Virus-cell interactions regulating induction of tumor necrosis factor alpha production in macrophages infected with herpes simplex virus.

Macrophages respond to virus infections by rapidly secreting proinflammatory cytokines, which play an important role in the first line of defense. Tumor necrosis factor alpha (TNF-alpha) is one of the major macrophage-produced cytokines. In this study we have investigated the virus-cell interactions responsible for induction of TNF-alpha expression in herpes simplex virus (HSV)-infected macrophages. Both HSV type 1 (HSV-1) and HSV-2 induced TNF-alpha expression in macrophages activated with gamma interferon (IFN-gamma). This induction was to some extent sensitive to UV treatment of the virus. Virus particles unable to enter the cells displayed reduced capacity to stimulate TNF-alpha expression but retained a significant portion which was abolished by HSV-specific antibodies. Recombinant HSV-1 glycoprotein D was able to trigger TNF-alpha secretion in concert with IFN-gamma. Sugar moieties of HSV glycoproteins have been reported to be involved in induction of IFN-alpha but did not contribute to TNF-alpha expression in macrophages. Moreover, the entry-dependent portion of the TNF-alpha induction was investigated with HSV-1 mutants and found to be independent of the tegument proteins VP16 and UL13 and partly dependent on nuclear translocation of the viral DNA. Finally, we found that macrophages expressing an inactive mutant of the double-stranded RNA (dsRNA)-activated protein kinase (PKR) produced less TNF-alpha in response to infectious HSV infection than the empty-vector control cell line but displayed the same responsiveness to UV-inactivated virus. These results indicate that HSV induces TNF-alpha expression in macrophages through mechanisms involving (i) viral glycoproteins, (ii) early postentry events occurring prior to nuclear translocation of viral DNA, and (iii) viral dsRNA-PKR.

Expression of TNF-alpha by herpes simplex virus-infected macrophages is regulated by a dual mechanism: transcriptional regulation by NF-kappa B and activating transcription factor 2/Jun and translational regulation through the AU-rich region of the 3' untranslated region.

Here we have investigated the regulation of TNF-alpha expression in macrophages during HSV-2 infection. Despite a low basal level of TNF-alpha mRNA present in resting macrophages, no TNF-alpha protein is detectable. HSV-2 infection marginally increases the level of TNF-alpha mRNA and protein in resting macrophages, whereas a strong increase is observed in IFN-gamma-activated cells infected with the virus. By reporter gene assay it was found that HSV infection augments TNF-alpha promoter activity. Moreover, treatment of the cells with actinomycin D, which totally blocked mRNA synthesis, only partially prevented accumulation of TNF-alpha protein, indicating that the infection lifts a block on translation of TNF-alpha mRNA. EMSA analysis showed that specific binding to the kappaB#3 site of the murine TNF-alpha promoter was induced within 1 h after infection and persisted beyond 5 h where TNF-alpha expression is down-modulated. Binding to the cAMP responsive element site was also induced but more transiently with kinetics closely following activation of the TNF-alpha promoter. Inhibitors against either NF-kappaB activation or the activating transcription factor 2 kinase p38 abrogated TNF-alpha expression, showing a requirement for both signals for activation of the promoter. This observation was corroborated by reporter gene assays. As to the translational regulation of TNF-alpha, the AU-rich sequence in the 3' untranslated region of the mRNA was found to be responsible for this control because deletion of this region renders mRNA constitutively translationable. These results show that TNF-alpha production is induced by HSV-2 in macrophages through both transcriptional and translational regulation.

Interferon (IFN)-gamma and Herpes simplex virus/tumor necrosis factor-alpha synergistically induce nitric oxide synthase 2 in macrophages through cooperative action of nuclear factor-kappa B and IFN regulatory factor-1.

The radical nitric oxide (NO) constitutes an important part of the innate immune response to many viruses, and among these notably Herpes simplex virus (HSV). We have previously shown that HSV/tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma synergistically induce NO production in macrophages, and here we have investigated the molecular mechanism underlying this phenomenon. The enhancement of NO production was regulated at the level of NO synthase 2 (NOS2, iNOS) transcription. The ISRE element of the NOS2 promoter, which binds IFN regulatory factor (IRF)-1, was essential both for full responsiveness to IFN-gamma and the synergistic response. The GAS motif, binding signal transducer and activator of transcription 1 (STAT1), did not contribute to the cross-talk with virus/TNF-induced signals, but was necessary for full responsiveness to IFN-gamma. The distal binding site for nuclear factor (NF)-kappa B was important for the cooperative response, while the proximal kappa B site was not involved in the cooperative promoter activation but played a role in full promoter inducibility. By ectopic expression of IRF-1 and NF-kappa B (p65), we found that these factors synergistically induce NO accumulation. Together, our results show that binding of IRF-1 and NF-kappa B to their respective sites in the distal domain of the NOS2 promoter, creates a potent trans-activating complex with the ability to induce NOS2 transcription synergistically in response to simultaneous HSV-2/TNF-alpha and IFN-gamma treatment.

CD4+ CD8+ (thymocyte-like) T lymphocytes present in blood and skin from patients with atopic dermatitis suggest immune dysregulation.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease expressed early in life. Disease development is primarily determined by as yet unknown genetic factors, leading to the accumulation of activated T lymphocytes in the skin. OBJECTIVES: To investigate the nature of these T cells. METHODS: T-cell lines could be established from AD skin biopsies, but not from normal skin or AD peripheral blood, when placed in RPMI 1640 medium with 10% human AB serum, antibiotics, and the T-lymphocyte growth factors interleukins 2 and 4. The cell lines were subjected to phenotypic analysis using a fluorescence-activated cell sorter and compared with lymphocytes from AD and normal control peripheral blood. RESULTS: T-cell lines from 22 of 24 consecutive skin biopsies taken from 24 adult patients with AD were established. All cells were T lymphocytes expressing several activation markers. A significant proportion of the lymphocytes had stable expression of a CD4+ CD8+ phenotype (26% +/- 6%; mean +/- SEM). Such double-positive T lymphocytes are normally only seen in the thymus and not in the peripheral immune system. CD4+ CD8+ cells in peripheral blood of the patients (12.5% +/- 3.3%) were also detected. CONCLUSIONS: We suggest that a basic pathophysiological change in AD may be a faulty maturation of the T-lymphocyte system, leading to skin inflammation with CD4+ CD8+ T lymphocytes resembling immature T cells. This is likely to lead to skewing of many immune reactions in the patients.

The impact of CMV on the respiratory burst of macrophages in response to Pneumocystis carinii.

Infection of human monocyte-derived macrophages with CMV decreased the respiratory burst when cells were stimulated with opsonized zymosan or Pneumocystis carinii (P. carinii). Such an effect, though smaller, was also seen with heat-inactivated CMV, but only when triggered by zymosan. The effect was most pronounced in cells obtained from CMV antibody-negative donors. Dexamethasone further reduced the respiratory burst, both in uninfected and CMV-infected cells. Interferon-gamma increased the response in uninfected cells and, to a lesser extend, in cells treated with heat-inactivated CMV, whereas no effect was seen with infective CMV. No overt productive infection or cytopathology could be detected, however, the monocytes incubated with infective but also heat-inactivated CMV formed clusters, a phenomenon that was equally pronounced in cultures from CMV antibody positive and negative-donors. These results might help explain the worse prognosis of P. carinii pneumonia in patients coinfected with CMV and receiving dexamethasone.

Inhibition of NO production in macrophages by IL-13 is counteracted by Herpes simplex virus infection through tumor necrosis factor-alpha-induced activation of NK-kappa B.

Interleukin (IL)-13 is known to antagonize many interferon (IFN)-gamma-activated functions in macrophages and among these, nitric oxide (NO) production. We have previously shown that this function of IL-13 is reduced in Herpes simplex virus type 2 (HSV-2)-infected macrophages. In the present study we show that IL-13 and IFN-gamma are indeed produced during infection of BALB/c mice with HSV-2. The lack of inhibitory function of IL-13 in infected macrophages, which was not overcome even at very high concentrations of IL-13, was not due to impaired IL-13 signalling, since virus infection did not affect IL-13-mediated activation of STAT6 (signal transducer and activator of transcription 6). Neutralizing tumour necrosis factor (TNF)-alpha antibodies, however, largely restored the effect of IL-13 on NO production in virus-infected macrophages. The same was observed after treatment of the cells with inhibitors of nuclear factor (NF)-kappa B activation, known to be involved in enhancement of IFN-gamma-induced NO production. Even though IL-13 reduced TNF-alpha secretion by 50%, this did not impair NF-kappa B activation in IFN-gamma-treated cells infected with HSV-2. The results indicate that TNF-alpha, secreted by virus-infected macrophages, activates NF-kappa B which impairs the IL-13-mediated inhibition of inducible NO synthase (iNOS) expression. This could imply that a sustained NO production would be focused to sites of active virus replication.

Herpes simplex virus type 2 infection of macrophages impairs IL-4-mediated inhibition of NO production through TNF-alpha-induced activation of NF-kappaB.

Nitric oxide (NO) production in macrophages by the interferon (IFN)-gamma-inducible NO synthase has been shown to play a role in clearance of viral infections. We have previously shown that IFN-gamma-induced NO production is augmented by herpes simplex virus type 2 (HSV-2) infection through autocrine tumour necrosis factor (TNF)-alpha secretion and is inhibited by interleukin (IL)-4. Here we investigated the effect of HSV-2 infection on the inhibitory function of IL-4. Virus infection of mouse J774A.1 macrophages strongly reduced the ability of IL-4 to inhibit IFN-gamma-induced NO production, even at very high IL-4 concentrations. The effect of HSV-2 infection did not involve the IL-4 signal transduction pathway through STAT6. IL-4 reduced virus-induced TNF-alpha secretion and nuclear factor (NF)-kappaB activation significantly, but less in cells concomitantly treated with IFN-gamma. Furthermore, neutralisation of residual TNF-alpha activity or inhibition of NF-kappaB activation largely restored the inhibitory effect of IL-4. The data show that inhibition of IFN-gamma-induced NO production by IL-4 is impaired by HSV-2 infection due to autocrine TNF-alpha-mediated NF-kappaB activation. We suggest that the described phenomenon might be beneficial for the host by limiting high and sustained NO production to infectious foci.

Interleukin-4-mediated inhibition of nitric oxide production in interferon-gamma-treated and virus-infected macrophages.

Upon interferon-gamma (IFN-gamma) stimulation, murine macrophages (Mphi) produce nitric oxide (NO) through expression of inducible nitric oxide synthase (iNOS). Interleukin (IL)-4 treatment, even delayed 12 h relative to IFN-gamma, antagonized this induction, whereas infection with herpes simplex virus type 2 (HSV-2) or treatment with tumour necrosis factor-alpha exerted a synergistic effect, which partly compensated for the antagonistic effect of IL-4. Neither IL-4 nor HSV-2 affected the IFN-gamma-activated Jak-STAT (Janus kinase-signal transducer and activator of transcription) pathway or altered the levels of IFN-gamma-induced interferon regulatory factor (IRF)-1 expression, which is STAT1-dependent and known to play a central role in IFN-gamma-mediated gene induction. The effect of IL-4 was completely dependent on de novo protein synthesis, indicating that a direct activation of latent inhibitors is not sufficient to explain the inhibitory effect of IL-4. Furthermore, IL-4 substantially augmented the IFN-gamma-induced expression of IRF-2, which is known to compete with IRF-1 for the DNA recognition site, ISRE (interferon-stimulated response element). Our findings could indicate that IL-4 suppresses IFN-gamma-stimulated iNOS transcription by elevating the level of IRF-2 which, through competition, prevents IRF-1 from binding to ISRE in the iNOS promoter. The virus-induced effects on iNOS and NO levels in IFN-gamma-stimulated Mphi do not seem to involve the Jak/STAT pathway or a differential expression of IRF-1 and IRF-2.

NF-kappaB activation is responsible for the synergistic effect of herpes simplex virus type 2 infection on interferon-gamma-induced nitric oxide production in macrophages.

Nitric oxide (NO), produced in interferon (IFN)-gamma-activated murine macrophages by the enzyme inducible nitric oxide synthase (iNOS), has been found to have antiviral properties. We have previously shown that herpes simplex virus type 2 (HSV-2) infection of macrophages synergistically enhances IFN-gamma-induced NO production, and we now extend these findings by providing evidence that virus-induced tumour necrosis factor (TNF)-alpha mediates activation of the transcription factor nuclear factor (NF)-kappaB, which in turn is responsible for the synergistic effect. HSV-2 infection and IFN-gamma stimulation of macrophages synergistically induced TNF-alpha secretion and nuclear translocation of NF-kappaB, which bound to a sequence corresponding to a kappaB site in the iNOS promoter. The effect of HSV-2 on NF-kappaB and NO production was eliminated when cells were treated with antibodies to TNF-alpha, and direct inhibition of NF-kappaB activation with pyrrolidinedithiocarbamate (PDTC) also blocked the effect of HSV-2 infection on NO production. The effect of the NF-kappaB activation inhibitor was not mediated through inhibition of the production of interferon regulatory factor (IRF)-1 or of TNF-alpha itself, and a possible alternative mechanism of activation of NF-kappaB through virus-induced activation of the kinase PKR was also ruled out. Thus, our data indicate that NF-kappaB activation, through virus-induced autocrine TNF-alpha secretion, is responsible for the synergistic effect of HSV-2 infection on IFN-gamma-induced NO production, and that such activation might constitute a mechanism by which high-output NO production is targeted to infectious foci.

Effect of IL-4 and IL-13 on IFN-gamma-induced production of nitric oxide in mouse macrophages infected with herpes simplex virus type 2.

Interleukin (IL)-4 and IL-13 share a wide range of activities. Prominent among these is the ability to antagonize many interferon (IFN)-gamma-induced activities. Here we demonstrate that IL-4 and IL-13 totally abrogate IFN-gamma-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA and protein synthesis in a murine macrophage cell line. IFN-gamma-treated cells infected with herpes simplex virus type 2 (HSV-2) or costimulated with tumor necrosis factor (TNF)-alpha showed an enhanced reactivity, which was only partially reduced by IL-4/13. The results indicate that IL-4 and IL-13 function by intervening with a step prior to iNOS transcription by antagonizing IFN-gamma-induced signal(s) without counteracting synergistic virus- or TNF-alpha-induced signals. The beneficial effect of a sustained NO production in foci of virus infection is suggested.

Acyclovir prophylaxis and fever during remission-induction therapy of patients with acute myeloid leukemia: a randomized, double-blind, placebo-controlled trial.

PURPOSE: A randomized, double-blind, placebo-controlled trial was performed to estimate the preventive effect of the antiherpetic drug acyclovir on fever, incidence of bacteremia, use of antibiotics, and presentation of infections in patients with acute myeloid leukemia (AML). PATIENTS AND METHODS: Ninety herpes simplex virus (HSV)-seropositive patients aged 18 to 84 years were included. Forty-five patients received acyclovir (800 mg by mouth daily) and 45 placebo. The patients were examined daily for 28 days from the initiation of remission-induction chemotherapy. RESULTS: Fever developed in all patients in both groups. Acyclovir prophylaxis postponed the development of an oral temperature > or = 38.0 degrees C by 3 days (95% confidence interval [CI], 1 to 4 days; P = .03) and the initiation of antibacterial treatment by 3 days (95% CI, 1 to 5 days; P = .008). The duration of fever, use of antibacterial treatment, incidence of bacteremia, and need for systemic antifungal therapy were not affected by acyclovir prophylaxis. At fever development, acyclovir prophylaxis affected the incidence and localization pattern of oral ulcers. Thus, in the acyclovir group, the number of nonfungal oral infections was reduced (relative risk, 0.45 [95% CI, 0.24 to 0.85]) and mainly located on the soft palate (relative risk, 2.49 [95% CI, 1.19 to 5.22]). CONCLUSION: Acyclovir prophylaxis has an impact on fever development, but not on the duration of fever or the need for antibiotics. It does not reduce the incidence of bacteremia, but the presentation of acute oral infections is changed.

Herpes simplex virus type 2 synergizes with interferon-gamma in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumour necrosis factor-alpha.

We have analysed the ability of herpes simplex virus type 2 (HSV-2) to induce nitric oxide (NO) production in resting BALB/c mouse peritoneal macrophages. In most experiments, macrophages produced very small amounts of NO upon infection with HSV-2. Mock virus preparations did not induce NO production, and virus inactivation experiments showed that infectious virus was required. Since interferon-gamma (IFN-gamma) is the prototype cytokine that is able to induce significant NO production in macrophages, we found it of interest to examine the influence of HSV-2 infection on the IFN-gamma-induced NO production. The virus exerted a synergistic effect on the IFN-gamma-induced NO release, which was accompanied by induction of the iNOS-gene as revealed by RT-PCR. This effect was largely dependent on the presence of infectious virus particles, since only a minor effect was seen with mock virus and inactivated virus preparations. From experiments with neutralizing antibodies to tumour necrosis factor-alpha (TNF-alpha) and IFN-alpha/beta it was concluded that the synergistic effect is dependent on autocrine secretion of TNF-alpha, which acts as a second signal and synergizes with IFN-gamma in NO production.

Influence of mercuric chloride on resistance to generalized infection with herpes simplex virus type 2 in mice.

The effect of mercuric chloride on resistance to generalized infection with herpes simplex virus type 2 (HSV-2) in mice was studied. The severity of the infection was evaluated by the amount of infectious virus in the liver. Mercury at a single dose of 20 micrograms aggravated the infection, and neither increasing the single dose to 80 micrograms nor giving repeated doses of 20 micrograms further intensified the infection. Examination of the course of infection after mercury exposure revealed an increased virus replication and dissemination during the first days of the infection, indicating that the early, nonspecific defence mechanisms were affected. Virus clearance and elimination, which is mediated by specific immunity, seemed not to be influenced. Examination of cells from the peritoneal cavity and of livers from virus-infected mice showed that mercury detectable by autometallography was exclusively found in mature peritoneal macrophages and in Kupffer cells of the liver. Inflammatory cells, recruited to the peritoneal cavity or infiltrating the infectious foci of the liver, did not show any mercury deposits. Attempts to demonstrate an effect in vivo of mercury on potential antiviral macrophage functions like interferon-alpha/beta (IFN-alpha/beta) and tumour necrosis factor-alpha (TNF-alpha) secretion and oxidative burst capacity were not successful, possibly because recruited, inflammatory cells, which have not been exposed to the high mercury concentrations at the site of injection, take over these functions of intoxicated macrophages.

Acyclovir given as prophylaxis against oral ulcers in acute myeloid leukaemia: randomised, double blind, placebo controlled trial.

OBJECTIVES: To evaluate (a) the prophylactic effect of the antiherpetic drug acyclovir on oral ulcers in patients with acute myeloid leukaemia receiving remission induction chemotherapy and thus (b), indirectly, the role of herpes simplex virus in the aetiology of these ulcers. DESIGN: Randomised, double blind, placebo controlled trial. SUBJECTS: 74 herpes simplex virus seropositive patients aged 18-84. Thirty seven patients received acyclovir (800 mg by mouth daily) and 37 placebo. The patients were examined daily for 28 days. MAIN OUTCOME MEASURES: Occurrence of herpes labialis, intraoral ulcers, and acute necrotising ulcerative gingivitis. RESULTS: The two populations were comparable in age, sex, type of antineoplastic treatment, and history of herpes labialis. Acute oral infections occurred in 25 of the acyclovir treated patients and 36 of the placebo treated patients (relative risk 0.69 (95% confidence interval 0.55 to 0.87)). This difference was due to a reduction in the incidence of herpes labialis (one case versus eight cases; relative risk 0.13 (0.02 to 0.95)), intraoral ulcers excluding the soft palate (one case versus 13 cases; relative risk 0.08 (0.01 to 0.56)), and acute necrotising ulcerative gingivitis (one case versus eight cases; relative risk 0.13 (0.02 to 0.95)). However, ulcers on the soft palate were diagnosed with similar frequency in the two groups. Isolation of herpes simplex virus type 1 in saliva was reduced from 15 cases in the placebo group to one case in the acyclovir group (relative risk 0.07 (0.01 to 0.48)). CONCLUSION: Intraoral ulcers excluding the soft palate are most often due to infection with herpes simplex virus, whereas ulcers on the soft palate have a non-herpetic aetiology. The findings suggest that acute necrotising ulcerative gingivitis may also be due to herpes simplex virus. Prophylaxis with acyclovir should be considered for patients with acute myeloid leukaemia during remission induction therapy.

Sterility of the uterine cavity.

In a prospective open study the sterility of the uterine cavity was evaluated in 99 women admitted for hysterectomy. The indications for hysterectomy were in most cases persistent irregular vaginal bleeding and fibromyomas of the uterus. Samples for both aerobic and anaerobic bacteria, Chlamydia trachomatis, yeasts and viruses were taken preoperatively from the apex of the vagina and cervical os. Immediately after hysterectomy the uterus was opened under sterile conditions and samples obtained from the isthmus and fundus of the uterine cavity for microbiological examination. Wet smears were taken from the same sites. Nearly a quarter of all the patients harbored one or more microorganisms in the uterus, mostly Gardnerella vaginalis, Enterobacter and Streptococcus agalactiae. We found that in a significant number of cases, the uterine cavity is colonized with potentially pathogenic organisms which may play a causative role in endometritis. The results indicate that inflammation of the uterine cavity should be evaluated by hysteroscopic examination before hysterectomy is undertaken in patients with persistent irregular vaginal bleeding.

Effect of mercuric chloride on macrophage-mediated resistance mechanisms against infection with herpes simplex virus type 2.

Macrophages play an important role in the early, nonspecific resistance to infection with herpes simplex virus. Mercuric chloride (HgCl2) accumulates in macrophages and has in certain concentrations a marked influence on the functional capacity of these cells. Therefore the influence of HgCl2 on resistance to generalized infection with herpes simplex virus type 2 (HSV-2) in mice and its effect on the HSV-2-induced activation of macrophages in vitro was examined. Mice injected intraperitoneally with HgCl2 24 h before infection with HSV-2 had more than 100 times higher virus titres in the liver 4 days after infection than mice not receiving any mercury. HgCl2 exerted a toxic effect on macrophages in vitro, which was especially pronounced during their adherence. Macrophages infected with HSV-2 were activated for an enhanced respiratory burst. This activation was abolished by treatment of the cells for 24 h with relatively low concentrations of HgCl2, resulting in macrophages with a potential to react with a respiratory burst comparable to that of uninfected cells. The HSV-2-induced activation of macrophages is mediated through the production and synergistic interaction of interferon-alpha/beta and tumour necrosis factor-alpha in an autocrine manner. The ability of these cytokines to activate macrophages and to interact synergistically was not affected by mercury. However the production by macrophages of both cytokines during the HSV-2 infection, but especially interferon-alpha/beta, which is essential for the activation, was reduced at low concentrations of HgCl2. Collectively these data indicate that mercury, by interfering with the early macrophage-production of cytokines, disables the early control of virus replication, leading to an enhanced infection.

Mannan-binding protein and bovine conglutinin mediate enhancement of herpes simplex virus type 2 infection in mice.

A broad range of plant lectins have recently been shown to inhibit the infectivity of herpes simplex virus type 1 (HSV-1) in vitro. We decided to investigate the role of mammalian lectins in infection with herpes simplex virus. Two lectins, conglutinin and mannan-binding protein (also called mannose-binding protein, MBP), belonging to the collectin family of lectins, were examined. Four week-old BALB/c mice were injected subcutaneously with 100 micrograms bovine conglutinin or 50 micrograms human MBP 1 day before intravenous infection with 5 x 10(4) PFU of herpes simplex virus type 2 (HSV-2). A three-fold increase in virus titre of the liver was observed on day 3 of the infection in the mice pretreated with conglutinin or MBP, whereas no effect was seen on days 1 and 5. In a standard plaque assay using Vero cells we were not able to demonstrate reproducibly either infection inhibition or infection enhancement, when virus was pre-incubated with differing concentrations of the collectins. The concentrations used were similar to those used by us in vivo, and by others in in vitro experiments showing inhibition of the infectivity of HSV-1 with plant lectins. In an ELISA with HSV-2 antigens captured on anti-HSV-2 antibodies, calcium-dependent and carbohydrate inhibitable binding of the collectins was observed. Our results indicate that the effect of endogenous mammalian collectins in vivo may not be neutralization as suggested by the data using plant lectins. Instead, the previously described opsonizing activity of the mammalian collectins may provide the virions with an alternative port of entry into cells leading to infection enhancement.

Comparison of the interaction of methyl mercury and mercuric chloride with murine macrophages.

The toxicity of organic methyl mercury was studied on murine macrophages in cell culture and compared to that of inorganic mercuric chloride. Long-term treatment of macrophage cultures with methyl mercury resulted in decreased cell viability in a concentration-dependent fashion. Experiments showed that 20 microM methyl mercury was highly toxic, causing cell death within a few days, while cultures exposed to lower levels were less severely affected. Comparison of the toxicity of organic and inorganic mercury by cell viability showed no difference between equimolar concentrations of methyl mercury and mercuric chloride. Furthermore, protein synthesis (interferon-alpha/beta) was reduced in a concentration dependent manner and had the same reduced magnitude in cells treated with either methyl mercury or mercuric chloride. However, impairment of random migration and phagocytosis of macrophages appeared at lower concentrations in cells exposed to methyl mercury than in cells exposed to mercuric chloride. Electron microscopy of cells exposed to methyl mercury revealed mercury deposits in lysosomes and dispersed in the cytoplasm and nuclei. The present study shows that methyl mercury and mercuric chloride impair cell viability and protein production in cell cultures at equimolar concentrations, while methyl mercury inhibits macrophage functions such as migration and phagocytosis at lower concentrations than mercuric chloride.

Bioactive and inactive forms of tumor necrosis factor-alpha in spinal fluid from patients with meningitis.

Twenty-four patients, including 12 with meningitis, were admitted with meningeal symptoms and fever. Their serum and spinal fluid tumor necrosis factor-alpha (TNF alpha) levels were determined by ELISA. TNF alpha immunoreactivity was found in spinal fluid of 10 meningitis patients and one nonmeningitis patient, whereas 7 sera, 5 from meningitis patients, contained TNF alpha. Levels were significantly higher in spinal fluids than in serum samples, and the TNF alpha bioactivity of spinal fluids and sera was considerably below predictions based on ELISA measurements. Gel filtration chromatography demonstrated the presence of both polymeric (greater than 200 kDa) and oligomeric (10-40 kDa) TNF alpha in spinal fluid. Significant bioactivity was obtained only from samples containing oligomeric cytokine. In agreement with previous in vitro findings, these results strongly indicate that bioactive TNF alpha oligomers form inactive polymers and monomers, which could contribute to the observed in vivo discrepancies between immunoreactive and bioactive protein. Finally, the data support the concept of local central nervous system production of TNF alpha in meningitis.