D J Loskutoff
Recent studies have suggested that T cells play a critical role in the pathogenesis of psoriasis. Guttate psoriasis is a well-defined form of psoriasis frequently associated with streptococcal throat infection. This study tested the hypothesis that T cells in acute guttate psoriasis skin lesions may be activated by streptococcal superantigens. Peripheral blood as well as lesional and perilesional skin biopsies were analyzed for T cell receptor V beta repertoire using monoclonal antibodies against 10 different V beta families. Skin biopsies from all patients with acute guttate psoriasis, but not skin biopsies from patients with acute atopic dermatitis or inflammatory skin lesions induced in normal subjects with sodium lauryl sulfate, demonstrated selective accumulation of V beta 2+ T cells (P < 0.05). The expansion of V beta 2+ T cells occurred in both the CD4+ and the CD8+ T cell subsets. Sequence analysis of T cell receptor beta chain genes of V beta 2-expressing T cells from skin biopsies of patients with guttate psoriasis showed extensive junctional region diversity that is more compatible with a superantigen rather than a conventional (nominal) antigen-driven T cell response. All streptococcal isolates from patients with guttate psoriasis secreted streptococcal pyrogenic exotoxin C, a superantigen known to stimulate marked V beta 2+ T cell expansion. These data support the concept that acute guttate psoriasis is associated with superantigenic stimulation of T cells triggered by streptococcal superantigen(s).
D Y Leung, J B Travers, R Giorno, D A Norris, R Skinner, J Aelion, L V Kazemi, M H Kim, A E Trumble, M Kotb
We studied glucocorticoid-induced muscle wasting and subsequent recovery in adult (7-mo-old) and old (22-mo-old) rats, since the increased incidence of various disease states may result in glucocorticoids hypersecretion in aging. Adult and old rats received dexamethasone in their drinking water and were then allowed to recover. Muscle wasting occurred more rapidly in old rats and the recovery of muscle mass was impaired, suggesting that glucocorticoids may be involved in the emergence of muscle atrophy with advancing age. According to measurements in incubated epitrochlearis muscles, dexamethasone-induced muscle wasting mainly resulted from increased protein breakdown in the adult, but from depressed protein synthesis in the aged animal. Increased expression of cathepsin D, m-calpain, and ubiquitin was observed in the muscles from both dexamethasone-treated adult and old rats. By contrast, the disappearance of the stimulatory effect of glucocorticoids on protein break-down in aging occurred along with a loss of ability of steroids to enhance the expression of the 14-kD ubiquitin carrier protein E2, which is involved in protein substrates ubiquitinylation, and of subunits of the 20 S proteasome (the proteolytic core of the 26 S proteasome that degrades ubiquitin conjugates). Thus, if glucocorticoids play any role in the progressive muscle atrophy seen in aging, this is unlikely to result from an activation of the ubiquitin-proteasome proteolytic pathway.
D Dardevet, C Sornet, D Taillandier, I Savary, D Attaix, J Grizard
The pathogenesis of the eosinophilia myalgia syndrome (EMS) remains unclear. Several abnormal constituents have been found in the L-tryptophan lots responsible for the illness, particularly, 1,1-ethylidenebis[L-tryptophan], also called peak E or EBT, and 3-phenylamino-alanine or peak 5. However, the role of these contaminants in the pathogenesis of EMS and in the development of fibrosis is unknown. We now report that peak E, a dimer of L-tryptophan, is a potent stimulus for human dermal fibroblast DNA and collagen synthesis. Peak E (0.1-1.0 microM) increased DNA synthesis up to four-fold (P = 0.0001) in a dose-dependent manner (r = 0.987). When added to monolayer cultures for 2 to 24 h, peak E (0.5 to 100 microM) caused a progressive, more than threefold increase in alpha 1(I) procollagen mRNA levels and collagenous protein. No increase in procollagen mRNA levels was found after the addition of another major L-tryptophan contaminant, peak 5, or with L-tryptophan itself. Transient transfection with a 2.5-kb alpha 1(I) procollagen promoter-luciferase construct showed that peak E causes a twofold upregulation of promoter activity (P = 0.022). Contraction of collagen gels, consisting of human dermal fibroblasts incorporated into a type I collagen lattice, was enhanced two-fold by exposure to peak E (P = 0.001). We conclude that a major constituent of contaminated batches of L-tryptophan, peak E, is a potent stimulus for fibroblast activation and collagen synthesis. This stimulatory action of peak E may provide a direct mechanism for the development of fibrosis in EMS.
H Takagi, M S Ochoa, L Zhou, T Helfman, H Murata, V Falanga
We examined the effect of urea on NaK2Cl cotransport in human erythrocytes. In erythrocytes from nine normal subjects, the addition of 45 mM urea, a concentration commonly encountered in uremic subjects, inhibited NaK2Cl cotransport by 33 +/- 7%. Urea inhibited NaK2Cl cotransport reversibly, and in a concentration-dependent fashion with half-maximal inhibition at 63 +/- 10 mM. Acute cell shrinkage increased, and acute cell swelling decreased NaK2Cl cotransport in human erythrocytes. Okadaic acid (OA), a specific inhibitor of protein phosphatase 1 and 2A, increased NaK2Cl cotransport by nearly 80%, suggesting an important role for these phosphatases in the regulation of NaK2Cl cotransport. Urea inhibited bumetanide-sensitive K influx even when protein phosphatases were inhibited with OA, suggesting that urea acted by inhibiting a kinase. In cells subjected to shrinking and OA pretreatment, maneuvers expected to increase the net phosphorylation, urea inhibited cotransport only minimally, suggesting that urea acted by causing a net dephosphorylation of the cotransport protein, or some key regulatory protein. The finding that concentrations of urea found in uremic subjects inhibited NaK2Cl cotransport, a widespread transport pathway with important physiological functions, suggests that urea is not only a marker for accumulation of other uremic toxins, but may be a significant uremic toxin itself.
J Lim, C Gasson, D M Kaji
The genetic and functional basis of phosphoribosylpyrophosphate synthetase (PRS) superactivity associated with purine nucleotide inhibitor-resistance was studied in six families with this X chromosome-linked purine metabolic and neurodevelopmental disorder. Cloning and sequencing of PRS1 and PRS2 cDNAs, derived from fibroblast total RNA of affected male patients by reverse transcription and PCR amplification, demonstrated that each PRS1 cDNA contained a distinctive single base substitution predicting a corresponding amino acid substitution in the PRS1 isoform. Overall, the array of substitutions encompassed a substantial portion of the translated sequence of PRS1 cDNA. Plasmid-mediated expression of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yielded recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance comparable to that found in cells of the respective patient. Kinetic analysis of recombinant mutant PRS1s showed that widely dispersed point mutations in the X chromosome-linked PRPS1 gene encoding the PRS1 isoform result in alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate. The functional consequences of these mutations provide a tenable basis for the enhanced production of phosphoribosylpyrophosphate, purine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity.
M A Becker, P R Smith, W Taylor, R Mustafi, R L Switzer
The colon, unlike most organs, is normally exposed to high concentrations of ammonia, a weak base which exerts profound and diverse biological effects on mammalian cells. The impact of ammonia on intestinal cell function is largely unknown despite its concentration of 4-70 mM in the colonic lumen. The human intestinal epithelial cell line T84 was used to model electrogenic Cl- secretion, the transport event which hydrates mucosal surfaces and accounts for secretory diarrhea. Transepithelial transport and isotopic flux analysis indicated that physiologically-relevant concentrations of ammonia (as NH4Cl) markedly inhibit cyclic nucleotide-regulated Cl- secretion but not the response to the Ca2+ agonist carbachol. Inhibition by ammonia was 25-fold more potent with basolateral compared to apical exposure. Ion substitution indicated that the effect of NH4Cl was not due to altered cation composition or membrane potential. The site of action of ammonia is distal to cAMP generation and is not due simply to cytoplasmic alkalization. The results support a novel role for ammonia as an inhibitory modulator of intestinal epithelial Cl- secretion. Secretory responsiveness may be dampened in pathological conditions associated with increased mucosal permeability due to enhanced access of lumenal ammonia to the basolateral epithelial compartment.
M Prasad, J A Smith, A Resnick, C S Awtrey, B J Hrnjez, J B Matthews
apo B is a structural constituent of several classes of lipoprotein particles, including chylomicrons, VLDL, and LDL. To better understand the role of apo B in the body, we have used gene targeting in embryonic stem cells to create a null apo B allele in the mouse. Homozygous apo B deficiency led to embryonic lethality, with resorption of all embryos by gestational day 9. Heterozygotes showed an increased tendency to intrauterine death with some fetuses having incomplete neural tube closure and some live-born heterozygotes developing hydrocephalus. The majority of male heterozygotes were sterile, although the genitourinary system and sperm were grossly normal. Viable heterozygotes had normal triglycerides, but total, LDL, and HDL cholesterol levels were decreased by 37, 37, and 39%, respectively. Hepatic and intestinal apo B mRNA levels were decreased in heterozygotes, presumably contributing to the decreased LDL levels through decreased synthesis of apo B-containing lipoproteins. Kinetic studies indicated that heterozygotes had decreased transport rates of HDL cholesterol ester and apo A-I. As liver and intestinal apo A-I mRNA levels were unchanged, the mechanism for decreased apo A-I transport must be posttranscriptional. Heterozygotes also had normal cholesterol absorption and a normal response of the plasma lipoprotein pattern to chronic consumption of a high fat, high cholesterol, Western-type diet. In summary, we report a mouse model for apo B deficiency with several phenotypic features that were unexpected based on clinical studies of apo B-deficient humans, such as embryonic lethality in homozygotes and neural tube closure defects, male infertility, and a major defect in HDL production in heterozygotes. This model presents an opportunity to study the mechanisms underlying these phenotypic changes.
L S Huang, E Voyiaziakis, D F Markenson, K A Sokol, T Hayek, J L Breslow
H Yoshida, T Mitarai, T Kawamura, T Kitajima, Y Miyazaki, R Nagasawa, Y Kawaguchi, H Kubo, I Ichikawa, O Sakai
apoE deficiency causes hyperlipidemia and premature atherosclerosis. To determine if macrophage-specific expression of apoE would decrease the extent of atherosclerosis, we expressed human apoE in macrophages of apoE-null mice (apoE-/-) and assessed the effect on lipid accumulation in cells of the arterial wall. Macrophage-specific expression of human apoE in normal mice was obtained by use of the visna virus LTR. These animals were bred with apoE-/- mice to produce animals hemizygous for expression of human apoE in macrophages in the absence of murine apoE (apoE-/-,hTgE+/0). Low levels of human apoE mRNA were present in liver and spleen and high levels in lung and peritoneal macrophages. Human apoE was secreted by peritoneal macrophages and was detected in Kupffer cells of the liver. Human apoE in the plasma of apoE-/-,hTgE+/0 mice (n = 30) was inversely correlated (P < 0.005) with the plasma cholesterol concentration. After 15 wk on a normal chow diet, atherosclerosis was assessed in apoE-/-,hTgE+/0 animals and in apoE-/-,hTgE0/0 littermates matched for plasma cholesterol level (approximately 450 mg/dl) and lipoprotein profile. There was significantly less atherosclerosis in both the aortic sinus and in the proximal aorta (P < 0.0001) in the animals expressing the human apoE transgene. In apo-E-/-,hTgE+/0 animals, which had detectable atherosclerotic lesions, human apoE was detected in the secretory apparatus of macrophage-derived foam cells in the arterial wall. The data demonstrate that expression of apoE by macrophages is antiatherogenic even in the presence of high levels of atherogenic lipoproteins. The data suggest that apoE prevents atherosclerosis by promoting cholesterol efflux from cells of the arterial wall.
S Bellosta, R W Mahley, D A Sanan, J Murata, D L Newland, J M Taylor, R E Pitas
Individuals heterozygous or homozygous for the variant aldehyde dehydrogenase (ALDH2) allele (ALDH2*2), which encodes a protein differing only at residue 487 from the normal protein, have decreased ALDH2 activity in liver extracts and experience cutaneous flushing when they drink alcohol. The mechanisms by which this allele exerts its dominant effect is unknown. To study this effect, the human ALDH2*1 cDNA was cloned and the ALDH2*2 allele was generated by site-directed mutagenesis. These cDNAs were transduced using retroviral vectors into HeLa and CV1 cells, which do not express ALDH2. The normal allele directed synthesis of immunoreactive ALDH2 protein (ALDH2E) with the expected isoelectric point. Extracts of these cells contained increased aldehyde dehydrogenase activity with low Km for the aldehyde substrate. The ALDH2*2 allele directed synthesis of mRNA and immunoreactive protein (ALDH2K), but the protein lacked enzymatic activity. When ALDH2*1-expressing cells were transduced with ALDH2*2 vectors, both mRNAs were expressed and immunoreactive proteins with isoelectric points ranging between those of ALDH2E and ALDH2K were present, indicating that the subunits formed heteromers. ALDH2 activity in these cells was reduced below that of the parental ALDH2*1-expressing cells. Thus, the ALDH2*2 allele is sufficient to cause ALDH2 deficiency in vitro.
Q Xiao, H Weiner, T Johnston, D W Crabb
In the mammalian renal proximal tubule, protein kinase A (PKA) plays an important role in mediating hormonal regulation of apical membrane Na/H exchanger activity. This exchanger is likely encoded by NHE-3. The present studies examined regulation of NHE-3 by PKA. NHE-3 was stably expressed in Na/H exchanger-deficient fibroblasts (AP-1/NHE-3 cells). PKA activation (0.1 mM 8-BrcAMP x 20 min) inhibited NHE-3 activity by 39% (P < 0.01) with no change in NHE-3 protein abundance in the plasma membrane. To define the structural requirements for PKA-mediated inhibition, full-length NHE-3 and a cytoplasmic domain-truncated mutant (NHE-3 delta cyto) were expressed in Xenopus laevis oocytes. 8-BrcAMP inhibited NHE-3 activity by 27% (P < 0.05), an effect that was blocked by 10(-7) M PKA inhibitor peptide. NHE-3 delta cyto had baseline activity similar to that of full-length NHE-3 but its activity was not regulated by 8-BrcAMP. The purified recombinant cytoplasmic domain of NHE-3 was phosphorylated in vitro by the catalytic subunit of PKA on serine residues. In AP-1/NHE-3 cells, NHE-3 was immunoprecipitated as a approximately 87-kD phosphoprotein. Addition of 0.1 mM 8-BrcAMP increased the phosphocontent of NHE-3 by threefold. In summary, acute activation of PKA inhibits NHE-3 activity, an effect that is likely mediated by phosphorylation of its cytoplasmic domain.
O W Moe, M Amemiya, Y Yamaji
The activation and differentiation of T cells require both antigen/MHC recognition and costimulatory signals. The present studies examined the role of B7-1 (CD80) and B7-2 (CD86) costimulation in the prototypic autoimmune disorder, experimental allergic encephalomyelitis (EAE). In adoptively transferred EAE, in vitro activation of myelin basic protein (MBP)-specific lymph node cells was inhibited by the combination of anti-CD80 plus anti-CD86, but not individually. However, in actively induced disease, one injection of anti-CD80 significantly reduced disease, while anti-CD86 exacerbated disease. Interestingly, one injection of CTLA-4Ig suppressed disease, while multiple injections resulted in enhanced disease. Thus, the costimulation provided by B7-1 molecules appears to be important for the development of encephalitogenic T cells. The enhanced disease caused by multiple injections of CTLA-4Ig or a single injection of anti-CD86 suggests an inhibitory function for CD86 interaction with its counterreceptors CD28 and CTLA-4 in EAE. Alternatively, these results are consistent with an essential timing requirement for the coordinated interaction of B7 and CD28 family receptors, and that disruption of this critical timing can have opposing results on the outcome of an immune response.
M K Racke, D E Scott, L Quigley, G S Gray, R Abe, C H June, P J Perrin
Respiratory epithelial cells play a crucial role in the inflammatory response during Pseudomonas aeruginosa infection in the lungs of patients with cystic fibrosis. In this study, we determined whether the binding of specific Pseudomonas gene products (pilin, flagellin) to their receptors on respiratory epithelial cells would result in production of the neutrophil chemoattractant IL-8. Piliated wild-type organisms, purified pili, or antibody to the pilin receptor (asialoGM1) evoked significant production of IL-8 by immortalized airway epithelial cells, whereas nonpiliated organisms were less able to bind to respiratory epithelial cells and stimulated much less IL-8 secretion (P < 0.01). A piliated, nonflagellated strain was also associated with decreased binding and a diminished level of IL-8 production when compared to wild-type organisms. Isogenic, nonadherent rpoN mutants, lacking pilin and flagellin, did not bind or elicit an IL-8 response. In addition, the IL-8 response was four-fold higher in a cystic fibrosis cell line compared with its corrected cell line. The Pseudomonas autoinducer, an exoproduct secreted during chronic infection, was found to stimulate IL-8 in a dose-dependent manner. P. aeruginosa adhesins, which are necessary for initial infection, directly stimulate IL-8 production by respiratory epithelial cells and therefore play a major role in the pathogenesis of Pseudomonas infection in patients with cystic fibrosis. The inflammatory response is subsequently perpetuated by Pseudomonas autoinducer which is secreted during chronic infection.
E DiMango, H J Zar, R Bryan, A Prince
Circulating antiphospholipid antibodies (aPL) are associated with a syndrome of thrombosis, recurrent fetal loss, and thrombocytopenia. We have demonstrated the activation of cultured human umbilical vein endothelial cells (HUVEC) by IgG from patients with anticardiolipin antibodies (aCL). Incubation of HUVEC for 4 h with purified IgG (100 micrograms/ml) from patients with high-titer aCL induced a 2.3-fold increase in monocyte adhesion over that seen in HUVEC incubated with IgG's from normal subjects. The effect of aCL was not attributable to LPS contamination, Fc receptors, or immune complexes. Monocyte adhesion was not induced when the aCL were added in serum-free media but was restored by the addition of purified beta 2GP1, previously described as a necessary cofactor for aCL reactivity. Purified rabbit polyclonal IgG raised against beta 2GP1 also induced monocyte adhesion when incubated with HUVEC. Preadsorption of patient serum with cardiolipin reduced monocyte adhesion by 60%. Immunofluorescent microscopy demonstrated that endothelial cells incubated with patient IgG expressed cell adhesion molecules, including E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1. These data support the hypothesis that aPL activate vascular endothelial cells, thereby leading to a pro-thrombotic state.
R Simantov, J M LaSala, S K Lo, A E Gharavi, L R Sammaritano, J E Salmon, R L Silverstein
We investigated the mechanisms of alphafetoprotein (AFP) transfer across the human placenta by correlating measurements of AFP transfer with cytochemical localization of AFP. Placental cotyledons were dually perfused in vitro with either the fetal or maternal perfusate containing umbilical cord plasma as a source of AFP. Steady state AFP clearance, corrected for release of endogenous AFP, was 0.973 +/- 0.292 microliter/min per gram in the fetal to maternal direction (n = 10), significantly higher (P < 0.02) than that in the maternal to fetal direction (n = 5; 0.022 +/- 0.013 microliter/min per gram). Clearance of a similarly sized protein, horseradish peroxidase was also asymmetric but clearance of the small tracer creatinine was not. Using a monoclonal antibody, we localized AFP to fibrinoid deposits in regions of villi with discontinuities of the syncytiotrophoblast, to cytotrophoblast cells in these deposits, to syncytiotrophoblast on some villi, and to trophoblast cells in the decidua. We conclude that AFP transfer in the placenta is asymmetric and that there are two available pathways for AFP transfer: (a) from the fetal circulation into the villous core and across fibrinoid deposits at discontinuities in the villous syncytiotrophoblast to enter the maternal circulation; and (b) AFP present in the decidua could enter vessels that traverse the basal plate.
P Brownbill, D Edwards, C Jones, D Mahendran, D Owen, C Sibley, R Johnson, P Swanson, D M Nelson
Effects of transplantable rat insulinomas (IN) and glucagonomas (GLU) on the endogenous pancreas were analyzed using morphometry, immunocytochemistry, in situ hybridization, and staining for apoptotic cells. Hyperinsulinemia (IN-rats) and hyper-GLP-1/glucagonemia (GLU-rats) were both associated with marked islet atrophy (67 and 76% of control average planimetrical islet area, respectively). Selective islet B cell inhibition of proinsulin (I and II) genes as well as of expression of the insulin gene transcription factor, IPF1/STF1, was found in IN-rats. Moreover, these islets were characterized by significant B cells apoptosis in the absence of infiltrating lymphocytes. In GLU-rats selective islet A cell inhibition was observed at the level of glucagon mRNA. These islets contained small, highly condensed but clearly active B cells with prominent IPF1/STF1-positive nuclei, surrounded by densely packed glucagon-negative cells with reduced cytoplasm. Furthermore, an active apoptotic process was found exclusively in the exocrine pancreas of GLU-rats. Thus, in IN-rats, islet B cell mass reduction is distinguished by non-immune-mediated programmed cell death, while GLU-rats exhibit A cell mass reduction by cytoplasmic retraction and selective exocrine apoptosis.
N Blume, J Skouv, L I Larsson, J J Holst, O D Madsen
Glucocorticoid-suppressible hyperaldosteronism is a dominantly inherited form of hypertension believed to be caused by the presence of a hybrid CYP11B1/CYP11B2 gene which has arisen from an unequal crossing over between the two CYP11B genes in a previous meiosis. We have studied a French pedigree with seven affected individuals in which two affected individuals also have adrenal tumors and two others have micronodular adrenal hyperplasia. One of the adrenal tumors and the surrounding adrenal tissue has been removed, giving a rare opportunity to study the regulation and action of the hybrid gene causing the disease. The hybrid CYP11B gene was demonstrated to be expressed at higher levels than either CYP11B1 or CYP11B2 in the cortex of the adrenal by RT-PCR and Northern blot analysis. In situ hybridization showed that both CYP11B1 and the hybrid gene were expressed in all three zones of the cortex. In cell culture experiments hybrid gene expression was stimulated by ACTH leading to increased production of aldosterone and the hybrid steroids characteristic of glucocorticoid-suppressible hyperaldosteronism. The genetic basis of the adrenal pathologies in this family is not known but may be related to the duplication causing the hyperaldosteronism.
L Pascoe, X Jeunemaitre, M C Lebrethon, K M Curnow, C E Gomez-Sanchez, J M Gasc, J M Saez, P Corvol
To determine the effects of loading on active and passive tensions, programmed cell death, superoxide anion formation, the expression of Fas on myocytes, and side-to-side slippage of myocytes, papillary muscles were exposed to 7-8 and 50 mN/mm2 and these parameters were measured over a 3-h period. Overstretching produced a 21- and a 2.4-fold increase in apoptotic myocyte and nonmyocyte cell death, respectively. Concurrently, the generation of reactive oxygen species increased 2.4-fold and the number of myocytes labeled by Fas protein 21-fold. Moreover, a 15% decrease in the number of myocytes included in the thickness of the papillary muscle was found in combination with a 7% decrease in sarcomere length and the inability of muscles to maintain stable levels of passive and active tensions. The addition of the NO-releasing drug, C87-3754, prevented superoxide anion formation, programmed cell death, and the alterations in active and passive tensions with time of overloaded papillary muscles. In conclusion, overstretching appears to be coupled with oxidant stress, expression of Fas, programmed cell death, architectural rearrangement of myocytes, and impairment in force development of the myocardium.
W Cheng, B Li, J Kajstura, P Li, M S Wolin, E H Sonnenblick, T H Hintze, G Olivetti, P Anversa
Vascular smooth muscle cell (VSMC) proliferation after arterial injury is important in the pathogenesis of a number of vascular proliferative disorders, including atherosclerosis and restenosis after balloon angioplasty. Thus, a better understanding of the molecular mechanisms underlying VSMC proliferation in response to arterial injury would have important therapeutic implications for patients with atherosclerotic vascular disease. The p21 protein is a negative regulator of mammalian cell cycle progression that functions both by inhibiting cyclin dependent kinases (CDKs) required for the initiation of S phase, and by binding to and inhibiting the DNA polymerase delta co-factor, proliferating cell nuclear antigen (PCNA). In this report, we show that adenovirus-mediated over-expression of human p21 inhibits growth factor-stimulated VSMC proliferation in vitro by efficiently arresting VSMCs in the G1 phase of the cell cycle. This p21-associated cell cycle arrest is associated both with significant inhibition of the phosphorylation of the retinoblastoma gene product (Rb) and with the formation of complexes between p21 and PCNA in VSMCs. In addition, we demonstrate that localized arterial infection with a p21-encoding adenovirus at the time of balloon angioplasty significantly reduced neointimal hyperplasia in the rat carotid artery model of restenosis. Taken together, these studies demonstrate the important role of p21 in regulating Rb phosphorylation and cell cycle progression in VSMC, and suggest a novel cytostatic gene therapy approach for restenosis and related vascular proliferative disorders.
M W Chang, E Barr, M M Lu, K Barton, J M Leiden
The expression of the collagen alpha 1(I) gene in activated stellate cells plays an important role during liver fibrogenesis. To identify the critical cis-elements of the collagen alpha 1(I) gene in stellate cells, we used transgenic animals bearing various collagen alpha 1(I) regulatory regions directing the expression of either a human growth hormone minigene or the bacterial beta-galactosidase gene. We found that collagen alpha 1(I)-human growth hormone transgene expression was constitutively high in tendon and skin, provided the transgene contained the -2.3 to -0.44 kb collagen regulatory region. However in the liver, expression was stimulated several-fold, as was the endogeneous gene, by the fibrogenic hepatotoxin carbon tetrachloride. This stimulation occurred whether the collagen 5' regulatory region extended -2.3, -1.6 or -0.44 kb, and in the presence or absence of much of the first intron (+292 to +1607 bp). In addition, the -0.44 kb 5' region was sufficient for high-level transgene expression in stellate cells, following their activation by culture on plastic. In contrast, in skin and tendon, high-level transcription of the collagen alpha 1(I) gene required the -2.3 to -0.44 kb 5' flanking region. Thus, two different cis-regulatory regions direct cell-specific transcription of the collagen alpha 1(I) gene in stellate cells and in skin and tendon.
K Houglum, M Buck, J Alcorn, S Contreras, P Bornstein, M Chojkier
Acid extrusion is essential for osteoclast (OC) activity. We examined Na+ and HCO3(-)-independent H+ extrusion in rat- and mouse OCs by measuring intracellular pH (pHi) changes, with the pHi indicator BCECF (biscarboxyethyl-5-(6) carboxyfluorescein) after H+ loading with an ammonium pulse. 90% of OCs attached to glass do not possess HCO3- and Na(+)-independent H(+)-extrusion (rate of pHi recovery = 0.043 +/- 0.007 (SEM) pH U/min, n = 26). In contrast, in OCs attached to bone, the pHi recovery rate is 0.228 +/- 0.011 pHi U/min, n = 25. OCs on bone also possess a NH(4+)-permeable pathway not seen on glass. The bone-induced H+ extrusion was inhibited by salmon calcitonin (10(-8) M, for 2 h), and was not present after pretreating the bone slices with the aminobisphosphonate alendronate (ALN). At ALN levels of 0.22 nmol/mm2 bone, H+ extrusion was virtually absent 12 h after cell seeding (0.004 +/- 0.002 pH U/min) and approximately 50% inhibition was observed at 0.022 pmol ALN/mm2 bone. The Na(+)-independent H+ extrusion was not inhibited by bafilomycin A1 (up to 10(-7) M), although a bafilomycin A1 (10(-8) M)-sensitive H+ pump was present in membrane vesicles isolated from these osteoclasts. These findings indicate that Na(+)-independent acid extrusion is stimulated by osteoclast attachment to bone and is virtually absent when bone is preincubated with ALN, or when osteoclasts are treated with salmon calcitonin.
Z Zimolo, G Wesolowski, G A Rodan
Although the source-sink relationship for impulse propagation in cardiac tissues has been demonstrated in vitro, there has been no verification of this hypothesis in humans. Accordingly, eight patients undergoing surgical division of their accessory pathways were studied. A 56-channel (7 x 8) bipolar plaque electrode array was placed over the atrioventricular groove on the accessory pathway and atrial fibrillation electrically induced. 10 episodes of QRS transition from consecutively preexcited to nonpreexcited complexes were analyzed. This showed that consecutively preexcited QRS complexes were always associated with uniform large atrial wavefronts. Immediately prior to QRS transition, four general types of changes were observed: (a) premature invasion by secondary wavefronts creating local conduction block (n = 5); (b) wavefront collision leading to wavefront curvature (n = 2); (c) transition from a uniform large atrial wavefront to multiple fractionated small wavefronts (n = 1); and (d) uniform atrial wavefronts "marching" into the accessory pathway refractory period (n = 2). We conclude that local atrial wavefront characteristics are important factors influencing impulse propagation through the accessory pathway. The findings that local wavefront collision, curvature, or fractionation often precede loss of accessory pathway conduction support the notion that source-sink relationship is an important determinant of the safety factor for impulse propagation in the human heart.
J J Ong, Y M Cha, J M Kriett, K Boyce, G K Feld, P S Chen
These studies were conducted to determine whether men and women differ with regards to their overnight postabsorptive (basal) and postprandial fatty acid kinetics. Systemic oleate turnover ([9,10(3)H]oleate) was measured before and after the consumption of a mixed meal. Leg and splanchnic free fatty acid (FFA) uptake and release were measured, allowing the calculation of upper-body subcutaneous FFA release. RESULTS: basal oleate flux was virtually identical in men and women (3.0 +/- 3 versus 2.9 +/- 0.4 mumol.kg FFM-1.min-1), however, oleate Ra suppressed more in women than in men following meal ingestion (0.5 +/- 0.1 versus 0.8 +/- 0.1 mumol.kg FFM-1.min-1, P < 0.05). The fractional contribution of basal, regional FFA release to total FFA flux was not significantly different between men and women. In contrast, oleate release by upper-body subcutaneous adipose tissue was significantly greater (30 +/- 5 vs 8 +/- 3 mumol/min, respectively, P < 0.01) in men than in women during the meal nadir of FFA flux, whereas splanchnic oleate release was a greater percentage (39 +/- 7% vs 20 +/- 3%, respectively, P < 0.05) of nadir oleate Ra in women than in men. Thus, normal weight men and women differ significantly in the postprandial regulation of adipose tissue lipolysis in that men's upper-body subcutaneous adipose tissue is more resistant to the antilipolytic effects of meal ingestion. Differential regulation of regional adipose tissue lipolysis could contribute to the gender based differences in body fat distribution.
M D Jensen
Human mononuclear phagocytes can modulate the turnover of extracellular matrix by producing metalloproteinases such as 92-kD gelatinase and interstitial collagenase as well as the tissue inhibitor of metalloproteinases (TIMP). We have previously reported that IL-4 and IFN gamma released by lymphocytes suppress metalloproteinase biosynthesis in macrophages without affecting TIMP production (Lacraz, S., L. Nicod, B. C. de Rochementeix, C. Baumberger, J. Dayer, and H. Welgus. 1992. J. Clin. Invest. 90:382-388.; Shapiro, S. D., E. J. Campbell, D. K. Kobayashi, and H. G. Welgus 1990. J. Clin. Invest. 86:1204-1210). Like IL-4, IL-10 is secreted by Th2 lymphocytes and is inhibitory to several macrophage functions. In the present study, IL-10 was tested and compared to IL-2, IL-4, IL-6, and IFN gamma for its capacity to modulate synthesis of 92-kD gelatinase, interstitial collagenase and TIMP in human macrophages and monocytes. We found that IL-10, just like IL-4, inhibited the production of 92-kD gelatinase and blocked LPS-, as well as killed Staphylococcus aureus-induced, interstitial collagenase production. The principal finding of this study, however, was that IL-10, in distinction to IL-4, produced a dose-dependent stimulation in the biosynthesis of TIMP-1. TIMP-2 production was not affected. IL-10 regulated the expression of 92-kD gelatinase and TIMP-1 at the pretranslational level. Furthermore, IL-10 regulation was cell type-specific, as it had no effect on the production of metalloproteinases or TIMP by human fibroblasts. In summary, IL-10 has a potent and unique effect upon tissue macrophages and blood monocytes by enhancing TIMP-1 production while decreasing metalloproteinase biosynthesis.
S Lacraz, L P Nicod, R Chicheportiche, H G Welgus, J M Dayer
Cytotoxic T lymphocytes (CTL) are important to the control of viral replication and their presence may be important to disease outcome. An understanding of the spectrum of proteins recognized by hepatitis C virus (HCV)-specific CTL and the functional properties of these cells is an important step in understanding the disease process and the mechanisms of persistent infection, which occurs in the majority of HCV-infected individuals. In this report we identify HCV-specific CTL responses restricted by the HLA class I molecules A2, A3, A11, A23, B7, B8, and B53. The epitopes recognized by these intrahepatic CTL conform to published motifs for binding to HLA class I molecules, although in some cases we have identified CTL epitopes for which no published motif exists. The use of vectors expressing two different strains of HCV, HCV-1 and HCV-H, revealed both strain-specific and cross-reactive CTL. These HCV-specific CTL were shown to produce cytokines including IFN-gamma, TNF-alpha, GM-CSF, IL-8, and IL-10 in an antigen- and HLA class I-specific manner. These studies indicate that the CTL response to HCV is broadly directed and that as many as five different epitopes may be targeted in a single individual. The identification of minimal epitopes may facilitate peptide-specific immunization strategies. In addition, the release of proinflammatory cytokines by these cells may contribute to the pathogenesis of HCV-induced liver damage.
M J Koziel, D Dudley, N Afdhal, A Grakoui, C M Rice, Q L Choo, M Houghton, B D Walker
Oxidative modification of lipoproteins is believed to be important in the genesis of atherosclerosis. We established cultures of smooth muscle cells (SMC) and exposed them to native LDL or oxidized LDL. Oxidized LDL, but not native LDL, was mitogenic as measured by incorporation of [3H]-thymidine into DNA. This effect was concentration dependent, averaged 288% of control, and was blocked by a platelet-activating factor (PAF) receptor antagonist. We hypothesized that phospholipids with PAF-like activity were generated during the oxidation of LDL. To test this hypothesis we extracted phospholipids from copper-oxidized LDL and assayed for PAF-like activity. Phospholipids extracted from oxidized LDL and purified by HPLC induced neutrophil adhesion equivalent to PAF (10 nM) and were mitogenic for smooth muscle cells. These effects were not seen with phospholipids extracted from native LDL and were blocked by two structurally different, competitive antagonists of the PAF receptor. The effects of these lipids were also abolished by pretreating them with PAF acetylhydrolase. Finally, we used Chinese hamster ovary cells that had seen stably transfected with a cDNA for the PAF receptor to confirm that phospholipids from oxidized LDL act via this receptor. We found that PAF (control) and the oxidized phospholipids each induced release of arachidonic acid from the transfected cells, but had no effect on wildtype Chinese hamster ovary cells, which lack the PAF receptor. This effect was also blocked by a PAF receptor antagonist. Thus, phospholipids generated during oxidative modification of LDL may participate in atherosclerosis by stimulating SMC proliferation and leukocyte activation.
J M Heery, M Kozak, D M Stafforini, D A Jones, G A Zimmerman, T M McIntyre, S M Prescott
Intermittent administration of PTH has been found to be an effective anabolic agent in cancellous bone. We have reported previously that combined treatment with PTH and estrogen in estrogen-deficient rats was beneficial in correcting established osteopenia. To determine if the beneficial effects of PTH therapy can be preserved by estrogen alone and whether PTH therapy can be effective in treating osteopenic subjects stabilized with estrogen, we have undertaken a "crossover" study in the rat model of estrogen-deficiency induced osteopenia. Six-month-old female rats were ovariectomized and after 5 wk treated for 8 wk with vehicle, 30 micrograms/kg per day of rPTH(1-34) plus 15 micrograms/kg per day of 17 beta-estradiol or 17 beta-estradiol alone. One group from each treatment regimen was then sacrificed and for an additional 8 weeks the remaining rats were (a) maintained on their previous treatment; (b) "crossed over" to their reciprocal treatment; or (c) administered vehicle only. At the end of this second 8-wk treatment period all rats were sacrificed. Bone mineral density of the distal femur, histomorphometric measurements of the proximal tibia and mechanical testing of the distal femur and selected vertebral bodies were performed. Our results demonstrated that (a) the gains in bone mass, trabecular connectivity and mechanical strength induced by PTH can be maintained by estrogen alone, but are reversed when both agents are withdrawn; and (b) rats with established osteopenia, maintained on estrogen treatment alone, can derive the full beneficial effects from the addition of PTH to the treatment at a later date. These data indicate that combined and/or sequential use of antiresorptive and anabolic agents may be a promising approach to the treatment of osteoporosis.
V Shen, R Birchman, R Xu, M Otter, D Wu, R Lindsay, D W Dempster
Previous studies in vivo have shown that IL-10 infusion can prevent lethal endotoxic shock. Mice deficient in the production of IL-10 (IL10T) were used to investigate the regulatory role of IL-10 in the responses to LPS in three experimental systems. In a model of acute endotoxic shock, it was found that the lethal dose of LPS for IL10T mice was 20-fold lower than that for wild type (wt) mice suggesting that endogenous IL-10 determines the amount of LPS which can be tolerated without death. The high mortality rate of IL10T mice challenged with modest doses of LPS was correlated to the uncontrolled production of TNF as treatment with anti-TNF antibody (Ab) resulted in 70% survival. Additional studies suggested that IL-10 mediates protection by controlling the early effectors of endotoxic shock (e.g., TNF alpha) and that it is incapable of directly antagonizing the production and/or actions of late appearing effector molecules (e.g., nitric oxide). We also found that IL10T mice were extremely vulnerable to a generalized Shwartzman reaction where prior exposure to a small amount of LPS primes the host for a lethal response to a subsequent sublethal dose. The priming LPS dose for IL10T mice was 100-fold lower than that required to prime wt mice implying that IL-10 is important for suppressing sensitization. In agreement with this assumption, IL-10 infusion was found to block the sensitization step. Interestingly, IL-10 was not the main effector of endotoxin tolerance as IL10T mice could be tolerized to LPS. Furthermore, IL-10 infusion could not substitute for the desensitizing dose of LPS. These results show that IL-10 is a critical component of the host's natural defense against the development of pathologic responses to LPS although it is not responsible for LPS-induced tolerance.
D J Berg, R Kühn, K Rajewsky, W Müller, S Menon, N Davidson, G Grünig, D Rennick
The kidney widely expresses membrane-associated complement regulatory proteins (membrane inhibitors of complement). The aim of this work was to evaluate the roles of these molecules in rat kidneys in vivo. To suppress functions of rat membrane inhibitors of complement, two mAbs, 512 and 6D1, were used. 5I2 and 6D1 inhibit functions of membrane inhibitors of complement at C3 level (rat Crry/p65) and C8/9 level (rat CD59), respectively. F(ab')2 fragment of 5I2 or 6D1 was perfused in the left kidneys, and perfusate was discarded from the renal vein. After perfusion, the left kidneys were connected to systemic circulation. In rats perfused with 5I2, mouse IgG was found in glomeruli, peritubular capillaries, vascular bundles, and tubules 15 min after recirculation. Binding of C3 and C5b-9 was evident in these areas. 1 d after perfusion with 5I2, cast formation, dilatation of tubular lumen, and tubular cell degeneration were observed. At day 4 through day 7, significant mononuclear cell infiltration and proximal tubule damage were observed. These changes were completely prevented by complement depletion. Rats perfused with 6D1 showed the binding of mouse IgG in the similar areas as 5I2, but C3 or C5b-9 deposition was not observed. Rats perfused with 6D1 or vehicle only did not show any pathology in the left kidneys. These results suggest that rat Crry/p65 plays protective roles against spontaneously occurring indiscriminate attack to tubulointerstitial tissues by autologous complement and that rat Crry/p65 is one of the important factors to maintain normal integrity of the kidney in rats.
A Nomura, K Nishikawa, Y Yuzawa, H Okada, N Okada, B P Morgan, S J Piddlesden, M Nadai, T Hasegawa, S Matsuo
In this study, we have identified the source of nitric oxide (NO) produced in the human inflammatory joints by analyzing expression of inducible NO synthase. In ex vivo organ cultures, both inflammatory synovium and cartilage from patients with rheumatoid arthritis produced NO. The NO production was suppressed by NG-monomethyl-L-arginine, an inhibitor of NO synthase. The amount of NO produced by the synovium correlated with the proportion of CD14+ cells in the corresponding tissue (r = 0.8, P < 0.05). Immunohistochemical analysis as well as in situ hybridization showed that inducible NO synthase was predominantly expressed in synovial lining cells, endothelial cells, chondrocytes, and to a lesser extent, in infiltrating mononuclear cells and synovial fibroblasts. The synovial lining cells and the infiltrating cells expressing inducible NO synthase were identified where CD14+ cells were located. Together with morphological features, this suggests that they are type A synoviocytes. NO production from freshly isolated synoviocytes and chondrocytes was up-regulated by in vitro stimulation with a combination of IL-TNF-beta, TNF-alpha, and LPS. In summary, the present results suggest that NO is produced primarily by CD14+ synoviocytes, chondrocytes, and endothelial cells in inflammatory joints of arthritides. NO production can be upregulated by cytokines present in inflamed joints. The increased NO production may thus contribute to the pathological features in inflammatory arthritides.
H Sakurai, H Kohsaka, M F Liu, H Higashiyama, Y Hirata, K Kanno, I Saito, N Miyasaka
Cyclic mechanical strain (1 Hz) causes a mitogenic response in neonatal rat vascular smooth muscle cells due to production and secretion of PDGF. In this study, the mechanism for sensing mechanical strain was investigated. Silicone elastomer strain plates were coated at varying densities with elastin, laminin, type I collagen, fibronectin, or vitronectin. Strain was applied by cyclic application of a vacuum under the dishes. Cells adhered, spread, and proliferated on each matrix protein, but the mitogenic response to strain was matrix dependent. Strain increased DNA synthesis in cells on collagen, fibronectin, or vitronectin, but not in cells on elastin or laminin. When strain was applied on matrices containing both laminin and vitronectin, the mitogenic response to strain depended upon the vitronectin content of the matrix. Fibronectin, in soluble form (0-50 micrograms/ml), and the integrin binding peptide GRGDTP (100 micrograms/ml) both blocked the mitogenic response to mechanical strain in cells grown on immobilized collagen. Neither soluble laminin nor the inactive peptide GRGESP blocked the response to strain. GRGDTP did not alter the mitogenic response to exogenous PDGF or alpha-thrombin but did prevent the secretion of PDGF in response to strain. Furthermore, GRGDTP, but not GRGESP, prevented strain-induced expression of a PDGF-A chain promoter 890 bp-chloramphenicol acetyltransferase construct that was transiently transfected into vascular smooth muscle cells. Finally, the response to strain was abrogated by antibodies to both beta 3 and alpha v beta 5 integrins but not by an antibody to beta 1 integrins. Thus interaction between integrins and specific matrix proteins is responsible for sensing mechanical strain in vascular smooth muscle cells.
E Wilson, K Sudhir, H E Ives
S K Singh, H J Binder, W F Boron, J P Geibel
We have isolated and purified outer membrane vesicles (OMV) from Borrelia burgdorferi strain B31 based on methods developed for isolation of Treponema pallidum OMV. Purified OMV exhibited distinct porin activities with conductances of 0.6 and 12.6 nano-Siemen and had no detectable beta-NADH oxidase activity indicating their outer membrane origin and their lack of inner membrane contamination, respectively. Hydrophobic proteins were identified by phase partitioning with Triton X-114. Most of these hydrophobic membrane proteins were not acylated, suggesting that they are outer membrane-spanning proteins. Identification of palmitate-labeled lipoproteins revealed that several were enriched in the OMV, several were enriched in the protoplasmic cylinder inner membrane fraction, and others were found exclusively associated with the inner membrane. The protein composition of OMV changed significantly with successive in vitro cultivation of strain B31. Using antiserum with specificity for virulent strain B31, we identified OMV antigens on the surface of the spirochete and identified proteins whose presence in OMV could be correlated with virulence and protective immunity in the rabbit Lyme disease model. These virulent strain associated outer membrane-spanning proteins may provide new insight into the pathogenesis of Lyme disease.
J T Skare, E S Shang, D M Foley, D R Blanco, C I Champion, T Mirzabekov, Y Sokolov, B L Kagan, J N Miller, M A Lovett
We hypothesized that hyperresponsiveness in asthma is caused by an impairment in the ability of inspiration to stretch airway smooth muscle. If the hypothesis was correct, we reasoned that the sensitivity to inhaled methacholine in normal and asthmatic subjects should be the same if the challenge was carried out under conditions where deep inspirations were prohibited. 10 asthmatic and 10 normal subjects received increasing concentrations of inhaled methacholine under conditions where forced expirations from a normal end-tidal inspiration were performed. When no deep inspirations were allowed, the response to methacholine was similar in the normal and asthmatic subjects, compatible with the hypothesis we propose. Completely contrary to our expectations, however, was the marked responsivity to methacholine that remained in the normal subjects after deep breaths were initiated. 6 of the 10 normal subjects had > 20% reduction in forced expiratory volume in one second (FEV 1) at doses of methacholine < 8 mg/ml, whereas there was < 15% reduction with 75 mg/ml during routine challenge. The ability of normal subjects to develop asthmatic responses when the modulating effects of increases in lung volume was voluntarily suppressed suggests that an intrinsic impairment of the ability of inspiration to stretch airway smooth muscle is a major feature of asthma.
G Skloot, S Permutt, A Togias
Immunoglobulins regulate the complement system by activating complement on foreign surfaces and diverting reactive complement proteins away from autologous cell surfaces. Based on this model, we explored the ability of Ig to balance complement activation versus control in a pig-to-primate cardiac xenotransplantation model in which the binding of xenoreactive antibodies of the recipient to graft blood vessels and the activation of complement cause hyperacute rejection. Human IgG added to human serum caused a dose-dependent decrease in deposition of iC3b, cytotoxicity, and heparan sulfate release when the serum was incubated with porcine endothelial cells. This decrease was not caused by alteration in antibody binding or consumption of complement but presumably reflected decreased formation of C3 convertase on the endothelial cells. Infusion of purified human IgG into nonhuman primates prevented hyperacute rejection of porcine hearts transplanted into the primates. As expected, the transplants contained deposits of recipient Ig and C1q but not other complement components. The inhibition of complement on endothelial cell surfaces and in the xenotransplantation model supports the idea that IgG regulates the classical complement pathway and supports therapeutic use of that agent in humoral-mediated disease.
J C Magee, B H Collins, R C Harland, B J Lindman, R R Bollinger, M M Frank, J L Platt
The mechanisms that regulate the expression of genes encoding extracellular matrix proteins in fibroblasts and other mesenchymal cells have remained elusive. Studies from several laboratories have indicated that Tax, a trans-regulatory protein from the human T cell leukemia virus type I not only augments viral gene expression but also triggers the expression of various cellular genes. Here, we examined the hypothesis that the expression of collagen genes may also be modulated by Tax. NIH-3T3 cells were simultaneously transfected with a Tax expressor plasmid and a chimeric construct containing regulatory sequences (-804 to +42 bp) of the alpha 1(I) procollagen gene (COL1A1) promoter. The results indicated that the promoter activity of the -804 to bp COL1A1 fragment increased up to 12-fold in cells expressing Tax. Deletion analysis revealed that the region of COL1A1 encompassing nucleotides -174 to -84 contained the Tax-responsive elements. A gene segment encompassing nucleotides -187 to -67, which contained this region, proved sufficient to confer Tax inducibility (2.5-fold) to a herpes simplex virus thymidine kinase promoter. Stably transfected NIH-3T3 cell clones that constitutively produce Tax displayed elevated levels of alpha 1(I) procollagen and fibronectin transcripts and increased production and accelerated processing of type I procollagen. These findings suggest that retroviral proteins may be involved in the pathogenesis of idiopathic diseases accompanied by collagen overproduction.
E Muñoz, D Suri, S Amini, K Khalili, S A Jiménez
Type 3 iodothyronine deiodinase (D3) catalyzes the conversion of T4 and T3 to inactive metabolites. It is highly expressed in placenta and thus can regulate circulating fetal thyroid hormone concentrations throughout gestation. We have cloned and expressed a 2.1-kb human placental D3 cDNA which encodes a 32-kD protein with a Km of 1.2 nM for 5 deiodination of T3 and 340 nM for 5' deiodination of reverse T3. The reaction requires DTT and is not inhibited by 6n-propylthiouracil. We quantitated transiently expressed D3 by specifically labeling the protein with bromoacetyl [125I]T3. The Kcat/Km ratio for 5 deiodination of T3 was over 1,000-fold that for 5' deiodination of reverse T3. Human D3 is a selenoenzyme as evidenced by (a) the presence of an in frame UGA codon at position 144, (b) the synthesis of a 32-kD 75Se-labeled protein in D3 cDNA transfected cells, and (c) the presence of a selenocysteine insertion sequence element in the 3' untranslated region of the mRNA which is required for its expression. The D3 selenocysteine insertion sequence element is more potent than that in the type 1 deiodinase or glutathione peroxidase gene, suggesting a high priority for selenocysteine incorporation into this enzyme. The conservation of this enzyme from Xenopus laevis tadpoles to humans implies an essential role for regulation of thyroid hormone inactivation during embryological development.
D Salvatore, S C Low, M Berry, A L Maia, J W Harney, W Croteau, D L St Germain, P R Larsen
M Suematsu, N Goda, T Sano, S Kashiwagi, T Egawa, Y Shinoda, Y Ishimura
Lipoprotein lipase (LPL) is a central enzyme in lipoprotein metabolism and is in part responsible for adipocyte lipid accumulation. Catecholamines are known to decrease the activity of LPL in adipocytes, and we have previously demonstrated that this inhibition occurs posttranscriptionally, with a prominent inhibition of LPL translation. To better characterize the inhibition of LPL translation, 3T3-L1 cells were differentiated into adipocytes, and exposed to epinephrine. Epinephrine induced a dose-dependent decrease in LPL synthesis using [35S]methionine incorporation, with no change in LPL mRNA levels, demonstrating translational regulation of LPL in this cell line. The poly A-enriched RNA from epinephrine-treated cells was translated well in vitro, and there was no difference in the polysome profiles from control and epinephrine-treated cells, suggesting that epinephrine did not affect mRNA editing, and did not induce an inhibition of translation initiation. To obtain evidence for the presence of an inhibitory factor, a cytoplasmic extract from control, and epinephrine-treated adipocytes was human. When compared to the control cell extract, the epinephrine-treated cell extract sharply inhibited LPL translation in vitro, yet had no effect on the translation of other mRNAs. Epinephrine-treated cells had fourfold more of this inhibitor activity than control cells, and this translation inhibition was partially reversed by heat treatment. To determine what region of the LPL mRNA was involved in the translation inhibition, different LPL constructs were synthesized. The inhibitory effect of the epinephrine-treated cell extract was dependent on the presence of the first 40 nucleotides of the 3' (untranslated region UTR) (nucleotides 1599-1638), whereas deletion of the 5' UTR and other areas of the 3' UTR had no effect on translation inhibition. When a sense RNA strand corresponding to this region was added to the in vitro translation reaction, it restored translation towards normal, suggesting that the sense strand was competing for a transacting binding protein. Thus, epinephrine-treated adipocytes produced a transacting factor, probably a protein, that interacted with a region on the LPL mRNA between nucleotides 1599 and 1638, resulting in an inhibition of translation. These studies add new insight into the hormonal regulation of LPL.
A Yukht, R C Davis, J M Ong, G Ranganathan, P A Kern
Respiratory syncytial virus (RSV) causes repeated infections thought to be due to an ineffective immune response. We examined the hypothesis that incomplete immunity may result, in part, from RSV-infected alveolar macrophage production of IL-10 which can interfere with the production of immunoregulatory cytokines. We also assessed whether RSV induced the expression of the 2',5' oligoadenylate (2-5A)-dependent RNase L, an endoribonuclease involved in the antiviral activities of interferons. Human alveolar macrophages were exposed to medium (uninfected control), RSV, LPS, and RSV + LPS then were assessed for expression of the cytokines TNF-alpha, IL-1 beta, IL-8, IL-10, as well as 2-5A-dependent RNase L. LPS up-regulated the expression of protein and mRNA for all cytokines. RSV stimulated the protein levels of TNF-alpha, did not alter IL-1 beta, and decreased IL-8. RSV markedly stimulated protein expression of IL-10 and 2-5A-dependent RNase L. RSV had minor effects on the steady state mRNA levels of TNF-alpha, IL-1 beta, and IL-8, yet potently induced IL-10. Cells costimulated with RSV + LPS demonstrated reduced protein and mRNA levels of TNF-alpha, IL-1 beta, IL-8 but synergistically increased IL-10 levels compared to RSV- or LPS-activated cells. Kinetic analysis indicated that RSV induced a delayed and sustained increase in IL-10 transcripts. Furthermore, RSV-infected alveolar macrophage supernatants suppressed IL-1 beta and IL-8 production by LPS-stimulated alveolar macrophages as did recombinant IL-10. Anti-IL-10 neutralized these effects. These studies indicate that RSV is capable of suppressing production of early immunoregulatory cytokines through induction of IL-10 perhaps mediated by 2-5A-dependent RNase L (or other endoribonucleases) accounting for the ineffective immune response to this virus.
J R Panuska, R Merolla, N A Rebert, S P Hoffmann, P Tsivitse, N M Cirino, R H Silverman, J A Rankin
Gene therapy used in the context of delivering a therapeutic gene(s) to chondrocytes offers a new approach for treating chondrocyte-mediated cartilage degradation associated with various human arthropathies including osteoarthritis. In this study, gene delivery to human osteoarthritis chondrocytes in monolayer culture was demonstrated using two adenoviral vectors (Ad.CMVlacZ and Ad.RSVntlacZ) carrying the Escherichia coli beta-galactosidase marker gene, and a third vector (Ad.RSV hIL-1ra) containing the cDNA for human interleukin-1 receptor antagonist. At an moi of 10(3) plaque-forming units/chondrocyte, > 90% of the infected cells stained positive for E. coli beta-galactosidase activity, indicating a high efficiency of transduction. Genetically modified chondrocytes were then transplanted onto the articular surface of osteoarthritic cartilage organ cultures with and without the underlying subchondral bone. Both in situ staining of the cartilage organ cultures for E. coli beta-galactosidase activity and examination by scanning electron microscopy indicated that the transplanted chondrocytes adhered and integrated into the articular surface and continued to express transgenic protein. Chondrocytes transduced with Ad.RSV hIL-1ra and seeded onto the surface of osteoarthritic cartilage secreted high levels of biologically active IL-1 receptor antagonist. The Ad.RSV hIL-1ra-treated cartilage samples were resistant to IL1-induced proteoglycan degradation over 10 d of sustained organ culture. These data demonstrate that transplantation of transduced chondrocytes onto the articular surface protects cartilage from IL-1-induced extracellular matrix degradation.
V M Baragi, R R Renkiewicz, H Jordan, J Bonadio, J W Hartman, B J Roessler
Excessive production of collagen type I is a major contributor to hepatic fibrosis. Activated (myofibroblastic), but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and alpha-smooth muscle actin expression. Therefore, stellate cell activation is a critical step in hepatic fibrosis. Here we show that quiescent stellate cells were activated by the generation of free radicals with ascorbate/FeSO4 and by malondialdehyde, a product of lipid peroxidation. In addition, stellate cell activation by collagen type I matrix and TGF alpha was blocked by antioxidants, such as d-alpha-tocopherol and butylated hydroxytoluene. Moreover, oxidative stress, TGF alpha and collagen type I markedly stimulated stellate cell entry into S-phase, NFkB activity, and c-myb expression, which were prevented by antioxidants. c-myb antisense oligonucleotide blocked the activation and proliferation of stellate cells induced by TGF alpha. Nuclear extracts from activated, but not from quiescent, stellate cells formed a complex with the critical promoter E box of the alpha-smooth muscle actin gene, which was disrupted by c-myb and NFkB65 antibodies, and competed by c-myb and NFkB cognate DNA. c-Myb expression was also stimulated in activated stellate cells in carbon tetrachloride-induced hepatic injury and fibrogenesis. This study indicates that oxidative stress plays an essential role, through the induction of c-myb and NFkB, on stellate cell activation.
K S Lee, M Buck, K Houglum, M Chojkier
These studies were performed to determine if the effects of angiotensin II infusion on the development of cardiac fibrosis could be modified by the chronic inhibition of nitric oxide synthase activity. NG-nitro-L-arginine-methyl ester (L-NAME) was administered to adult Wistar rats in drinking water (40 mg/kg per d). Although blood pressure was maintained at hypertensive levels after 2 wk, cardiac hypertrophy or fibrosis did not occur. Angiotensin II, given for 3 d at a dose which induced little or no blood pressure elevation and minimal if any fibrosis, caused significant fibrosis when given to a rat pretreated for 2 wk with L-NAME. This marked fibrosis did not occur if angiotensin II was given shortly after L-NAME treatment was begun or briefly after discontinuation of L-NAME. The fibrosis that occurred with combined treatment was characterized by increased immunodetectable fibronectin, the presence of inflammatory cells within interstitial and perivascular regions, and increased steady state mRNA levels for matrix genes and atrial natriuretic protein. The data indicated a regulatory role for nitric oxide in modulating the angiotensin II-induced cardiac fibrosis and suggest a potentially important autocrine or paracrine role for nitric oxide in fibroblast proliferation.
J Hou, H Kato, R A Cohen, A V Chobanian, P Brecher
Neurologic diseases are important complications of measles. The role of virus infection of the central nervous system as well as the route of virus entry has been unclear. Five autopsied cases of individuals who died with severe acute measles 3-10 d after the onset of the rash were studied for evidence of viral involvement of the central nervous system. In all cases, in situ hybridization and RT-PCR in situ hybridization techniques showed endothelial cell infection. Immunoperoxidase staining with an anti-ferritin antibody revealed a reactive microgliosis. These data suggest that endothelial cells in the brain are frequently infected during acute fatal measles. This site of infection may provide a portal of entry for virus in individuals who subsequently develop subacute sclerosing panencephalitis or measles inclusion body encephalitis and a target for immunologic reactions in post-measles encephalomyelitis.
L M Esolen, K Takahashi, R T Johnson, A Vaisberg, T R Moench, S L Wesselingh, D E Griffin
The atheroprotective effects of estrogen are well documented, but the mechanisms responsible for these effects are not well understood. To study the role of physiologic (nanomolar) estrogen levels on the arterial response-to-injury, we applied a mouse carotid artery injury model to ovariectomized C57BL/6J mice. Mice were treated with vehicle (-E2, n = 10) or 17 beta-estradiol (+E2, n = 10) for 7 d, subjected to unilateral carotid injury, and 14 d later contralateral (normal = NL) and injured carotids from -E2 and +E2 animals were pressure fixed, harvested, and analyzed by quantitative morphometry. E2 levels in +E2 mice were consistently in the nanomolar range (2.1-2.5 nM) at days 0, 7, and 14. At 14 d, measures of both intimal and medial area were markedly increased in the -E2 group: (-E2 vs NL, P < 0.05 for both), but were unchanged from normal levels in the +E2 group (+E2 vs NL, P = NS and +E2 vs -E2, P < 0.05 for both). Cellular proliferation, as assessed by bromodeoxyuridine (BrdU) labeling, was significantly increased over NL in the -E2 mice, but this increase was markedly attenuated in the estrogen replacement group (total BrdU positive cells/section: NL = 6.4 +/- 4.5; -E2 = 113 +/- 26, +E2 = 40 +/- 3.7; -E2 vs NL, P < 0.05; +E2 vs NL, P = NS; -E2 vs +E2, P < 0.05). These data (a) demonstrate significant suppression of the mouse carotid response-to-injury by physiologic levels of estrogen replacement; (b) support the utility of this model in the study of the biologic effects of estrogen on the vascular-injury response; and (c) suggest a direct effect of estrogen on vascular smooth muscle cell proliferation in injured vessels.
T R Sullivan Jr, R H Karas, M Aronovitz, G T Faller, J P Ziar, J J Smith, T F O'Donnell Jr, M E Mendelsohn
Glucose homeostasis is controlled by a glucose sensor in pancreatic beta-cells. Studies on rodent beta-cells have suggested a role for GLUT2 and glucokinase in this control function and in mechanisms leading to diabetes. Little direct evidence exists so far to implicate these two proteins in glucose recognition by human beta-cells. The present in vitro study investigates the role of glucose transport and phosphorylation in beta-cell preparations from nondiabetic human pancreata. Human beta-cells differ from rodent beta-cells in glucose transporter gene expression (predominantly GLUT1 instead of GLUT2), explaining their low Km (3 mmol/liter) and low VMAX (3 mmol/min per liter) for 3-O-methyl glucose transport. The 100-fold lower GLUT2 abundance in human versus rat beta-cells is associated with a 10-fold slower uptake of alloxan, explaining their resistance to this rodent diabetogenic agent. Human and rat beta-cells exhibit comparable glucokinase expression with similar flux-generating influence on total glucose utilization. These data underline the importance of glucokinase but not of GLUT2 in the glucose sensor of human beta-cells.
A De Vos, H Heimberg, E Quartier, P Huypens, L Bouwens, D Pipeleers, F Schuit
Somatostatin (SRIF) regulates secretion from several endocrine cell types. SRIF inhibits both insulin and glucagon secretion and reduces insulin gene expression. However, whether SRIF inhibition of glucagon secretion from the pancreatic alpha cell is mediated via pertussis toxin-sensitive G-proteins is not presently known, nor has it been determined whether SRIF can regulate glucagon gene expression. Consequently, we performed studies in the transformed islet cell line HIT-T15 to determine whether the inhibitory effect of SRIF on glucagon exocytosis is preserved in this cell line, whether this effect is mediated through a pertussis toxin-sensitive mechanism, and whether SRIF has an inhibitory effect on glucagon gene expression. Confocal microscopy with immunostaining revealed that 15-25% of HIT-T15 cells contained glucagon. In static incubations forskolin (FSK, 1 microM) increased glucagon secretion 3.6 +/- 0.9-fold (P < 0.01) and mixed amino acids (15 mM) increased glucagon secretion 2.8 +/- 0.4-fold (P < 0.01). Addition of SRIF significantly inhibited both forskolin- and amino acid-stimulated secretion. Maximal inhibition of both FSK- and amino acid-stimulated secretion occurred at SRIF concentrations > or = 10(-8) M and these inhibitory effects were completely prevented by pertussis toxin pretreatment. In addition to inhibiting glucagon secretion, SRIF significantly reduced both basal and FSK-stimulated glucagon mRNA levels and this reduction in glucagon mRNA was completely prevented by the addition of cyclic AMP analogue. Glucagon gene promoter activity, as assessed by transient transfection experiments, was stimulated 2.1 +/- 0.25-fold by forskolin (P < 0.01). This effect was significantly inhibited by SRIF (71 +/- 4% reduction from FSK alone, P < 0.04) suggesting that SRIF inhibition of the glucagon promoter may, at least in part, account for the observed decrease in glucagon mRNA levels. These studies uniquely demonstrate that glucagon secretion from the HIT-T15 cell line is inhibited by SRIF through a pertussis toxin-sensitive mechanism and that SRIF also inhibits glucagon gene expression in part by reducing glucagon promoter activity. These findings indicate that SRIF can coordinately regulate glucagon delivery by the alpha cell both at the level of gene expression and hormone exocytosis.
D M Kendall, V Poitout, L K Olson, R L Sorenson, R P Robertson
Neuropeptide Y (NPY) levels are increased in plasma and tumors of patients with pheochromocytoma. The present study was designed to evaluate plasma and tissue NPY levels simultaneously as well as to study its release and expression in patients with either adrenal or extraadrenal pheochromocytomas. Plasma NPY levels were higher (P < 0.01) in patients with adrenal tumors than in matched normal subjects and patients with extraadrenal tumors. NPY levels were also higher (P < 0.05) in adrenal than in extraadrenal tumors. Bioactive NPY (1-36) was the predominant form in plasma and tumors of patients with adrenal pheochromocytomas. In contrast, patients with extraadrenal pheochromocytomas had an abundance of NPY fragments. NPY mRNA was abundant in 11 of 13 adrenal tumors but in only 1 of 6 extraadrenal tumors. Moreover, NPY was coreleased with NE with manipulation of adrenal but not extraadrenal tumors. These findings indicate that increased NPY gene expression in adrenal pheochromocytomas accounts for the greater biosynthesis and storage of NPY in these tumors and that increased release of NPY results in elevated plasma NPY. Factors regulating NPY gene expression in pheochromocytoma and the role of NPY in the clinical manifestations of the disease remain to be elucidated.
P deS Senanayake, J Denker, E L Bravo, R M Graham
A thiazide-sensitive Na-Cl cotransporter contributes importantly to mammalian salt homeostasis by mediating Na-Cl transport along the renal distal tubule. Although it has been accepted that thiazide-sensitive Na-Cl cotransport occurs predominantly along the distal convoluted tubule in rats and mice, sites of expression in the rabbit have been controversial. A commonly accepted model of rabbit distal nephron transport pathways identifies the connecting tubule, not the distal convoluted tubule, as the predominant site of thiazide-sensitive Na-Cl cotransport. The thiazide-sensitive Na-Cl cotransporter has been cloned recently. The present experiments were designed to localize sites of thiazide-sensitive Na-Cl cotransporter mRNA expression along the rabbit distal nephron. Nonradioactive in situ hybridization with a thiazide-sensitive Na-Cl cotransporter probe was combined with immunocytochemistry with an antibody that recognizes distal convoluted tubule cells and with a Na+/Ca2+ exchanger antibody that recognizes only connecting tubule cells. The results indicate that thiazide-sensitive Na-Cl cotransporter mRNA is highly expressed by cells of the distal convoluted tubule and not by connecting tubule cells. Segments that stain with the Na+/Ca2+ exchanger antibody (connecting tubules) do not demonstrate thiazide-sensitive Na-Cl cotransporter mRNA expression. We conclude that the predominant site of thiazide-sensitive Na-Cl cotransporter mRNA expression in rabbit distal nephron is the distal convoluted tubule and that sites of mRNA expression of electroneutral Na and Cl transport are similar in rabbits, rats, and mice.
S Bachmann, H Velázquez, N Obermüller, R F Reilly, D Moser, D H Ellison
Recent studies from this laboratory have demonstrated that angiotensin II (Ang II) stimulates the expression of plasminogen activator inhibitor 1 (PAI-1) in cultured endothelial cells. This response does not appear to be mediated via an interaction with either the AT1 or the AT2 receptor subtype. Since a novel angiotensin receptor has been identified in a variety of tissues that specifically binds the hexapeptide Ang IV (Ang II, [3-8]), we therefore examined the effects of Ang IV on the expression of PAI-1 mRNA in bovine aortic endothelial cells. Ang IV stimulated dose- and time-dependent increases in the expression of PAI-1 mRNA. The effect of Ang IV (10 nM) was not inhibited by Dup 753 (1.0 microM), a highly specific antagonist of the AT1 receptor, or by PD123177 (1.0 microM), a highly specific antagonist of the AT2 receptor. In contrast, the AT4 receptor antagonist, WSU1291 (1.0 microM), effectively prevented PAI-1 expression. Although larger forms of angiotensin (i.e., Ang I, Ang II, and Ang III) are capable of inducing PAI-1 expression, this property is lost in the presence of converting enzyme or aminopeptidase inhibitors. These results indicate that the hexapeptide Ang IV is the form of angiotensin that stimulates endothelial expression of PAI-1. This effect appears to be mediated via the stimulation of an endothelial receptor that is specific for Ang IV.
D M Kerins, Q Hao, D E Vaughan
We have recently discovered bovine and human vascular smooth muscle cells (SMCs) express a novel constitutive Nuclear Factor-kappa B (NF-kappa B)/Rel-like activity (Lawrence, R., L.-J. Chang, U. Siebenlist, P. Bressler, and G.E. Sonenshein. 1994. J. Biol. Chem. 269:28913-28918), here termed SMC-Rel. Since cytomegalovirus (CMV) infection of human vascular SMCs has been implicated in aberrant SMC proliferation during post-angioplasty restenosis, we tested the role of NF-kappa B/Rel activity in transactivation of the CMV immediate early (ie) promoter. The basal CMV ie promoter linked to three wild-type, but not mutant, copies of its NF-kappa B element was active in bovine aortic SMCs. The anti-oxidants N-acetyl cysteine (NAC) or pentoxifylline (PTX), which are used clinically to reduce NF-kappa B/Rel activity, inhibited NF-kappa B driven promoter transactivation, and SMC-Rel binding activity. Treatment with either NAC or PTX was observed to slow the growth of the SMCs in a dose dependent fashion. Microinjection of either purified I kappa B-alpha, a naturally occurring specific inhibitor of NF-kappa B/Rel activity, or double-stranded oligonucleotides harboring wild type, but not non-binding mutants of NF-kappa B elements selectively inhibited SMC proliferation. Thus constitutive NF-kappa B/Rel activity appears essential for proliferation of vascular SMCs and might be a novel target for therapeutic intervention for restenosis.
R E Bellas, J S Lee, G E Sonenshein
Despite ample evidence that the kidney can both produce and use appreciable amounts of glucose, the human kidney is generally regarded as playing a minor role in glucose homeostasis. This view is based on measurements of arteriorenal vein glucose concentrations indicating little or no net release of glucose. However, inferences from net balance measurements do not take into consideration the simultaneous release and uptake of glucose by the kidney. Therefore, to assess the contribution of release and uptake of glucose by the human kidney to overall entry and removal of plasma glucose, we used a combination of balance and isotope techniques to measure renal glucose net balance, fractional extraction, uptake and release as well as overall plasma glucose appearance and disposal in 10 normal volunteers under basal postabsorptive conditions and during a 3-h epinephrine infusion. In the basal postabsorptive state, there was small but significant net output of glucose by the kidney (66 +/- 22 mumol.min-1, P = 0.016). However, since renal glucose fractional extraction averaged 2.9 +/- 0.3%, there was considerable renal glucose uptake (2.3 +/- 0.2 mumol.kg-1.min-1) which accounted for 20.2 +/- 1.7% of systemic glucose disposal (11.4 +/- 0.5 mumol.kg-1.min-1). Renal glucose release (3.2 +/- 0.2 mumol.kg-1.min-1) accounted for 27.8 +/- 2.1% of systemic glucose appearance (11.4 +/- 0.5 mumol.kg-1.min-1). Epinephrine infusion, which increased plasma epinephrine to levels observed during hypoglycemia (3722 +/- 453 pmol/liter) increased renal glucose release nearly twofold (5.2 +/- 0.5 vs 2.8 +/- 0.1 mol.kg-1.min-1, P = 0.01) so that at the end of the infusion, renal glucose release accounted for 40.3 +/- 5.5% of systemic glucose appearance and essentially all of the increase in systemic glucose appearance. These observations suggest an important role for the human kidney in glucose homeostasis.
M Stumvoll, U Chintalapudi, G Perriello, S Welle, O Gutierrez, J Gerich
A Redlitz, A K Tan, D L Eaton, E F Plow