclooxygenase-2 expression. Cells were preincubated for 30 minutes with interleukin-1 receptor antagonist and then challenged with interleukin-1 alpha. Northern and Western analyses revealed that interleukin-1 receptor antagonist blocked interleukin-1 alpha-induced expression of cyclooxygenase-2 messenger ribonucleic acid transcripts and the subsequent appearance of cyclooxygenase-2 protein. Interleukin-1 receptor antagonist had no effect on the constitutive expression of cyclooxygenase-1 messenger ribonucleic acid and protein. Interleukin-1 receptor antagonist failed to alter prostaglandin E2 formation in response to tumor necrosis factor-alpha, indicating that the antagonist is specific for interleukin-1 family cytokines. Finally, interleukin-1 receptor antagonist acted as a partial agonist in some experiments in that relatively high concentrations (> 100 ng/ml) caused a modest increase in prostaglandin E2 and F2 alpha production. CONCLUSIONS These data indicate that interleukin-1 receptor antagonist is a potent inhibitor of interleukin-1-induced arachidonic acid metabolism and could possibly serve as an endogenous or exogenous modulator of interleukin-1 action in the endometrial epithelium.

Insulin and glucose modulate glucose transporter messenger ribonucleic acid expression and glucose uptake in trophoblasts isolated from first-trimester chorionic villi

OBJECTIVE Our purpose was to determine the effects of insulin and glucose on glucose transport and expression of GLUT1 glucose transporter messenger ribonucleic acid in first-trimester human trophoblast-like cells. STUDY DESIGN First-trimester human trophoblast-like cells were maintained as a continuous cell line. For 2[3H]deoxy-D-glucose uptake and messenger ribonucleic acid studies the cells were incubated in the presence or absence of insulin (10(-7) to 10(-11) mol/L) or D-glucose (0 to 50 mmol/L) for 0 to 24 hours. Glucose transport was measured by incubating cells with 0.1 mmol/L 2[3H]deoxy-D-glucose for 5 minutes. Specific uptake was determined by incubating companion cultures with 10 mumol/L cytochalasin B. The cells were then solubilized with sodium hydroxide and the radioactivity counted. Data were expressed as nanomoles of 2[3H]deoxy-D-glucose transported per milligram of protein per 5 minutes and analyzed by one-way analysis of variance with post hoc testing by the method of Tukey. GLUT1 messenger ribonucleic acid was measured by Northern blotting of total ribonucleic acid samples hybridized to a phosphorus 32-labeled complementary deoxyribonucleic encoding the rat GLUT1 glucose transporter. As a control for loading efficiency, blots were stripped and rehybridized to a 40-mer phosphorus 32-labeled beta-actin oligonucleotide probe. RESULTS Insulin treatment resulted in a dose-dependent increase in the transport of 2[3H]deoxy-D-glucose at 24 hours (p < 0.001 at 10(-7) mol/L). This change was first detected at 12 hours of incubation. These data closely paralleled the insulin-induced increase in GLUT1 messenger ribonucleic acid seen in Northern blots. In contrast to insulin, increasing concentrations of D-glucose did not change the transport of 2[3H]deoxy-D-glucose. However, when cells were incubated in low concentrations of D-glucose (0 or 1 mmol/L), an enhancement in the uptake of 2[3H]deoxy-D-glucose (p < 0.001) was observed. Kinetic studies indicated that D-glucose augmentation of 2[3H]eoxy-D-glucose uptake was significant at 9 hours (p < 0.05). The effects of D-glucose on GLUT1 messenger ribonucleic acid expression paralleled the uptake of 2[3H]deoxy-D-glucose, although the modulation of GLUT1 messenger ribonucleic acid levels by glucose was much less pronounced than in insulin-treated cells. CONCLUSION Although it has been assumed that the placenta has a limited role in influencing glucose transport to the fetus, our in vitro data demonstrate that both insulin and glucose can modulate glucose transport at the cellular level of the placental trophoblast. Thus maternal insulin and glycemic status may influence the expression of GLUT1, the major trophoblast glucose transporter protein, therefore directly affecting first-trimester placental glucose transport. These in vitro data may help explain the association between maternal glucose abnormalities and impaired fetal development during the first trimester when placental GLUT1 messenger ribonucleic acid expression is at its peak.

Tumour necrosis factor-α induces cyclo-oxygenase-2 gene expression in first trimester trophoblasts: suppression by glucocorticoids and NSAIDs

Tumour necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine which stimulates the synthesis and release of prostaglandins (PGs) in several in vitro and in vivo models of preterm labour. While TNF-alpha simulated PG production has been described in decidual, amnion and myometrial cells, to date no studies have focused on the role of TNF-alpha in the stimulation of arachidonic acid metabolism in placental trophoblast cells. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in PG biosynthesis and is expressed de novo during cellular activation by cytokines. To test whether TNF-alpha alters expression of COX-2, trophoblasts from first trimester chorionic vili were cultured as a continuous cell line and treated with TNF-alpha alone or with TNF-alpha and dexamethasone (Dex). Total RNA and protein were extracted from the trophoblasts and subjected to Northern and immunoblot analysis, respectively. Northern blots were hybridized with a 32P-labelled probe encoding the COX-2 cDNA and immunoblots were incubated with anti-COX-2 antibodies. There was a time- and dose-dependent increase in COX-2 mRNA and protein expression in cells stimulated with TNF-alpha. The effect of TNF-alpha on COX-2 mRNA and protein expression was inhibited by dexamethasone (Dex). To examine the production of PGE2 and PGF(2 alpha), specific RIAs were performed on culture media from similarly stimulated cells. PG accumulation after TNF-alpha stimulation occurred in a time- and dose-dependent fashion with a similar inhibition of PG accumulation after Dex exposure. To be certain that TNF-alpha stimulated PGE2 production was, indeed, a result of COX-2 induction, RIAs were carried out with the COX-2-selective inhibitor NS-398. Cells stimulated with the NS-398 after TNF-alpha exposure demonstrated suppression of TNF-alpha-stimulated PGE2 formation. The results suggest that TNF-alpha elicits part of its pathophysiologic effects in preterm labour via alterations in COX-2 gene expression within the placental microenvironment.

Disruption of murine cardiac allograft acceptance by latent cytomegalovirus.

Cytomegalovirus (CMV) reactivation is a well‐described complication of solid organ transplantation. These studies were performed to (1) determine if cardiac allograft transplantation of latently infected recipients results in reactivation of CMV and (2) determine what impact CMV might have on development of graft acceptance/tolerance. BALB/c cardiac allografts were transplanted into C57BL/6 mice with/without latent murine CMV (MCMV). Recipients were treated with gallium nitrate induction and monitored for graft survival, viral immunity and donor reactive DTH responses. Latently infected allograft recipients had ∼80% graft loss by 100 days after transplant, compared with ∼8% graft loss in naïve recipients. PCR evaluation demonstrated that MCMV was transmitted to cardiac grafts in all latently infected recipients, and 4/8 allografts had active viral transcription compared to 0/6 isografts. Latently infected allograft recipients showed intragraft IFN‐α expression consistent with MCMV reactivation, but MCMV did not appear to negatively influence regulatory gene expression. Infected allograft recipients had disruption of splenocyte DTH regulation, but recipient splenocytes remained unresponsive to donor antigen even after allograft losses. These data suggest that transplantation in an environment of latent CMV infection may reactivate virus, and that intragraft responses disrupt development of allograft acceptance.

Antiviral prevention of sepsis induced cytomegalovirus reactivation in immunocompetent mice.

INTRODUCTION Immunocompetent patients can reactivate latent cytomegalovirus (CMV) during critical illness and reactivation is associated with significantly worse outcomes. Prior to clinical trials in humans to prove causality, we sought to determine an optimal antiviral treatment strategy. METHODS Mice latently infected with murine CMV (MCMV) received a septic reactivation trigger and were randomized to receive one of four ganciclovir regimens or saline. Lungs were evaluated for viral transcriptional reactivation and fibrosis after each regimen. Influences of ganciclovir on early sepsis-induced pulmonary inflammation and T-cell activation were studied after sepsis induction. RESULTS All ganciclovir regimens reduced measurable MCMV transcriptional reactivation, and 10mg/day for 7 or 21 days was most effective. Lower dose (5mg/kg/day) or delayed therapy was associated with significant breakthrough reactivation. Higher doses of ganciclovir given early were associated with the lowest incidence of pulmonary fibrosis, and delay of therapy for 1 week was associated with significantly worse pulmonary fibrosis. Although bacterial sepsis induced activation of MCMV-specific pulmonary T-cells, this activation was not influenced by ganciclovir. CONCLUSION These results suggest that antiviral treatment trials in humans should use 10mg/kg/day ganciclovir administered as early as possible in at-risk patients to minimize reactivation events and associated pulmonary injury.

Interleukin-1β induces cyclooxygenase-2 in cultured human decidual cells

PROBLEM: The purpose of this study was to examine the hypothesis that interleukin‐1β (IL‐1β)‐elicited increases in decidual prostaglandin E2and F2α (PGE2 and PGF2α) biosynthesis are due to the de novo expression of the inducible isoform of cyclooxygenase (i.e., COX‐2).

Proinflammatory cytokines interact synergistically with epidermal growth factor to stimulate PGE2 production in amnion-derived cells

Recent evidence has implicated cytokines and growth factors in the initiation of parturition in women. In the present study, the amnion-derived cell line WISH was used to determine whether proinflammatory cytokines (interleukins 1 beta, 6, and 8, tumor necrosis factor-alpha, and granulocyte/macrophage colony stimulating factor) could amplify epidermal growth factor-induced prostaglandin E2 production. WISH cells were preincubated with cytokines (0.0001-10 ng/ml) for 60 min and then challenged with EGF (10 ng/ml) for 4 hrs after which PGE2 production was measured by radioimmunoassay. EGF, IL-1 beta and TNF-alpha alone caused a dose-dependent increase in PGE2 production, while IL-6, IL-8 and GM-CSF were ineffective over the dose range tested. When cells were preincubated with IL-1 beta or TNF-alpha, there was a dose-dependent potentiation of EGF-induced PGE2 production that was greater than the sum of EGF alone and IL-1 beta or TNF-alpha alone. In each case, the minimum dose of IL-1 beta or TNF-alpha which amplified EGF-induced PGE2 production was 0.1 ng/ml (p less than 0.05, Students t-test). These data show that low concentrations of IL-1 beta or TNF-alpha may serve to amplify EGF-mediated PGE2 biosynthesis in amnion-derived cells and suggest that cytokines may modulate EGF function in responsive cells.

Transient CD8‐memory contraction: a potential contributor to latent cytomegalovirus reactivation

It is clear that latent CMV can reactivate in immunocompetent individuals, but the mechanism triggering such reactivations remains unclear. Recent clinical data suggest that reactivation can be subverted by CMV‐specific T‐memory. We therefore monitored CMV‐specific T cells in immunocompetent mice with latent mCMV after a known reactivation trigger (LPS). LPS induced transient systemic contraction of mCMV‐specific CD8 memory that was followed by transcriptional reactivation. Subsequent recovery of mCMV‐specific T cells coincided with resumption of latency. These data suggest that bacterial antigen encounters can induce transient T‐memory contraction, allowing viral recrudescence in hosts latently infected with herpes family viruses.