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Background: In the monocrotaline (MCT)-treated rat, there is marked stimulation of DNA synthesis and megalocytosis of pulmonary arterial endothelial cells (PAECs) within 3 to 4 days, followed by pulmonary hypertension (PH) 10 to 14 days later. Growing evidence implicates caveolin-1 (cav-1) in plasma membrane rafts as a negative regulator of promitogenic signaling. We have investigated the integrity and function of endothelial cell-selective cav-1alpha/raft signaling in MCT-induced PH., Methods and Results: Although PH and right ventricular hypertrophy developed by 2 weeks after MCT, a reduction in cav-1alpha levels in the lung was apparent within 48 hours, declining to approximately 30% by 2 weeks, accompanied by an increase in activation of the promitogenic transcription factor STAT3 (PY-STAT3). Immunofluorescence studies showed a selective loss of cav-1alpha and platelet endothelial cell adhesion molecule-1 in the PAEC layer within 48 hours after MCT but an increase in PY-STAT3. PAECs with cav-1alpha loss displayed high PY-STAT3 and nuclear immunostaining for proliferating cell nuclear antigen (PCNA). Biochemical studies showed a loss of cav-1alpha from the detergent-resistant lipid raft fraction concomitant with hyperactivation of STAT3. Moreover, cultured PAECs treated with MCT-pyrrole for 48 hours developed megalocytosis associated with hypo-oligomerization and reduction of cav-1alpha, hyperactivation of STAT3 and ERK1/2 signaling, and stimulation of DNA synthesis., Conclusions: MCT-induced disruption of cav-1alpha chaperone and scaffolding function in PAECs likely accounts for diverse alterations in endothelial cell signaling in this model of PH.

The cellular physiology of signal transducer and activator of transcription protein family (STAT) transcription factors includes activation by Tyr-phosphorylation (PY) in cytokine and growth factor receptor complexes at the level of plasma membrane rafts, subsequent cytoplasmic transit and nuclear import, and transcriptional regulation of target genes, followed by dephosphorylation and export back to the cytoplasm. The ubiquitous protein tyrosine phosphatase (PTP) called "T-cell protein tyrosine phosphatase" has been reported to mediate Tyr-dephosphorylation of both interferon-gamma (IFN-gamma)-induced PY-STAT1 and interkleukin-6 (IL-6)-induced PY-STAT3 in some cell lines. To test whether the same PTP regulated both PY-STAT1 and PY-STAT3 in human hepatocytes we used orthovanadate (VO(4); 0.01-1.0mM) as a PTP-inhibitory probe and evaluated the kinetics of PY-STAT3 and PY-STAT1 accumulation, nuclear trafficking, and dephosphorylation following cytokine (IL-6 or IFN-gamma) stimulation of Hep3B cells. As evaluated using DNA binding or Western blotting assays, in IL-6-treated hepatocytes VO(4) had a modest enhancing effect on peak levels of cytoplasmic and nuclear PY-STAT3 reached by 1h and on their subsequent decline. In contrast, in the same cells and at the same time, VO(4) caused a marked and continuing increase in cytoplasmic and nuclear levels of PY-STAT1 which, by 4h, were 5- to 10-fold higher than peak levels reached in VO(4)-free, IL-6-treated cells. Prolonged treatment of cells with VO(4) alone (for 4-8h) replicated this markedly selective enhancement of PY-STAT1 levels. Consistent with this selectivity, shorter term VO(4) treatment (1-2h) markedly increased PY-STAT1 levels in all cellular compartments of IFN-gamma-treated cells by >10-fold. The unexpected selectivity in the effects of VO(4) on PY-STAT1 compared to that on PY-STAT3 levels in Hep3B cells suggests that, at least in these hepatocytes, the regulation of PY-STAT1 and PY-STAT3 likely involves distinct protein tyrosine phosphatase mechanisms.

We and others have recently obtained data suggesting that cytokine-STAT signaling in many different cell-types is a chaperoned pathway initiated at the level of specialized plasma membrane microdomains called "rafts" (the "raft-STAT signaling hypothesis"). These findings are of broad significance in that all cytokines and growth factors initiate signaling in target cells by interacting with respective cell-surface receptors. The new data suggest that raft microdomains represent the units of function at the cell-surface through which ligand-stimulated STAT signaling is initiated. Moreover, recent evidence shows the involvement of chaperone proteins in regulating the STAT signaling pathway. These chaperones include the human homolog of the tumorous imaginal disc 1 protein (hTid1) which associates with Janus kinase 2 (JAK2) at the level of the plasma membrane, heat shock protein 90 (HSP90) which associates with STAT3 and STAT1 proteins in caveolin-1-containing raft and cytoplasmic complexes, and glucose regulated protein 58 (GRP58/ER-60/ERp57), a thiol dependent protein-disulfide isomerase, found in association with STAT3 "statosome" complexes in the cytosol and in the raft fraction. We suggest a function of the HSP90 chaperone system in preserving IL-6/STAT3 signaling in liver cells in the context of fever. The identification and function of protein partners associated with specific STAT species in rafts and in cytosolic complexes, and in the efficient departure of cytokine-activated STATs from the cytosolic face of rafts towards the cell nucleus are now areas of active investigation.

Interleukin-6 (IL-6) initiates STAT3 signaling in plasma membrane rafts with the subsequent transit of Tyr-phosphorylated STAT3 (PY-STAT3) through the cytoplasmic compartment to the nucleus in association with accessory proteins. We initially identified caveolin-1 (cav-1) as a candidate STAT3-associated accessory protein due to its co-localization with STAT3 and PY-STAT3 in flotation raft fractions, and heat shock protein 90 (HSP90) due to its inclusion in cytosolic STAT3-containing 200-400-kDa complexes. Subsequent immunomagnetic bead pullout assays showed that STAT3, PY-STAT3, cav-1, and HSP90 interacted in plasma membrane and cytoplasmic complexes derived from uninduced and stimulated Hep3B cells. This was a general property of STAT3 in that these interactions were also observed in alveolar epithelial type II-like cells, lung fibroblasts, and pulmonary arterial endothelial cells. Exposure of Hep3B cells to the raft disrupter methyl-beta-cyclodextrin for 1-10 min followed by IL-6 stimulation for 15 min preferentially inhibited the appearance of PY-STAT3 in the cav-1-enriched sedimentable cytoplasmic fraction, suggesting that these complexes may represent a trafficking intermediate immediately downstream from the raft. Because IL-6 is known to function in the body in the context of fever, the possibility that HSP90 may help preserve IL-6-induced STAT3 signaling at elevated temperature was investigated. Geldanamycin, an HSP90 inhibitor, markedly inhibited IL-6-stimulated STAT3 signaling in Hep3B hepatocytes cultured overnight at 39.5 degrees C as evaluated by DNA-shift assays, trafficking of PY-STAT3 to the nucleus, cross-precipitation of HSP90 by anti-STAT3 polyclonal antibody, and reporter/luciferase construct experiments. Taken together, the data show that IL-6/raft/STAT3 signaling is a chaperoned pathway that involves cav-1 and HSP90 as accessory proteins and suggest a mechanism for the preservation of this signaling during fever.

Glucose-regulated protein 58 (GRP58/ER-60/ERp57), best known as a chaperone in the endoplasmic reticulum lumen, was previously identified by us as one of several accessory proteins in the S100 cytosol fraction of human hepatoma Hep3B cells that was differentially coshifted by anti-Stat3 antibody in an antibody-subtracted differential protein display assay. In the present study, the association between GRP58 and Stat3 in different cytoplasmic compartments was evaluated using cross-immunoprecipitation and cell-fractionation techniques. In the S100 cytosol fraction, three different anti-GRP58 polyclonal antibodies (pAb) cross-immunoprecipitated Stat3 (but not Stat1), and, conversely, anti-Stat3 pAb cross-immunoprecipitated GRP58. Both cytosolic Stat3 and GRP58 eluted during Superose-6 gel-filtration chromatography in complexes of size 200-400 kDa (statosome I), and anti-Stat3 pAb cross-immunoprecipitated GRp58 from these FPLC elution fractions. Using differential sedimentation and density equilibrium flotation methods, Stat3 and GRP58 were observed to be coassociated with cytoplasmic membranes enriched for the plasma membrane marker 5' nucleotidase but not with those containing the endoplasmic reticulum marker BiP/GRP78. The Stat3 and GRP58-containing plasma membrane fraction also contained Stat1, Stat5b, and gp130. Stat activation by orthovanadate caused the accumulation of PY-Stat3 in the GRP58-containing plasma membrane fraction. However, this PY-Stat3 was DNA-binding deficient. Likewise, excess exogenous recombinant human GRP58 prepared using a baculovirus expression system preferentially inhibited Stat3 DNA-binding activity in the S100 cytosol, suggesting that GRP58 may sequester activated Stat3. The new data confirm the association between GRP58 and Stat3 in cytosolic 200-400-kDa statosome I complexes and show that both GRP58 and Stat family members coassociate in the plasma membrane compartment. We suggest that the chaperone GRP58 may regulate signaling by sequestering inactive and activated Stat3.

STAT transcription factors signal from the plasma membrane to the nucleus in response to growth factors and cytokines. We have investigated whether plasma membrane "rafts" are involved in cytokine-activated STAT signaling. Cytokine-free human hepatoma Hep3B cells or cells treated with interleukin-6 (IL-6) or orthovanadate (a general activator of STATs) were fractionated, and plasma membrane raft fractions were obtained by equilibrium sedimentation or flotation through discontinuous sucrose gradients using either non-detergent or detergent-based (saponin or Triton X-100) methods. By Western blotting the plasma membrane raft fractions obtained using either non-detergent or detergent-based methods contained significant amounts of STAT1 and STAT3 (up to approximately 10% of the total cytoplasmic amount) as well as the integral raft proteins caveolin-1 and flotillin-1, the IL-6-receptor signal transducing chain gp130, the interferon-gamma receptor alpha chain (IFN-gammaRalpha), and the chaperone glucose-regulated protein 58 (GRP58/ER-60/ERp57). Upon activation of signaling by IL-6 or orthovanadate the respective Tyr-phosphorylated STAT species were now also observed in the membrane raft fraction but in a form deficient in DNA binding. The data show pre-association of STATs with plasma membrane rafts in flotation fractions, which also contained caveolin-1 and flotillin-1, and suggest that Tyr phosphorylation may not in itself be sufficient to cause the departure of PY-STATs from plasma membrane rafts. Methyl-beta-cyclodextrin, which sequesters cholesterol and disrupts plasma membrane rafts, markedly inhibited IL-6- and IFN-gamma-induced STAT signaling. Signaling through specialized raft microdomains may be a general mechanism operating at the level of the plasma membrane through which cytokines and growth factors activate STAT species (the "raft-STAT signaling hypothesis").

Objective: Enolase is a dimeric cytoplasmic enzyme whose double gamma isoenzyme, neuron-specific enolase, is predominantly found in neuronal and neuroendocrine tissues. Cell injury causes its release into the blood and cerebrospinal fluid (CSF). Neuron-specific enolase has been measured in the serum and CSF of adults and full-term asphyxiated neonates as a marker of neurological injury. We recently observed an elevation of neuron-specific enolase in the amniotic fluid of women whose neonates subsequently developed intraventricular hemorrhage or periventricular leukomalacia. The purpose of our study was to establish reference values of neuron-specific enolase in the amniotic fluid as a function of gestational age., Methods: A total of 110 amniotic fluid samples, obtained primarily for genetic studies (16-20 weeks, n = 22), for evaluation of preterm labor (21-35 weeks, n = 66) and for fetal lung maturity studies (36-40 weeks, n = 22), were analyzed for neuron-specific enolase. Samples were from women who subsequently delivered term neonates with normal neurological examinations or who delivered preterm neonates with normal neurosonograms up to the 7th day of life. Descriptive statistics and non-parametric correlations were used for analysis., Results: There was no correlation between gestational age and concentration of neuron-specific enolase (Spearman's r = 0.059, p = 0.63). The overall mean neuron-specific enolase value was 2.5 +/- 1.39 microg/l. The highest value obtained was 6 microgl. Of the 110 women, 105 (95.5%) had neuron-specific enolase values of less than 5 microg/l, while five (4.5%) had values ranging from 5 to 6 microg/l., Conclusions: The amniotic fluid level of neuron-specific enolase does not change as a function of gestational age. These stable levels may have utility in the evaluation of cases with fetal neurological injury.

The binding of a large number of cytokines and growth factors to their cognate receptors on the surface of mammalian-cell plasma membrane activates a signalling cascade involving the cytoplasmic STAT-family proteins, which is characterized by the nuclear translocation of a cytokine- or growth factor-specific subset of the cytoplasmic pool of the respective tyrosine- and serine-phosphorylated STAT proteins and the consequent transcriptional activation of specific target genes. In the standard model of cytokine-induced STAT signalling such as that elicited by various interferons and interleukins, it is thought that STAT proteins are recruited to the cytoplasmic side of the cell-surface receptor complex from within a monomeric cytosolic pool, and upon tyrosine-phosphorylation by respective Janus kinase family members, dimerize and translocate to the nucleus. The mechanisms which determine and regulate the recruitment of cytosolic STAT proteins to the plasma membrane-receptor complex, the transit of "activated" STATs through the expanse of the cytoplasmic compartment from the plasma membrane to the nuclear pore region, and the transit of STATs through the nuclear pore complex into the nuclear compartment, remain largely unknown. New data from different laboratories suggests consideration of a model for STAT signalling in which STAT proteins function in the cytoplasm not only as free monomers and dimers but as part of heteromeric complexes ("statosomes"), with accessory proteins which may serve to present specific STATs to the plasma membrane-receptor complex, and to chaperone "activated" STATs through the cytoplasmic compartment toward the nucleus and then into the nuclear compartment.

An increase in circulating levels of proinflammatory cytokines has been proposed as an important pathogenic factor contributing to cardiac injury during chronic heart failure. To determine whether plasma levels of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) increase during pacing-induced heart failure, we paced the hearts of seven dogs at 210 beats/min for 3 weeks and at 240 beats/min for an additional week to induce severe clinical signs of cardiac decompensation. Hemodynamic measurements and blood samples from the aorta and coronary sinus (CS) were taken at control, at 3 weeks, and in end-stage failure. Decompensated heart failure occurred at 29 +/- 1.8 days, when left ventricular (LV) end-diastolic pressure was 25 +/- 1.3 mmHg, LV systolic pressure was 92 +/- 4 mmHg, mean arterial pressure was 77 +/- 3 mmHg, and dP/dtmax was 1219 +/- 73 (all P < 0.05 vs control). Arterial concentration of IL-6 was 12 +/- 4.0 U/ml at control, 11 +/- 2.7 U/ml at 3 weeks, and 10 +/- 1.7 U/ml in end-stage failure (NS). At the same time points, IL-6 in CS plasma was 12 +/- 3.5, 13 +/- 2.8 and 11 +/- 2.4 U/ml, respectively (NS vs control and vs arterial concentrations). TNF-alpha did not reach detectable concentrations in arterial or CS blood at any time. TNF-alpha and IL-6 concentrations did not increase in arterial blood, were not released in the CS from the heart during the development of pacing-induced heart failure, and can not universally be implicated in the pathogenesis of all forms of cardiac dysfunction. Our findings are consistent with other data from patients in which severe heart failure was not associated with increased levels of circulating cytokines.

In the standard model of cytokine-induced signal transducer and activator of transcription (STAT) protein family signaling to the cell nucleus, it is assumed that STAT3 is recruited to the cytoplasmic side of the cell surface receptor complex from within a cytosolic monomer pool. By using Superose-6 gel-filtration chromatography, we have discovered that there is little monomeric STAT3 (91 kDa) in the cytosol of liver cells (human hepatoma Hep3B cell line and rat liver). The bulk of STAT3 (and STAT1, STAT5a, and -b) was present in the cytosol as high molecular mass complexes in two broad distributions in the size range 200-400 kDa ("statosome I") and 1-2 MDa ("statosome II"). Upon treatment of Hep3B cells with interleukin-6 (IL-6) for 30 min (i) cytosolic tyrosine-phosphorylated STAT3 was found to be in complexes of size ranging from 200-400 kDa to 1-2 MDa; (ii) a small pool of monomeric STAT3 and tyrosine-phosphorylated STAT3 eluting at 80-100 kDa was observed, and (iii) most of the cytoplasmic DNA-binding competent STAT3 (the so-called SIF-A "homodimer") co-eluted with catalase at 230 kDa. In order to identify the protein components of the 200-400-kDa statosome I cytosolic complexes, we used the novel technique of antibody-subtracted differential protein display using anti-STAT3 antibody. Eight polypeptides in the size range from 20 to 114 kDa co-shifted with STAT3; three of these (p60, p20a, and p20b) were co-shifted in an IL-6-dependent manner. In-gel tryptic fragmentation and mass spectroscopy identified the major IL-6-dependent STAT3-co-shifted p60 protein as the chaperone GRP58/ER-60/ERp57. Taken together, these data (i) emphasize the absence of a detectable STAT3 monomer pool in the cytosol of cytokine-free liver cells as posited by the standard model, and (ii) suggest an alternative model for STAT signaling in which STAT3 proteins function in the cytoplasm as heteromeric complexes with accessory scaffolding proteins, including the chaperone GRP58.

Objective: To determine the relationship between amniotic fluid (AF) neuron-specific enolase and the development of neonatal intraventricular hemorrhage and periventricular leucomalacia., Methods: Thirty-nine AF samples, obtained from women in preterm labor between 24 and 32 weeks' gestation, were analyzed for neuron-specific enolase. All women delivered preterm neonates who had neurosonograms on the 3rd and 7th days of life. The results of the neurosonograms were used to divide the study population first into normal and abnormal groups, then into normal, minor, and major brain lesion groups. The groups were compared for the median neuron-specific enolase, proportion with values of 6 microg/L or more, and other demographic characteristics., Results: There were no differences between the groups' maternal and neonatal characteristics. However, the abnormal group had significantly higher median value of neuron-specific enolase than the normal group (9.5 microg/L and 2.0 microg/L, respectively; P < .001). The median neuron-specific enolase levels for the major, minor, and normal groups were 9.75 microg/L, 6.5 microg/L and 2.0 microg/L, respectively (P < .001). The optimum cutoff point, with a sensitivity of 89% and specificity of 100%, was 6 microg/L; 89% of the abnormals had values of 6 microg/L or more, compared with none of the normals (P < .001). The risk of developing intraventricular hemorrhage or periventricular leucomalacia was 11.5 times greater when AF neuron-specific enolase levels were 6 microg/L or more., Conclusion: Amniotic fluid neuron-specific enolase is a useful marker of neonatal neurologic injury.

> Objective: To investigate the relationship between amniotic fluid interleukin-6 levels and the development of periventricular leukomalacia and intraventricular hemorrhage in the preterm neonate and to compare the value of amniotic fluid interleukin-6 with amniotic fluid culture and histologic chorioamnionitis in the prediction of periventricular leukomalacia and intraventricular hemorrhage. Methods: 119 women, between 20 and 34 weeks gestation, in preterm labor with intact membranes, underwent transabdominal amniocentesis. Amniotic fluid was cultured for aerobic and anaerobic bacteria, Ureaplasma urealyticum and Mycoplasma hominis. Amniotic fluid interleukin-6 levels were determined by enzyme-linked immunosorbent assay. The placentas were examined for histopathologic evidence of inflammation. Where the birth weight was <2,000 g, transfontanelle cranial sonography was performed on the 3rd and 7th days of life for diagnosis of periventricular leukomalacia and intraventricular hemorrhage. Student's t test, the Mann-Whitney U test, likelihood ratio chi2, logistic regression, and receiver-operator characteristic curve were used for analysis. Results: 33 women were excluded from the analysis because they delivered at other institutions. The neonates of 33 women did not have sonography because they weighed >2,000 g at birth. Two neonates died before sonography was performed; four neonates who weighed <2,000 g at birth did not have sonography. In the definitive study group of 47 women, those with neonates who developed periventricular leukomalacia and intraventricular hemorrhage (n = 14) had higher median amniotic fluid interleukin-6 levels (42,795 pg/ml versus 8,020 pg/ml; P = 0.009), more positive amniotic fluid cultures (64% vesus 21%; P < 0.003), and a shorter median amniocentesis-to-delivery interval (16 h versus 24 h; P = 0.045) than women (n = 33) who delivered neonates without periventricular leukomalacia or intraventricular hemorrhage. The groups did not differ in gestational age at admission (P = 0.15), birth weight (P = 0.09), or histologic chorioamnionitis (P = 0.37). An amniotic fluid interleukin-6 level >/=20,000 pg/ml had a sensitivity of 71% and a specificity of 70% compared with a sensitivity of 69% and specificity of 79% for amniotic fluid culture, and a sensitivity of 71% and specificity of 42% for histologic chorioamnionitis in the prediction of periventricular leukomalacia and intraventricular hemorrhage. Women with amniotic fluid interleukin-6 levels >/=20,000 pg/ml (n = 20) had more neonates with periventricular leukomalacia or intraventricular hemorrhage than women with amniotic fluid interleukin-6 levels <20,000 pg/ml (n = 27) (50% versus 15%; P = 0.009). They also were of lower birth weight (P = 0.02), had more neonatal morbidity (P = 0.01), had more positive amniotic fluid cultures (P = 0.01), and more histologic chorioamnionitis (P = 0.02). Logistic regression analysis demonstrated that amniotic fluid interleukin-6 was an independent risk factor for the development of periventricular leukomalacia and intraventricular hemorrhage (odds ratio, 5.81; 95% confidence interval, 1.02-33.16; P = 0.05) after controlling for gestational age, birth weight, histologic chorioamnionitis, and amniotic fluid culture (odds ratio, 7.94; 95% confidence interval 1.22-51.77; P = 0.03). Conclusions: In women in preterm labor with intact membranes amniotic fluid interleukin-6 is useful in predicting neonatal periventricular leukomalacia and intraventricular hemorrhage.

The influence of p53 on cytokine-triggered Janus kinase-STAT signaling was investigated in human hepatoma Hep3B cell lines engineered to constitutively express the temperature-sensitive Val135 mutant of p53. In comparison to the parental p53-free Hep3B cells, these p53-Val135-containing Hep3B cell lines displayed a reduced response to IL-6 at the wild-type-like p53 temperature (32.5 degrees C). In these cells, IL-6 induced a marked reduction in the immunologic accessibility of cytoplasmic and nuclear STAT3 and STAT5 within 20 to 30 min that lasted 2 to 4 h (STAT-masking) provided that the cells had been previously cultured at 32.5 degrees C for at least 18 to 20 h. The onset of IL-6-induced STAT-masking required protein tyrosine kinase, protein tyrosine phosphatase, proteasomal, phospholipase C, and mitogen-activated protein kinase kinase 1 activities. The maintenance of IL-6-induced STAT-masking was dependent on continued signaling through the phosphatidylinositol-dependent phospholipase C pathway. Despite a reduction in IL-6-induced STAT3 DNA binding activity in the nuclear compartment during STAT-masking, there was increased and prolonged accumulation of tyrosine-phosphorylated STAT3 in both the cytoplasmic and nuclear compartments, indicating that the capacity of tyrosine-phosphorylated STAT3 to bind DNA was reduced during STAT-masking. Thus, IL-6-induced STAT-masking, as dramatically evident on immunomicroscopy, is a visible consequence of a novel cellular process by which a p53-Val135-induced gene product(s) regulates the association of masking protein(s) with and the DNA-binding capacity of STAT3.

Transport of IL-6 in blood is fundamental to the biology of this cytokine. In the present study, IL-6 transport, immunological reactivity, and biological availability were investigated in blood from melanoma patients subjected to different active specific immunization regimens (an anti-idiotypic mAb immunization protocol (mAb-keyhole limpet hemocyanin (KLH)-Calmette-Guerin bacillus (BCG), an autologous anti-cancer vaccine protocol (AAAP), or both). Sera were subjected to Sephadex G-200 gel filtration chromatography, and the structure and biological activity of IL-6 complexes in the eluate fractions were probed using five IL-6 ELISAs and two bioassays. Sera from patients administered mAb-KLH+BCG followed by AAAP contained three distinct classes of IL-6 eluting at 30, 200, and 450 kDa, each with its characteristic ELISA reactivity and bioactivity: the 30- and 450-kDa complexes were bioactive in the B9 and Hep3B assays, but the 200-kDa complex was not. The 30- and 450-kDa IL-6 complexes were preferentially reactive in the 7IL6/5IL6 ELISA, the 200-kDa IL-6 complexes were preferentially reactive in the 4IL6/5IL6 ELISA, while the three commercial ELISAs (R&D, Endogen, and Genzyme) detected essentially only the 30-kDa IL-6. In contrast, 1) sera from AAAP patients contained biologically active 30- and 450-kDa IL-6 complexes, while 2) sera from mAb-KLH+BCG patients contained 200-kDa IL-6 complexes inactive in ex vivo bioassays. Both the 450- and 200-kDa complexes included soluble IL-6R, with the 200-kDa complexes additionally containing ligand-occupied anti-IL-6 and anti-soluble IL-6R IgG. The data indicate the existence of specific mechanisms that regulate the transport and function of IL-6 in vivo.

The relationship between plasma nitrite, nitrate, and nitric oxide (NOx), cytokines, and cardiac and vascular dysfunction after lipopolysaccharide (LPS) was studied in chronically instrumented anesthetized dogs. LPS was administered (1 mg/kg i.v.), and hemodynamics were recorded at baseline, every 15 min for 1 h, and every hour for an additional 14 h. Dramatic reductions in mean arterial pressure (-48 +/- 6%), cardiac output (-40 +/- 8%), stroke volume (-42 +/- 9%), and first derivative of left ventricular pressure (LV dP/dt, -38 +/- 7%) were seen within 1 h after injection of endotoxin. Cardiac output was not different from control by 9 h, whereas mean arterial pressure (-19 +/- 7%), stroke volume (-32 +/- 8%), and LV dP/dt (-21 +/- 10%) remained significantly depressed from control. Total peripheral resistance was not significantly different from control. Therefore, the hypotension appears to be due to a reduction in cardiac function and not to vasodilation. Levels of plasma NOx were not different from control until 4 h after LPS reached levels 597 +/- 126% higher than control at 15 h. In vitro production of nitrite by coronary microvessels was also elevated, supporting our in vivo findings. In contrast, production of tumor necrosis factor-alpha and interleukin-6 occurred shortly after endotoxin injection, reaching peak levels at 45 and 150 min, respectively. Our data suggest that inducible nitric oxide synthase induction occurred after LPS injection. It is unlikely that nitric oxide contributed significantly to the hypotension and cardiac dysfunction early in our study, whereas cardiodepressive cytokines, particularly tumor necrosis factor-alpha, may be important. In contrast, the hemodynamic effects seen late after injection of endotoxin may be the result of an overproduction of nitric oxide, since there was a sixfold increase in plasma NOx levels at this time and a marked production of nitric oxide in isolated coronary microvessels in vitro.

Hepatoma Hep3B cell lines stably expressing a temperature-sensitive p53 species (p53-Val-135) displayed a reduced response to interleukin-6 (IL-6) when cultured at the wild-type (wt) p53 temperature (Wang, L., Rayanade, R., Garcia, D., Patel, K., Pan, H., and Sehgal, P. B. (1995) J. Biol. Chem. 270, 23159-23165). We now report that in such cultures IL-6 caused a rapid (20-30 min) and marked loss of cellular immunostaining for STAT3 and STAT5, but not for STAT1. The loss of STAT3 and STAT5 immunostaining was transient (lasted 120 min) and tyrosine kinase-dependent, and even though the loss was blocked by the proteasome inhibitors MG132 and lactacystin it was not accompanied by changes in cellular levels of STAT3 and STAT5 proteins suggesting that IL-6 triggered a rapid masking but not degradation of these transcription factors. STAT3 and STAT5 masking was accompanied by a reduction in IL-6-induced nuclear DNA-binding activity. The data suggest that p53 may influence Jak-STAT signaling through a novel indirect mechanism involving a wt p53-dependent gene product which upon cytokine addition is activated into a "STAT-masking factor" in a proteasome-dependent step.

Investigators have traditionally thought of the class of inflammation- and injury-associated cytokines in large part as "free" entities in the peripheral circulation. In the case of interleukin-6 (IL-6), the cytokine can be found in blood in complexes of molecular mass 400-500, 150-200, and 25-35 kDa in association with binding proteins that can include soluble IL-6 receptor (sIL-6R), anti-IL-6, and anti-sIL-6R IgG, and others. Sustained high levels of different particular IL-6 complexes are observed in the human circulation in cancer patients subjected to particular active anticancer immunotherapy regimens. In the "chaperoned" state, circulating IL-6 complexes display differential immunoreactivity in different ELISAs and possess differential biological activity as assayed ex vivo. The discovery of "chaperoned" circulating IL-6 in humans points to a new level of modulation of cytokine function, that of regulated bioavailability of IL-6 in vivo.

The ability of p53 species (wild-type and mutant) to modulate the "differentiated" response of human hepatoma cell lines Hep3B and HepG2 to interleukin-6 (IL-6) was investigated. Transient transfection experiments were carried out in Hep3B and HepG2 cell cultures in which IL-6 was used to activate a beta-fibrinogen (beta Fib) enhancer/reporter construct containing two copies of the 36-base pair IL-6-response element (IL-6RE) (p beta FibCAT). Cotransfection with constitutive expression vectors for wild-type (wt) human or murine p53 inhibited the activation of the p beta FibCAT reporter by IL-6 in both Hep3B and HepG2 cells. Several mutant p53 species either did not inhibit the activation of p beta FibCAT or up-regulated the response. Hepatoma cell lines stably expressing the Val-135 temperature-sensitive mutant of murine p53 (wt-like at 32.5 degrees C and mutant-like at 37 degrees C) were derived from Hep3B cells and tested for the temperature-sensitive phenotype of their ability to synthesize and secrete fibrinogen and alpha 1-antichymotrypsin in response to IL-6. In an experimental protocol in which the parental Hep3B cells did not show a significant difference in plasma protein secretion at the two temperatures, hepatoma line 3 (p53Val-135+) had a greater response to IL-6 at 37 degrees C than parental Hep3B cells, while line 3 cells had a reduced response to IL-6 at 32.5 degrees C. Similarly, hepatoma lines 1 and 2 (both p53Val-135+) had reduced IL-6 responsiveness at 32.5 degrees C, whereas line 22 (transfected with pSVneo alone) and the parental Hep3B cells did not. These data indicate that mutations in p53 contained in tumor cells can modulate the "differentiated" response of these cells to cytokines.

The p53 mutant Val135 is widely considered to have a wild-type (wt) phenotype at 32.5 degrees C, but not at 37 degrees C. The ability of wt murine p53 and its Val135 mutant to modulate transcription from the muscle-specific creatine kinase promoter (-3.3 kb pMCK), from a reporter construct containing two copies of the p53-binding DNA element from within MCK (p50-2), and from the interleukin-6 (IL-6) promoter (pIC225) was evaluated in transient transfection experiments in CV1 and HeLa cells. In CV1 cells, wt p53 was confirmed to activate the pMCK and p50-2 reporters, but to repress the IL-6 promoter. However, although in these cells p53 Val135 had the expected wt-like phenotype with respect to activation of the p50-2 reporter at 32.5 degrees C (32.5 degrees C > 37 degrees C), this mutant had little effect on expression from pMCK at either temperature, and activated rather than repressed the IL-6 promoter at 32.5 degrees C. In HeLa cells, although wt p53 activated p50-2 but repressed the MCK and IL-6 promoters, p53 Val135 activated all three reporters. Unexpectedly, in these cells the upregulation of p50-2 and pIC225 was basically temperature-independent, and that of pMCK was inversely ts (37 degrees C > 32.5 degrees C). The novel ts properties of p53 Val135 show that this mutant is not always wt-like at 32.5 degrees C but exhibits strong cell-type and promoter-dependent differences in its ts phenotype for transcriptional modulation.

In the baboon or the mouse, a stimulus such as LPS, TNF, or IL-1 typically led to a rapid induction of circulating IL-6, the levels peaked by 2 to 3 h and then declined to near-baseline values by 6 to 8 h. Administration to baboons or mice of "neutralizing" anti-IL-6 mAb followed by an IL-6 inducer led to a marked and sustained increase in circulating IL-6 levels. IL-6 Ag, IL-6 biologic activity, neutralizing anti-IL-6 mAb, and IL-6/anti-IL-6-mAb complexes could all be observed for an extended period of time (beyond 8 h) in the circulation of such animals. Nevertheless, in mice, if the anti-IL-6 mAb had been administered before the IL-6 inducer, there was a reduction in the in vivo IL-6-induced stimulation of fibrinogen levels, indicating that most of the intravascular IL-6 was not readily available for eliciting hepatocyte effects under these experimental conditions. Intraperitoneal administration into mice of mixtures of murine rIL-6 or human rIL-6 together with their respective anti-IL-6 mAb led to a marked increase in the appearance and longevity in the peripheral circulation of the exogenously administered murine or human rIL-6 species in a biologically active form. Varying the ratio of human rIL-6 to anti-human IL-6 mAb indicated that a molar ratio of 1:1 was sufficient for the ability of mAb to chaperone IL-6 in the murine circulation. Human rIL-6 mixed with "neutralizing" mAb in the approximate ratio 1:1 elicited an enhanced fibrinogen response in vivo in the mouse; an IL-6:mAb ratio of 1:125 led to a reduction in the fibrinogen response even though the levels of circulating B9 bioactivity and of human rIL-6-Ag were maximal under these conditions. Gel-filtration chromatographic and Western blotting analyses of IL-6 present in vivo in the mAb-free baboon revealed that although the IL-6 Ag was largely present in high molecular mass complexes of size 400 kDa in association with soluble IL-6 receptor, the B9 bioactivity was largely of low molecular mass (20 kDa). In contrast, in the anti-IL-6 mAb-treated baboon or mouse, the IL-6 Ag and bioactivity were both largely in complexes of 200 kDa. Thus, the binding of IL-6 in the intravascular compartment to other proteins, anti-IL-6 mAb in the present studies, gives IL-6 unexpected biochemical and pharmacologic properties in vivo.

Problem: The purpose of this study was to determine if amniotic fluid concentrations of the interleukin-6 (IL-6) are of value in diagnosis of microbial invasion of the amniotic cavity and in the prediction of failure of tocolysis, preterm delivery and perinatal morbidity and mortality., Method: Amniotic fluid was obtained by transabdominal amniocentesis from 146 consecutive patients admitted with the diagnosis of preterm labor and intact membranes. Fluid was cultured for aerobic and anaerobic bacteria as well as for mycoplasmas. Amniotic fluid IL-6 levels were measured using a monoclonal antibody-based enzyme-linked immunosorbent assay with a sensitivity of 0.03 ng/ml. Logistic regression and Cox's proportional hazards model were used to examine the effect of several variables on dichotomous outcomes or interval to delivery., Results: Patients with a positive amniotic fluid culture had a significantly higher amniotic fluid IL-6 concentrations than patients with a negative culture (median 91.2 ng/ml, range 0.9 to 437 ng/ml versus median 0.4 ng/ml, range < 0.3 to 195 ng/ml, respectively; P < .0001). An amniotic fluid IL-6 concentration of greater than or equal to 11.3 ng/ml had a sensitivity of 93.3% (14 of 15) and a specificity of 91.6% (120 of 131). All patients with an amniotic fluid IL-6 concentration above 11.3 ng/ml and a negative amniotic fluid culture (N = 11) delivered preterm and all placenta available for examination (N = 7) had histologic evidence of chorioamnionitis. Amniotic fluid concentrations of IL-6 were an independent predictor of preterm delivery, amniocentesis-to-delivery interval and neonatal morbidity and mortality. Moreover, IL-6 concentrations added significant information to the prediction of these outcomes to that provided only by clinical information such as cervical dilatation, gestational age at admission or at delivery., Conclusion: IL-6 is a sensitive and rapid test for the detection of microbial invasion of the amniotic cavity and for identifying women at risk for spontaneous preterm delivery and neonates at risk for morbidity and mortality.

Constitutive up-regulation of interleukin-6 (IL-6) gene expression is observed in many neoplastic cell lines. The contribution of mutations in p53 to the up-regulation of the IL-6 promoter was evaluated in transient transfection experiments. In HeLa cells, wild-type (wt) human or murine p53 preferentially repressed the IL-6 promoter. The p53 mutants Val-135 and Phe-132 up-regulated IL-6 promoter activity in these cells at both 32.5 and 37 degrees C. The temperature-sensitive Val-135 mutant was not only not inhibitory or "wt-like" at the lower temperature, but had gained a transcriptional activator phenotype which was temperature-independent in HeLa cells. The functional DNA target for transcriptional modulation of the IL-6 promoter by p53 species included the multiple cytokine- and second messenger-response element (-173 to -145); point mutations in the transcription factor C/EBP beta-binding site within the second messenger-response element largely blocked the ability of p53 mutants Val-135 and Phe-132 to up-regulate this promoter. The up-regulation of IL-6 promoter constructs by co-transfection into HeLa cells of a C/EBP beta constitutive expression vector was blocked in a dominant negative manner by wt p53. In contrast, the p53 mutants Val-135 and Phe-132 further enhanced C/EBP beta-mediated up-regulation of IL-6 promoter constructs. The modulation of C/EBP beta function by p53 species provides a basis for the involvement of p53 not only in the regulation of cytokine synthesis but also in the altered responsiveness of tumor cells to cytokines.

The biochemical nature of endogenous interleukin-6 (IL-6) as it exists in human serum or plasma was investigated. Serum from a patient following bone marrow (BM) transplantation and fresh plasma samples from patients with epidermolysis bullosa or psoriasis, as well as from normal volunteers, were fractionated through G-200 columns and each of the eluted fractions assayed for IL (interleukin)-6 content using enzyme-linked immunosorbent assays (ELISAs) based on the monoclonal antibody (mAb) pairs IG61/5IL6 or 4IL6/5IL6 and in the B9 hybridoma growth factor bioassay. The IG61/5IL6 ELISA and the B9 assay detected IL-6 in BM serum almost exclusively of molecular mass approximately 20 kDa. In contrast, the 4IL6/5IL6 ELISA detected strong IL-6 immunoreactivity in complexes of size 100-150 and 400-500 kDa. IL-6 present in the 100-150- and 400-500-kDa complexes was purified by immunoaffinity chromatography through a 5IL6 mAb column. The 5IL6 mAb immunoaffinity column eluate of the respective pools from BM serum contained IL-6 at concentrations approaching 1 microgram/ml as characterized by Western blotting. Sufficient IL-6 and associated proteins were purified by 5IL6 mAb immunoaffinity column chromatography of the 100-150-kDa complex from 0.8 ml of BM serum to allow (i) verification of three of the polypeptides as IL-6 by amino-terminal sequencing (estimate of IL-6 in original serum sample: 5-10 micrograms/ml), (ii) identification by amino acid sequencing of the "associated" proteins as complement factor C3b (carboxyl-terminal of the alpha-chain), complement factor C4b (gamma-chain), C-reactive protein, and albumin, and (iii) detection of an "associated" polypeptide consistent with the soluble IL-6 receptor. Taken together, these data establish that IL-6 is present at unexpectedly high concentrations in human blood in novel biochemical complexes that include other plasma proteins, which in turn, can camouflage IL-6 immunoreactivity and bioactivity as measured in conventional assays.

The potential involvement of cytokines in acute graft-versus-host disease led us to analyze interleukin-6 in serial serum sets from 22 allogeneic marrow recipients who developed either grade 3 or 4 GVHD (n = 10), grade 2 GVHD (n = 6), or grade 1 or no diagnosed GVHD (n = 6). A total of 279 serial serum samples taken three times weekly before day 35 were analyzed. Maximum IL-6 levels were greater than 40 U/ml (range, 40-1536 U/ml), 11-40 U/ml, and less than or equal to 10 U/ml for six, eleven, and five patients, respectively. Serum IL-6 peaks were temporally related to onset of GVHD, onset of a syndrome of hepatorenal dysfunction (HRD), or bilateral lung infiltration. Eight of ten patients who developed grade 3 or 4 GVHD overall had IL-6 maxima of greater than 10 U/ml an average of 1.5 +/- 1.8 days before the clinical onset. Fifteen of 17 patients with peak IL-6 levels greater than 10 U/ml developed symptoms of hepatic and renal dysfunction within three days of the peak, while none of five patients with less than or equal to 10 U/ml of Il-6 developed HRD. Regression analysis demonstrated a linkage between the log magnitudes of the serum IL-6 peaks and onset of either GVHD or HRD within three days (P = 0.001). Furthermore, IL-6 peaks tended to precede GVHD onset for the 10 patients whose GVHD onset and IL-6 peak were within three days of each other (P = 0.02). These results, confirmed by both specific bioassay and by IL-6 ELISA, support the idea that acute GVHD in humans involves a cytokine cascade that includes production of IL-6 in addition to the previously reported involvement of tumor necrosis factor alpha and interferon-gamma.

Uterine stromal (USC) and uterine epithelial (UEC) cells were isolated from immature and mature mice to determine their ability to secrete interleukin-6 (IL-6) in response to ovarian steroids, IL-1 alpha, and soluble products produced by the heterologous cell type. In addition, the effect of IL-6 on embryo attachment and outgrowth in vitro was determined. UEC cultured on nitrocellulose filter inserts in a polarized manner secreted IL-6 with a 2.5- to 5-fold apical vs. basal preference, as determined by a B9 hybridoma cell proliferation assay and enzyme-linked immunosorbent assay. The hormonal status of animals at the time uteri were removed did not influence subsequent secretion of IL-6, as UEC isolated from immature, diestrous, and estrous stage mice exhibited both a similar amount and had a similar apical preference for secretion of IL-6. The addition of 17 beta-estradiol (E) to UEC cultures markedly inhibited total IL-6 secretion, but did not affect vectorial secretion. The inhibitory effect of E on IL-6 secretion by UEC was consistent with an apparent decrease in IL-6 transcript observed by a reverse transcriptase polymerase chain reaction assay. Other transcripts detected by this assay in UEC included IL-1 alpha, but not IL-1 beta or tumor necrosis factor-alpha. Secretion of IL-6 by UEC was not stimulated by IL-1 alpha, conditioned medium from USC, or coculture with USC. USC secreted IL-6, and while this also was inhibited by E, progesterone was more effective in this regard at physiological concentrations. In addition, there was a synergistic effect of E plus progesterone on inhibition of IL-6 secretion by USC. Secretion of IL-6 by USC was stimulated by IL-1 alpha, and coculture studies demonstrated the ability of UEC to stimulate a several-fold increase in IL-6 secretion by USC. The cytokine transcripts detected in USC cultures included IL-6 and IL-1 alpha, but not IL-1 beta. Transcripts for tumor necrosis factor-alpha were present in USC only after culture with IL-1 alpha. IL-6 added to blastocysts on laminin-coated tissue culture wells resulted in a transient inhibition of the rate of blastocyst attachment and, to a greater extent, an inhibition of the rate of embryo outgrowth. In addition, IL-6 inhibited the size of embryo outgrowths at 24 and 48 h of culture.(ABSTRACT TRUNCATED AT 400 WORDS)

The cytokine interleukin-6 (IL-6) is the major phosphoprotein secreted by human fibroblasts induced with interleukin-1 alpha (IL-1 alpha). We have determined that Ser54 is the predominant site of phosphorylation on the fibroblast-derived IL-6 polypeptide; the amino acid motif surrounding this site is reminiscent of the target site for the Golgi-associated protein (casein) kinase. It has been shown earlier that the IL-6 polypeptide follows the classical secretory pathway where N-linked glycosylation is detectable within the first 15 minutes of labeling with [35S]-methionine and O-linked glycosylation occurs between 15-30 minutes after the start of polypeptide synthesis. Pulse-chase experiments using [32P]-orthophosphate or [35S]-methionine as tracers indicated that phosphorylation of IL-6 occurred prior to its O-glycosylation suggesting that the de novo synthesized IL-6 polypeptide is rapidly, perhaps even cotranslationally, phosphorylated at an intravesicular site (in the endoplasmic reticulum and/or Golgi). When IL-1 alpha-induced fibroblasts were exposed to cycloheximide there was enhancement of the net de novo synthesis and secretion of IL-6 as followed by [35S]-methionine labeling ("superinduction") but the secreted cytokine was no longer phosphorylated as monitored by [32P] labeling. Thus, phosphorylation of the IL-6 polypeptide is not an obligatory requirement for secretion of this cytokine. Furthermore, IL-6 phosphorylation is inhibited by cycloheximide through a mechanism different from the drug's effects on polypeptide synthesis per se.

Recent years have seen the discovery and molecular characterization of a bewildering array of cytokines and hematopoietic growth factors--and an even more complex description of their overlapping functions. The molecular cloning of a wide array of the cell-surface receptors for these cytokines has led to the recognition of classes of structurally related receptor superfamilies. The functional receptors for many of these cytokines (e.g., interleukin (IL)-2R and IL-6R) involve two distinct subunits; strikingly, the same beta subunit can interact with distinct alpha subunits to constitute the receptor for different cytokines (e.g., those for IL-3, IL-5, and granulocyte monocyte colony-stimulating factor). Considerable progress has also been made in defining the molecular mechanisms that underlie clinically relevant cytokine-related phenomena. As an example, the molecular mechanism by which glucocorticoids inhibit IL-6 gene expression has been shown to include the occlusion of the inducible enhancer and the basal promoter elements in the IL-6 promoter. Acute rejection episodes in renal transplant patients are accompanied by increases in serum IL-6 levels; the administration of glucocorticoids during these episodes leads to a rapid and marked decrease in IL-6 levels. It appears that the serum IL-6 level may be a useful diagnostic and prognostic indicator in the transplant patient.

It was found that IL-1 is produced in livers and spleens of mice shortly after the i.v. injection of a sublethal or lethal Listeria monocytogenes inoculum. In sublethally infected mice, IL-1 was present in infected livers and spleens by the end of the first day of infection. Thereafter, the amounts of IL-1 in these organs increased and decreased in concordance with bacterial numbers. IL-1 was not present in the peripheral circulation of mice during sublethal listeriosis, but was present in the blood late in lethal infection. Evidence showing that IL-1 plays a role in antibacterial resistance early in listeriosis was obtained through the use of 35F5 mAb that binds to the murine type I IL-1R and functions to block IL-1 alpha and IL-1 beta actions. Blockade of the type I IL-1R by the 35F5 mAb results in greatly enhanced bacterial growth in the livers and spleens of mice that had received a sublethal Listeria inoculum. Consistent with the exacerbation of listeriosis caused by 35F5 mAb, but in contrast to the effect of 35F5 mAb in other murine models, 35F5 mAb-treated mice exhibit markedly elevated levels of IL-6 in their circulation and infected organs.

Cyclosporine (CSA) decreases lymphokine synthesis and keratinocyte proliferation in vitro, but its in vivo mechanism of action in treating recalcitrant psoriasis is incompletely understood. Ten psoriasis patients were treated with CSA (2-7.5 mg/kg/d) with clinical improvement in nine of 10 patients. Skin biopsies before and after 1-3 months of CSA treatment were studied for evidence of immune and keratinocyte activation using immunoperoxidase and Northern blotting analysis. The number of activated, IL-2 receptor+ T cells in plaques after CSA treatment was reduced in all patients by a mean of 60%. Seven of 10 patients showed a decrease in keratinocyte HLA-DR expression; five of seven showed a decrease in gamma-IP-10 immunoreactivity, suggesting a decline in gamma interferon levels in plaques after CSA therapy. We studied the effect of CSA treatment in vivo on TGF-alpha, IL-6, and keratin K16 expression, three markers of keratinocyte growth activation. Expression of keratinocyte TGF-alpha and IL-6, which are elevated in active psoriatic epidermis, did not change in these patients after CSA treatment. The majority of patients (five of eight) continued to express the hyperproliferative keratin K16 after CSA treatment. Our results suggest that the predominant direct mechanism of action of Cyclosporine in vivo is a diminution of T-cell activation in plaques, with attendant decreased lymphokine production.

A growing body of evidence suggests a role for cytokines in the mechanisms responsible for preterm parturition associated with intrauterine infection. Interleukin 6, a polyfunctional cytokine that is secreted by tissues in the feto-maternal interface in response to microbial products, has been implicated in the host response to intrauterine infection. The purpose of this study was to establish whether measurement of amniotic fluid concentrations of interleukin 6 could be of value in the diagnosis of microbial invasion of the amniotic cavity. Fluid was obtained by transabdominal amniocentesis from patients with preterm labour and intact chorioamniotic membranes and cultured for aerobic and anaerobic bacteria and mycoplasmas. Interleukin 6 concentrations were determined by an ELISA validated for human amniotic fluid. An interleukin 6 concentration above 11.2 ng/ml had a 93.7% sensitivity and a 92.3% specificity in the diagnosis of intra-amniotic infection. Moreover, patients with an amniotic fluid interleukin 6 level above 11.2 ng/ml and a negative amniotic fluid culture failed to respond to tocolysis, delivered a preterm infant and showed histological evidence of chorioamnionitis, and their neonates were at risk for congenital infections.

The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell elements in various B- and T-cell malignancies and in some carcinomas and sarcomas; many of these neoplasms have been shown to be associated with a mutated p53 gene. The possibility that wild-type (wt) p53, a nuclear tumor-suppressor protein, but not its transforming mutants might serve to repress IL-6 gene expression was investigated in HeLa cells. We transiently cotransfected these cells with constitutive cytomegalovirus (CMV) enhancer/promoter expression plasmids overproducing wt or mutant human or murine p53 and with appropriate chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoter elements of human IL-6, c-fos, or beta-actin genes or of porcine major histocompatibility complex (MHC) class I gene in pN-38 to evaluate the effect of the various p53 species on these promoters. Murine and human wt p53 derived from pCMVNc9 and pC53-SN3, respectively, strongly repressed the IL-6 (promoter position -225 to +13), c-fos (-711 to +42), beta-actin (-3400 to +912), and MHC (-528 to -38) promoters in serum-induced HeLa cells; additionally, IL-6 promoter/CAT transcription unit constructs induced by IL-1, phorbol ester, or pseudorabies virus were also repressed by wt human and murine p53. The murine transforming mutant p53 (pCMVc5) was less active in repressing the IL-6, c-fos, beta-actin, and MHC promoter constructs. The human p53 mutant derived from pC53-SCX3 was also less active than the wt protein in repressing the IL-6, c-fos, beta-actin, and MHC promoters, except that serum-induced IL-6/CAT expression was equally repressed by both human wt and mutant p53. In similar transient transfection experiments in HeLa cells, overexpression of the wt human retinoblastoma susceptibility gene product, RB, was found to repress the serum-induced IL-6 (-225 to +13), c-fos (-711 to +42), and beta-actin (-3400 to +912) promoters but not the PRV-induced IL-6 (-110 to +13) or the serum-induced MHC (-528 to -38) promoters. These observations identify transcriptional repression as a property of p53 and suggest that p53 and RB may be involved as transcriptional repressors in modulating IL-6 gene expression during cellular differentiation and oncogenesis.

Transfection of HeLa cells with cDNA vectors expressing the wild-type human glucocorticoid receptor (GR) enabled dexamethasone to strongly repress cytokine- and second messenger-induced expression of cotransfected chimeric reporter genes containing transcription regulatory DNA elements from the human interleukin 6 (IL-6) promoter. Deletion of the DNA-binding domain or of the second Zn finger or a point mutation in the Zn catenation site in the second finger blocked the ability of GR to mediate repression of the IL-6 promoter. Unexpectedly, deletion of the first Zn finger, a point mutation in the Zn-catenation site in the first finger, or one in the steroid-specificity domain at the base of the first finger converted GR into a dexamethasone-responsive activator that enhanced basal and interleukin 1-induced IL-6 promoter function. These first-finger mutants of GR also mediated dexamethasone-responsive enhancement of expression of the herpesvirus thymidine kinase-chloramphenicol acetyltransferase (TK-105-CAT and TK-80-CAT) reporter genes but not of the murine mammary tumor virus long terminal repeat-CAT or the c-fos-CAT (pFC700) reporter genes. Wild-type GR was able to specifically bind to DNA fragments containing glucocorticoid response element sequences in both the murine mammary tumor virus and IL-6 promoters, albeit weakly to the latter, in a sequential DNA-binding immunoprecipitation assay. The first-finger mutants of GR, however, were inactive in this assay. Thus, mutations in the first Zn finger unmask unusual promoter-specific activation properties of GR that may not require direct high-affinity binding of the mutant GR to target DNA.

Natural human interleukin-6 (IL-6) characterized under completely denaturing conditions consists of a set of differentially modified phosphoglycoproteins of molecular mass in the range from 23 to 30 kDa ("25-kDa" O-glycosylated species and "30-kDa" O- and N-glycosylated species). The 25-kDa O-glycosylated IL-6 (which contains only Ser- or Thr-GalNAc-Gal-NeuNAc and thus should not bind wheat germ or lentil lectins) bound to and was eluted from a wheat germ lectin affinity column by GlcNAc and from a lentil lectin affinity column by methyl-alpha-D-Man suggesting that the 25-kDa IL-6 species formed heteromeric complexes with the N-glycosylated 30-kDa IL-6. In non-denaturing gels (0.2% Nonidet P-40-polyacrylamide gel electrophoresis (PAGE)), even under reducing conditions (15 mM dithiothreitol or 1 M beta-mercaptoethanol and heating), fibroblast-derived IL-6 migrated as a predominant complex of mass approximately 85 kDa and additional minor 45-65-kDa complexes. Little IL-6 was detected in the size range 23-30 kDa. Elution of the major 85-kDa complex and re-electrophoresis through sodium dodecyl sulfate-PAGE revealed that it represented a heteromeric aggregate of the 25- and 30-kDa IL-6 species; the 45-65-kDa complexes were largely composed of the 25-kDa protein. The bulk of fibroblast-derived IL-6 eluted in the size range 45-85 kDa from a Sephadex G-200 gel filtration column further indicating that fibroblast-derived IL-6 was largely multimeric even in dilute solutions. Functionally, the high molecular mass IL-6 fractions from the G-200 column were less active in the B9 hybridoma growth factor assay than the lower molecular mass fractions but appeared to be equally active in the Hep3B hepatocyte-stimulating factor assay. Taken together, the data indicate that natural human IL-6 exists as a multimeric aggregate with varying biological activity.

Human interleukin-6 (IL-6) secreted by cytokine- or endotoxin-induced fibroblasts, monocytes, keratinocytes, endometrial stromal cells, and endothelial cells, when analyzed under denaturing and reducing conditions, consists of a set of differentially modified phosphoglycoproteins of molecular mass in the range from 23 to 30 kD (a set of at least three O-glycosylated 23- to 25-kD species and a set of at least three N- and O-glycosylated 28- to 30-kD species). The 23- to 25-kD and 28- to 30-kD fibroblast-derived IL-6 species have been separately purified to homogeneity with the use of a combination of lectin and immunoaffinity chromatography. Glycosidase digestion experiments on such purified preparations confirmed that almost all human fibroblast-derived IL-6 species were O-glycosylated; additionally, the 28- to 30-kD species were N-glycosylated. Amino acid sequencing revealed that the major amino terminus in the fibroblast-derived 23- to 25-kD O-glycosylated IL-6 was at Ala28 whereas the major amino terminus in the 28- to 30-kD N- and O-glycosylated IL-6 was at Val30, suggesting that targeting of newly synthesized IL-6 polypeptides into the two different processing pathways in fibroblasts may be keyed to differences in the signal peptide cleavage site. Unexpectedly, IL-6 "constitutively" secreted by the Epstein-Barr virus (EBV)-infected human and primate (tamarin) B-cell lines designated sfBJAB and sfBT, respectively, consisted of a major apparently unglycosylated 21-kD species and a minor 25-kD N-glycosylated species.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-6 (IL-6) causes an epithelial to fibroblastoid conversion and an increase in the motility of human ductal breast carcinoma cell lines ZR-75-1 and T-47D. Although IL-6 decreases DNA synthetic activity in these cell lines, the IL-6-induced alterations in cell shape and motility occur independently of inhibition of DNA synthesis per se. Whereas tumor necrosis factor alpha (TNF-alpha) inhibits DNA synthesis in T-47D cells, it does not cause an epithelial-fibroblastoid conversion or other major morphological changes and does not increase cell motility; TNF-alpha rapidly lyses a majority of ZR-75-1 cells. Furthermore, the DNA synthesis inhibitors 5-fluoro-2'-deoxyuridine (FUDR) and methotrexate (MTX) also do not cause effects mimicking the action of IL-6 on cell structure and motility. Transforming growth factors alpha and beta 1, acidic and basic fibroblast growth factors, epidermal growth factor, and insulin-like growth factor-1 (TGF-alpha, TGF-beta 1, aFGF, bFGF, EGF, and IGF-1) have little or no effect on breast cancer cell morphology, which serves to exclude the possibility that the IL-6-induced changes are a consequence of induction of these growth factors by IL-6. 12-O-tetradecanoyl phorbol-13-acetate (TPA) but not 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP) induces changes in the morphology and associative behavior of ZR-75-1 cells that are similar but not identical to those caused by IL-6. The TPA-induced alterations are not blocked by anti-IL-6 neutralizing antibodies; staurosporine inhibits the TPA-induced cell alterations but not those induced by IL-6. IL-6 and TPA used together have a phenotypic effect that greatly exceeds that of either agent alone and results in extensive cell scattering in less than 1 day. These findings are consistent with the hypothesis that IL-6 and TPA induce similar morphological changes and cell scattering via independent pathways.

Epithelial cells both produce and are affected by interleukin-6 (IL-6). Experiments with an adenocarcinoma-derived cell line (HeLa) reveal that activation of the transfected human IL-6 promoter occurs largely through two partially overlapping second messenger (cAMP, phorbol ester)- and cytokine (IL-1, TNF, serum)-responsive enhancer elements (MRE 1, -173 to -151 and MRE II, -158 to -145). MRE I contains the typical GACGTCA cAMP and phorbol ester-responsive (CRE-TRE) motif, whereas MRE II defines a new CRE/TRE motif that contains an imperfect dyad repeat. The mechanism of dexamethasone-mediated repression of IL-6 gene expression in epithelial cells involves occlusion of the entire MRE enhancer region and of the core-promoter elements (TATA-box and RNA start site) by ligand-activated glucocorticoid receptor. Enhanced levels of IL-6 expression are observed in many solid tumors and in the hyperproliferative (and glucocorticoid-suppressible) lesions of psoriasis. In cell culture, IL-6 enhances, inhibits, or has no effect on the proliferation of epithelial cells depending upon the cell-type examined. IL-6 enhances proliferation of keratinocytes but inhibits that of breast carcinoma cell lines ZR-75-1 and T-47D. In these breast carcinoma cells, IL-6 elicits a major change in cell phenotype which is characterized by a fibroblastoid morphology, enhanced motility, increased cell-cell separation, and decreased adherens type junctions (desmosomes and focal adhesions). The new data identify IL-6 as a regulator of epithelial cell growth and of cell-cell association.

Intranasal inoculation of type 5 adenovirus (Ad5) produced pneumonia in mice even though the virus did not replicate. To induce the pneumonia, however, a large viral infectious dose was required--i.e., 10(10) plaque-forming units. Four strains of inbred mouse were studied (C57BL/6N, C57BL/10ScN, CBA/N, and C3H/N): all showed similar inflammatory responses, although the greatest infiltration occurred in the C57BL/6N mice. The pathological response to Ad5 infection resembled that previously described in cotton rats: it consisted of overlapping early and late phases, and the infiltration contained primarily lymphocytes and monocytes/macrophages with a scattering of polymorphonuclear leukocytes. The prominent early phase and the presence of polymorphonuclear leukocytes suggested that induction of cytokines may play an important role in the pathogenesis of this pneumonia. Assays showed the appearance of tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and IL-6 in the infected mouse lungs concomitant with the developing early-phase infiltration. Only IL-6 was found in the peripheral blood. IL-6 reached maximum titers 6-24 hr after infection, whereas maximum levels of TNF-alpha and IL-1 were attained 2-3 days after infection. Specific RNAs for each of these cytokines were demonstrated in the infected lungs. To test the hypothesis that a cytotoxic T-cell response was responsible for the second phase, which primarily consisted of a perivascular and peribronchial infiltration of lymphocytes, Ad5 was used to infect C57BL/10ScN Nu/Nu and parent mice. The nude mice showed a normal early-phase response, but essentially no peribronchial and only minimal perivascular infiltrations occurred.

The participation of interleukin-6 (IL-6) in the pathophysiology of normal and abnormal human parturition was evaluated by determining IL-6 concentrations in amniotic fluid (AF). Biologically active IL-6 was determined (in U/ml) using the B9 hybridoma growth factor assay, while the concentrations of immunoreactive IL-6 species (in pg/ml) were assessed using a monoclonal antibody (moAb)-based ELISA. Two hundred and twenty-seven AF samples from women in normal labor and from those presenting with a clinical diagnosis of premature rupture of membranes (PROM) were assayed. In selected instances, IL-6 levels were evaluated simultaneously in AF and in maternal and fetal plasma. Women with a normal pregnancy had low titers of biologically active IL-6 in AF both at midtrimester (group 1, n = 27; median IL-6 concentration = 16 U/ml) and at term (group 2, n = 33; median = 15 U/ml). There was an increase in the IL-6 bioactivity in AF from women in normal labor at term (group 3, n = 40; median = 74 U/ml; p less than 0.001). In order to distinguish between the relative contributions of parturition per se and of intrauterine infection to the elevation of biologically active IL-6 levels in AF, IL-6 titers were compared in four different groups of women with PROM.(ABSTRACT TRUNCATED AT 250 WORDS)

We report that TNF, IL-6, and IFN-alpha/beta are produced by mice during either sublethal or lethal Listeria monocytogenes infections. The quantities of these cytokines in infected spleens increase and decrease in concordance with bacterial numbers in these organs. While all of these cytokines were present in Listeria-infected spleens, only IL-6 and IFN-alpha/beta were found in the peripheral circulation. Inasmuch as TNF has been reported to be responsible for the production of IL-6 in vivo following the inoculation of a lethal dose of the Gram-negative bacterium, Escherichia coli (Fong et al., 1989. J. Exp. Med. 170: 1627), experiments were undertaken to determine whether IL-6 production elicited by the Gram-positive bacterium, L. monocytogenes, was also TNF-dependent. It was found that the passive immunization of mice with neutralizing antibodies specific for TNF shortly before i.v. injection of a lethal or sublethal Listeria inoculum resulted in the complete neutralization of endogenously produced TNF, and in the progressive multiplication of bacteria in infected organs. It was also found that the anti-TNF IgG treatment resulted in a progressive increase in the amounts of Listeria-induced IL-6 present in spleen and blood, until the death of the host. These findings indicate that Listeria-induced IL-6 production in mice occurs primarily through a TNF-independent pathway, and correlates directly with the severity of the infection.

Interleukin-6 (IL-6) is the major systemic mediator of the early host response to infection and injury (the "acute phase response"). Furthermore, IL-6 is often detected in the peripheral circulation and in the local neoplastic tissue in cancer patients. IL-6 has distinctive effect on epithelial cells depending upon the cell type examined. IL-6 enhances proliferation of normal human keratinocytes without affecting cell morphology. In contrast, IL-6 inhibits the proliferation of ductal breast carcinoma cell lines T-47D, ZR-71-1 and MCF-7. In addition to, but independent of, the inhibition of cell proliferation, IL-6 induces a cellular phenotype in the typically epitheliod T-47D and ZR-75-1 cells, which is characterized by fibroblastoid morphology, increased cell-cell separation even within preformed colonies, decreased adherens type junction formation (desmosomes and focal adhesions), and enhanced motility. Time-lapse cinemicrography of T-47D and wild-type ZR-75-1 cells reveals increased local movement of IL-6-treated cells and also movement of these cells over considerable distances. The effects of IL-6 on breast cancer cell proliferation and motility are reversible by removal of IL-6 from the culture medium. Time-lapse cinemicrography reveals that in clone B ZR-75-1 cells, which are not sensitive to the DNA synthesis-inhibitory effect of IL-6 or to its cell-separating effect on preformed colonies, IL-6 can still block rapid readherence of post-mitotic cells to their neighbors and to the substratum leading to enhanced dispersal of cancer cells into the culture medium. In wild-type ZR-75-1 cells, 12-O-tetradecanoyl phorbol ester (TPA) exerts a cell-scattering effect on breast cancer cells without inhibiting cell proliferation. Combined treatment with IL-6 and TPA produces a cell-scattering effect that greatly exceeds in magnitude and speed the phenotypic change elicited by either reagent alone. Staurosporine blocks cell-scattering caused by TPA but not that caused by IL-6 suggesting that IL-6 and TPA elicit similar phenotypic changes in breast cancer cells via different pathways. Taken together, these findings identify a previously unrecognized property of IL-6, that of enhancing cell motility.

Retroviral-mediated gene transfer was employed to introduce an IL-1 alpha cDNA into an IL-6-dependent murine B-cell line. Bone marrow metastases and bone lesions were frequently observed following intravenous injection of these B cells into syngeneic mice. Because the retroviral vector also contained the neomycin phosphotransferase gene, metastatic cells could be easily recovered from bone marrow by addition of G418 to the culture medium. Interestingly, the metastatic B cells were found to retain their IL-6 dependency through several transplant generations. By comparison, intravenous injection of autonomously-growing B-cell lines generated in vitro by retroviral introduction of an IL-6 cDNA rarely resulted in bone marrow metastases. These results demonstrate that abrogation of growth factor dependency is neither necessary nor sufficient for the in vivo growth and dissemination of tumor cells in this experimental system. It is proposed that the increased metastasis of the IL-1 alpha-producing B-cells to bone marrow is due to alterations in cell adhesion molecules. The B-cell bone marrow metastasis model described here may be useful for studies of bone marrow homing and for evaluation of therapeutic regimens for multiple myeloma.

The feedback inhibition of interleukin-6 (IL-6) gene expression by glucocorticoids represents a regulatory link between the endocrine and immune systems. The mechanism of the efficient repression of the IL-6 promoter by dexamethasone (Dex) was investigated in HeLa cells transiently transfected with plasmid constructs containing different IL-6 promoter elements linked to the herpesvirus thymidine kinase gene (tk) promoter and the bacterial chloramphenicol acetyltransferase gene (cat) and cotransfected with cDNA vectors constitutively expressing either the active wild-type or inactive mutant human glucocorticoid receptor (GR). The induction by interleukin-1, tumor necrosis factor, phorbol ester, or forskolin of IL-6-tk-cat chimeric constructs containing a single copy of the IL-6 DNA segment from -173 to -151 (MRE I) or from -158 to -145 (MRE II), which derive from within the multiple cytokine- and second-messenger-responsive enhancer (MRE) region, was strongly repressed by Dex in a wild-type GR-dependent fashion irrespective of the inducer used. The induction by pseudorabies virus of an IL-6 construct containing the IL-6 TATA box and the RNA start site ("initiator" or Inr element) but not the MRE region was also repressed by Dex in the presence of wild-type GR. DNase I footprinting showed that the purified DNA-binding fragment of GR bound across the MRE, the TATA box, and the Inr site in the IL-6 promoter; this footprint overlapped that produced by proteins present in nuclear extracts from uninduced or induced HeLa cells. Imperfect palindromic nucleotide sequence motifs moderately related to the consensus GR-responsive element (GRE) motif were present at the Inr, the TATA box, and the MRE II site in the IL-6 promoter; although MRE I and a GR-binding site between -201 and -210 in IL-6 both lacked a discernible inverted repeat motif, their sequences showed considerable similarity with negative GRE sequences in other Dex-repressed genes. Surprisingly, chimeric genes containing MRE II, which lacks a recognizable GACGTCA cyclic AMP- and phorbol ester-responsive motif, were strongly induced by both phorbol ester and forskolin, suggesting that MRE II (ACATTGCACAATCT) may be the prototype of a novel cyclic AMP- and phorbol ester-responsive element. Taken together, these observations suggest that ligand-activated GR represses the IL-6 gene by occlusion not only of the inducible IL-6 MRE enhancer region but also of the basal IL-6 promoter elements.

IL-6 is a multifunctional cytokine which is produced by a variety of cells. Therefore it was examined whether anti-CD3-induced T cell activation was associated with the induction of functionally relevant IL-6 in human monocyte accessory cells. Significantly increased amounts of IL-6 were detected in supernatants of anti-CD3-treated PBMC. Stimulation of FACS-sorted greater than 98% pure monocyte accessory cells, but not of highly purified T cells with anti-CD3, resulted in an increased IL-6 production. Furthermore, anti-CD3 significantly enhanced IL-6 mRNA expression in monocyte accessory cells. IL-6 production was not limited to anti-CD3, inasmuch as equivalent IL-6 stimulation could be achieved with a mouse IgG2a isotype control antibody. In contrast to solid phase-bound mouse IgG2a, the soluble form of this antibody failed to induce IL-6 secretion indicating a requirement for Fc gamma RI receptor cross-linking. Moreover, this property may be specific for the Fc gamma RI receptor inasmuch as mouse IgG1 antibodies binding to the Fc gamma RII receptor did not significantly enhance IL-6 production. The role of IL-6 being an additional signal in T cell activation was confirmed by the finding that an anti-IL-6 antiserum was able to suppress anti-CD3-induced T cell activation. These data indicate that binding of anti-CD3 to Fc gamma RI may generate an activation signal towards the monocyte accessory cell leading to the production and secretion of monocyte IL-6, which in turn augments T cell activation, and also may be relevant to a variety of antibody-mediated immune responses against viral and bacterial infections.

The cytokine interleukin-6 (IL-6) has emerged as a major systemic alarm signal which appears to be produced by essentially every injured tissue. Recent evidence points to the skin, particularly the injured skin, as one of the major sites of IL-6 production. The hallmark of IL-6 gene regulation is its induction by inflammation-associated cytokines, bacterial products, virus infection, and activation of any of the three major signal transduction pathways (diacylglycerol-, cAMP-, and Ca(++)-activated). Many of these inducers act largely through a 23-bp "multiple-response element" in the IL-6 promoter. Different cell types, including keratinocytes, secrete multiple post-translationally modified forms of IL-6. This cytokine, in turn, plays a key role in activating a variety of local and systemic host defense mechanisms that are aimed at limiting tissue injury. Thus, IL-6 elicits major changes in the biochemical, physiologic, and immunologic status of the host (e.g., the "acute phase" plasma protein response; activation of B, T, and NK-cell function). IL-6 enhances the proliferation of human keratinocytes and of many B-cell lines but inhibits that of certain carcinoma cell lines; nevertheless, IL-6 can enhance the motility of these carcinoma cells. Elevated levels of IL-6 are observed in human body fluids during acute and chronic infections, neoplasia, autoimmune diseases, and psoriasis and following third-degree burns. It is likely that IL-6 produced by cellular elements in the skin represents an important means of communication between the external environment and the millieu interieur.

To evaluate whether IL-6 participates in the host response to intrauterine infection, we studied IL-6 bioactivity and isoforms in amniotic fluid (AF). Two different assays for IL-6 were used: the hepatocyte stimulating factor assay (in Hep3B2 cells) and the SDS-PAGE/immunoblot assay. IL-6 determinations were performed in 205 AF samples. Samples were obtained from patients in the midtrimester of pregnancy (n = 25), at term with no labor (n = 31), at term in active labor (n = 40), and from patients in preterm labor (n = 109). Higher AF IL-6 levels were observed in women in preterm labor with intraamniotic infection than in women in preterm labor without intraamniotic infection (median = 375 ng/ml, range = 30-5000 ng/ml vs. median = 1.5 ng/ml, range = 0-500, respectively, P less than 0.0001). The 23-25- and 28-30-kD IL-6 species could be readily detected in SDS-PAGE immunoblots performed directly on 10-microliters aliquots of AF from patients with intraamniotic infection. Among women in preterm labor with culture-negative AF, those who failed to respond to subsequent tocolytic treatment had higher AF IL-6 concentrations than those who responded to therapy (median = 50 ng/ml vs. median = 1.2 ng/ml, respectively, P less than 0.05). Only low levels of IL-6 were detected in AF obtained from normal women in the midtrimester and third trimester of pregnancy. Decidual tissue explants obtained from the placentas of women undergoing elective cesarean section at term without labor (n = 11) produced IL-6 in response to bacterial endotoxin. In a pilot study, AF IL-6 was determined in 56 consecutive women admitted with preterm labor. All patients (n = 10) with elevated AF IL-6 (cutoff = 46 ng/ml) delivered a premature neonate. 4 of these 10 patients had positive AF cultures for microorganisms. These studies implicate IL-6 in the host response to intrauterine infection and suggest that evaluation of AF IL-6 levels may have diagnostic and prognostic value in the management of women in preterm labor.