O-glycosylation of the alpha-subunit does not limit the assembly of chorionic gonadotropin alpha beta dimer in human malignant and nonmalignant trophoblast cells.

Biosynthetic experiments were carried out in cultures of human malignant trophoblast cells (the JAR cell line) and in explants of normal first trimester human placental tissue to test the hypothesis that the O-glycosylation of the glycoprotein hormone alpha-subunit at Thr-39 regulates the assembly of the CG alpha beta dimer. This modification of alpha has been shown to ablate its ability to combine in vitro with the beta-subunit of bovine LH and might explain why JAR cells and placental explants secrete uncombined alpha- and beta-subunits in addition to the hCG alpha beta dimer. We have previously detected an O-linked carbohydrate chain at Thr-39 in preparations of secreted free alpha-subunit, but not dimer CG alpha from JAR culture medium. We report here evidence that the O-glycosylation of alpha does not regulate the biosynthetic assembly of the hCG dimer in cultures of JAR choriocarcinoma cells or first trimester placental explants. The intracellular precursor forms of alpha and beta that accumulate in the endoplasmic reticulum and combine in that compartment are not yet modified with O-linked carbohydrate, as determined by measurements of their [3H]galactosamine content after biosynthetic labeling of amino sugars with [3H]glucosamine. Furthermore, only half of the free alpha-subunit secreted by JAR cells and less than 10% of free alpha secreted by 10-week-old placental explants received the O-linked chain. This was shown by determining the ratio of the unglycosylated and glycosylated forms of the tryptic peptide from free alpha that contains the O-glycosylation site (residues 36-42). Based on these findings, we make the following conclusions. 1) O-Glycosylation of alpha-subunit is a late event in the secretory pathway of trophoblasts compared to the rapid combination in the rough endoplasmic reticulum of hCG subunit precursors to form alpha beta dimer. 2) Association of alpha with beta precludes the subsequent addition of the glycan to alpha at Thr-39. 3) The alpha molecules that fail to combine with beta in the endoplasmic reticulum are substrates for the later addition of O-linked carbohydrate, presumably in the Golgi complex, but only a fraction of the free alpha molecules are modified with O-linked carbohydrate.

The effect of carbonyl cyanide trifluoromethoxyphenylhydrazone and methylamine on the processing and secretion of the glycoprotein hormone chorionic gonadotropin by human choriocarcinoma cells.

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.

Conformational intermediates in the production of the combinable form of the beta-subunit of chorionic gonadotropin.

Human trophoblastic cells synthesize and secrete hCG as well as uncombined forms of the alpha- and beta-subunits of hCG. We have previously reported that the rate-limiting step in alpha beta-dimer assembly in cultured JAR choriocarcinoma cells is a conformational change in beta-subunit accompanied by the formation of intramolecular disulfide bonds. We now report on the intermediate steps in the acquisition of this combinable conformation by the beta-subunit. The earliest biosynthetically labeled form of beta detected in JAR cells is a precursor termed p beta 1 that lacks at least one of the intramolecular disulfide bonds found in mature beta-subunit, that does not combine with alpha-subunit, and that does not react with a monoclonal antibody specific for free beta. The p beta 1 precursor rapidly assumes (within 5 min) a new conformation termed p beta 2 that, in contrast to p beta 1, migrates more slowly on nonreduced sodium dodecyl sulfate-polyacrylamide gels, combines with alpha to form the hCG dimer, and reacts with the monoclonal anti-free beta antibody. Pulse-chase kinetic experiments support the following sequence of events: p beta 1----uncombined p beta 2----combined p beta 2. The transition of p beta 1 to uncombined p beta 2 involves the formation of at least one intramolecular disulfide bond coincident with the conformational shift of the p beta molecule. Furthermore, treatment of the nonreduced subunits with trypsin releases a [35S]cysteine-labeled peptide from p beta 1, but not from either form of p beta 2. This peptide presumably contains one of the two crucial cysteine residues that participate in forming the disulfide bond that distinguishes p beta 1 from the p beta 2 forms. Dimer p beta 2 differs from both p beta 1 and uncombined p beta 2 in that it contains an O-linked N-acetylgalactosamine, which represents the first step in the formation of the O-linked glycans of beta-subunit. Dimer p beta 2 is, therefore, the most fully processed and kinetically the latest of the three p beta forms that appear in JAR cell lysates. We conclude that formation of an appropriate array of intramolecular S-S bonds accompanies the acquisition of a combinable conformation of beta-subunit, and we have identified intermediate steps in the pathway leading to this conformational change. The data suggest that it is the achievement of this conformation by beta-subunit that limits the alpha beta combination reaction rather than the amount or conformation of alpha-subunit.

Identification and characterization of subpopulations of the free alpha-subunit that vary in their ability to combine with chorionic gonadotropin-beta.

The free (uncombined) alpha-subunit of hCG is secreted in excess over alpha beta dimer from both malignant and nonmalignant trophoblast cells and is secreted ectopically from a variety of other malignant cell types. The free alpha-subunits from various sources are distinguishable from those that combine because they migrate more heterogeneously and more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than dimer alpha. We have previously identified three posttranslational modifications that may contribute to the altered mobility of the free alpha-subunit and to its inability to combine with the beta-subunit: 1) preferential phosphorylation of the free alpha-subunit, 2) O-glycosylation of free alpha, and 3) differences in the processing of the asparagine-linked oligosaccharides between the free and combinable forms. We have purified three populations of the alpha-subunit from the JAR choriocarcinoma cell line and from ChaGo, a bronchogenic carcinoma cell line that ectopically synthesizes only the alpha-subunit, in order to identify the posttranslational modifications that contribute to the altered mobility on SDS-PAGE. Fractionation of the oligosaccharides released from the alpha forms with peptide N-glycosidase has shown that the faster migrating alpha forms on SDS-PAGE have less completely processed oligosaccharide chains. Twenty-two to 25% of the JAR free alpha and 35-41% of the ChaGo alpha forms that migrate the fastest on SDS-PAGE recombine with beta in an in vitro recombination assay under conditions where 62% of the dimer alpha form recombines. In contrast, only 5-12% and 16-21% of the JAR free alpha and ChaGo alpha forms, respectively, that migrate the slowest on SDS-PAGE recombine with beta. The form of JAR free alpha least capable of combining with beta contains on O-linked glycan on Thr-39. This same site is a substrate for phosphorylation by JAR cells. However, most of ChaGo alpha fails to recombine with beta even though ChaGo alpha contains little O-linked carbohydrate. These results suggest that the larger asparagine-linked complex glycans on the slower migrating alpha forms are the major limiting factor for subunit combination. Although these modifications may not be rate limiting for combination in the rough endoplasmic reticulum, they may prevent dimerization of the free subunits later in the secretory pathway.

Pulmonary ferritin: differential effects of hyperoxic lung injury on subunit mRNA levels.

Ferritin is an iron storage protein that is regulated at the transcriptional and transcriptional levels, resulting in a complex mixture of tissue- and condition-specific isoforms. The protein shell of ferritin is composed of 24 subunits of two types (heavy or light), which are encoded by two distinct and independently regulated genes. In the present studies, the isoform profile for lung ferritin differed from other tissues (liver, spleen, and heart) as determined by isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Lung ferritin was composed of equal amounts of heavy and light subunits. Differences in isoform profiles were the result of tissue-specific differential expression of the ferritin subunit genes as demonstrated by Northern blot analyses. Like heart ferritin, lung ferritin exhibited a low iron content that did not increase extensively in response to iron challenge, which contrasts with ferritins isolated from liver or spleen. When animals were exposed to hyperoxic conditions (95% oxygen for up to 60 h), ferritin heavy subunit mRNA levels did not markedly change at any of the investigated time points. In contrast, ferritin light subunit mRNA increased severalfold in response to hyperoxic exposure. Investigation of the cytoplasmic distribution of ferritin mRNA showed that a substantial portion was associated with the ribonucleoprotein (RNP) fraction of the cytosol, suggesting that a pool of untranslated ferritin mRNA exists in the lung. Upon hyperoxic insult, all ferritin light subunit mRNA pools (RNP, monosomal, polysomal) were elevated, although a specific shift from RNP to polysomal pools was not evident. Therefore, the increase in translatable ferritin mRNA in response to hyperoxia resulted from transcriptional rather than specific translational activation. The observed pattern of light chain-specific transcriptional induction of ferritin is consistent with the hypothesis that hyperoxic lung injury is at least partially iron mediated.

Attenuation of oxidant-induced lung injury by 21-aminosteroids (lazaroids): correlation with the mRNA expression for E-selectin, P-selectin, ICAM-1, and VCAM-1.

We compared the effects of treatment with methylprednisolone or the 21-aminosteroids, U-74389 and U-74006F (Tirilizad mesylate), on hyperoxic lung injury and the associated expression of mRNA for several adhesion molecules in rats. Inhalation of > 95% oxygen for up to 72 hr in Sprague-Dawley rats produced a marked increase in lung weight and an accumulation of fluid in the thorax when compared with air-breathing controls. Hyperoxia also induced a marked neutrophil-rich influx of inflammatory cells into the bronchial lumen as measured by bronchoalveolar lavage. Neutrophil numbers in bronchoalveolar lavage fluid peaked after 60 hr of exposure to s 95% oxygen; this was associated with a marked upregulation of mRNA for the adhesion molecules P-selectin and E-selectin but not VCAM-1. mRNA for ICAM-1 was constitutively expressed at high levels in both air-breathing controls and in the lungs of rats exposed to high concentrations of oxygen. Pretreatment with the 21-aminosteroids reduced hyperoxic lung damage and improved survival times in animals exposed to > 95% oxygen. However, treatment with methylprednisolone significantly decreased survival times. Treatment with U-74389 did not significantly (p > 0.05) inhibit the BAL neutrophilia and did not significantly (p > 0.05) reduce hyperoxia-induced increases in mRNA expression for P-selectin and E-selectin. The inhibition of hyperoxic lung damage coupled with improved survival seen in treated animals suggests that 21-aminosteroids may provide valuable treatments for pulmonary disorders in which oxidant damage has been implicated.

Endogenous brain cytokine mRNA and inflammatory responses to lipopolysaccharide are elevated in the Tg2576 transgenic mouse model of Alzheimer's disease.

Beta-amyloid (Abeta) plaques have been shown to induce inflammatory changes in Alzheimer's disease brains. Cortical, but not cerebellar tissue from 16-month-old Tg2576 (Tg+) mice showed significant increases in interleukin (IL)-1alpha (2.2-fold), IL-1beta (3.4-fold), tumor necrosis factor-alpha (3.9-fold), and monocyte chemoattractant protein-1 (2.5-fold) mRNA levels compared to controls (Tg-). These changes were not apparent in 6-month-old Tg+ mice except for TNF-alpha. mRNA levels of glial fibrillary acidic protein and complement components, C1qA and C3 were also elevated in aged mice. Lipopolysaccharide (LPS) (25 microg/mouse, i.v.) induced a significantly greater production of IL-1beta protein in the cortices and hippocampi of Tg+ vs. Tg- mice at 1, 2, 4, and 6 h. Experiments in 6-month-old mice showed that not only was there less cytokine produced compared to 16-month-old mice, but the exacerbated cytokine response to LPS in Tg+ mice was not apparent. Higher levels of Abeta1-40 were measured in the cortices of 6- and 16-month-old Tg+ mice at 4-6 h after LPS, which returned to baseline after 18 h. We demonstrate that Abeta plaques elicit inflammatory responses in Tg2576 mice that are further exacerbated when challenged by an exogenous inflammatory insult, which may serve to amplify degenerative processes.

Pattern of cytokine and adhesion molecule mRNA in hapten-induced relapsing colon inflammation in the rat.

We examined the mRNA expression of cytokines, chemokines, integrins, and selectins in colon lesions of rat colitis with a semi-quantitative RT-PCR assay. Rat colitis was induced by trinitrobenzene sulfonic acid (TNBS) in 50% ethanol. Within 24 h, an acute inflammation occurred with hyperemia, edema, necrosis and an intense infiltration of granulocytes in the mucosa. The lesion proceeded into a T-lymphocyte/monocyte-driven chronic inflammation for two weeks and healed in 6 weeks. An acute inflammation recurred at the same site when the recovered animals were systemically injected with TNBS. We isolated RNA from colon tissue at 24 h, 1, 2, 4, 6 weeks after TNBS treatment and from the relapsed animals. The mRNA for cytokines IL-1beta, IL-6, IL-10 and the chemokines CINC, MIP-1alpha, MCP-1 were significantly (P < 0.05) elevated and persisted for 2 weeks, decreased in 6 weeks and increased again during relapse. IFN-gamma mRNA stayed at control levels initially, but increased dramatically in the second weeks of chronic inflammation as well as in relapse. The mRNA levels of adhesion molecules, ICAM-1, VCAM-1, the mucosal homing integrin beta7 as well as P- and E-selectin were greatly enhanced between 1 and 3 weeks. The data showed that the chronically inflamed tissue expresses a time-dependent changing pattern of TH1 cytokines and adhesion molecules that maintain the infiltration and activation of inflammatory cells and tissue injury.

Interactions between interleukin-1 and basic fibroblast growth factor on articular chondrocytes. Effects on cell growth, prostanoid production, and receptor modulation.

The interactions of interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (bFGF) with monolayer cultures of rabbit articular chondrocytes were examined. Both agonists resulted in a synergistic increase in prostanoid production, to levels higher than the maximal level for either protein alone. The synergy was time- and dose-dependent, was augmented in serum-free medium, and was blocked by indomethacin or a polyclonal antibody to IL-1 beta. The proteins had opposite effects on cell growth, and IL-1 beta completely blocked the mitogenic effect of bFGF. Pretreatment of cells with IL-1 beta induced a down-regulation in the number of the bFGF high-affinity receptors. Pretreatment of cells with bFGF increased the number of IL-1 receptors, which was dependent on messenger RNA synthesis.

Role of disulfide bond formation in the folding of human chorionic gonadotropin beta subunit into an alpha beta dimer assembly-competent form.

The malignant trophoblastic cell line JAR was used as a model system to study protein folding in intact cells. We have used this model previously to identify conformational intermediates in the production of an assembly-competent form of the human chorionic gonadotropin beta subunit (Ruddon, R. W., Krzesicki, R. F., Norton, S. E., Beebe, J. S., Peters, B. P., and Perini, F. (1987) J. Biol. Chem. 262, 12533-12540). The earliest biosynthetic precursor of the human chorionic gonadotropin beta subunit detectable in JAR cells pulse labeled for 2 min is p beta 1, a form that lacks half of the six intrachain disulfide bonds observed in the fully processed dimer form of beta and that does not combine with the alpha subunit. p beta 1 is rapidly (t1/2 approximately 4 min) converted into p beta 2, which has a full complement of intrachain disulfide bonds and does combine with the alpha subunit. In this study, we have identified the three late forming disulfide bonds involved in the transition of p beta 1 into the assembly-compete form, p beta 2. The last three disulfide bonds to form are those between cysteines 9 and 90, 23 and 72, and 93 and 100. These were identified in JAR cell lysates that had been pulse labeled with [35S]cysteine for 2 or 5 min followed by trapping of the cysteine thiols with iodoacetic acid before immunopurification of the beta subunit forms. Immunopurified p beta 1 was treated with trypsin under nonreducing conditions to liberate [35S]cysteine-containing peptides from the disulfide-linked beta core polypeptide. These tryptic peptides were then separated by high performance liquid chromatography and sequenced to determine the location of the carboxymethyl-[35S]cysteine residues. The three late forming disulfide bonds are most likely the ones involved in stabilizing the conformation of the beta subunit that is required for combination with alpha to form the biologically functional alpha beta heterodimer.

Phosphorylation of the secreted, free alpha subunit of human chorionic gonadotropin.

Phosphorylation of secretory proteins is an uncommon event. In this manuscript, the phosphorylation of human chorionic gonadotropin, a glycoprotein hormone secreted by the JAR choriocarcinoma cell line, is described. Labeling of JAR cells with 32PO4 indicates that both the intracellular and the secreted forms of the free alpha subunit are phosphorylated. Although the secreted alpha beta dimer also incorporates 32PO4, there is little detectable phosphorylation of the intracellular precursors of alpha beta dimer, suggesting that dimer phosphorylation occurs as a late event in post-translational processing. In addition, phorbol 12-myristate 13-acetate markedly stimulates the phosphorylation of both intracellular and secreted forms of free alpha subunit and to a lesser extent of secreted alpha beta dimer. In vitro assays, using homogenates of JAR cells as a source of protein kinase activity, indicate that the uncombined alpha subunit is preferentially phosphorylated. The phosphorylation sites are on serine and threonine residues in the alpha subunit.

A kinetic comparison of the processing and secretion of the alpha beta dimer and the uncombined alpha and beta subunits of chorionic gonadotropin synthesized by human choriocarcinoma cells.

Human choriocarcinoma cells (JAR) synthesize the alpha and beta subunits of the glycoprotein hormone chorionic gonadotropin (hCG) (R.W. Ruddon, C.A. Hanson, A. H. Bryan, G.J. Putterman, E.L. White, F. Perini, K. S. Meade, and P.H. Aldenderfer (1980) J. Biol. Chem. 255, 1000-1007). In addition to the hCG dimer (alpha beta), JAR cells secrete uncombined alpha and beta subunits into the culture medium (L.A. Cole, R.J. Hartle, J.A. Laferla, and R.W. Ruddon (1983) Endocrinology 113, 1176-1178). Pulse-chase studies with [35S]methionine or [3H]mannose were carried out in order to compare free alpha, free beta, and the alpha beta dimer with regard to the kinetics of synthesis, N-linked oligosaccharide processing, and secretion and to determine the kinetics of alpha-beta subunit combination. A panel of three antisera was used to immunoprecipitate directly the free subunits and the alpha beta dimer sequentially from the same cell lysates and culture media. The alpha subunit of hCG was synthesized in a slight molar excess (1.2-1.5-fold) over the beta subunit, and alpha beta dimer was rapidly formed by combination of the intracellular alpha and beta precursors. Dimer formation was already apparent in JAR cells following a 10-min biosynthetic labeling incubation with [35S]methionine. The combination of subunits ceased by 30 min of chase even though 51% of alpha and 44% of beta remained free within the cells. Combination of the alpha and beta precursors had occurred before their N-linked oligosaccharides were processed beyond the Man8GlcNAc2 structure. The initial trimming of glucosyl and mannosyl units from the high-mannose oligosaccharides of the hCG precursors occurred more rapidly for free alpha and CG-alpha than for free beta and CG-beta. JAR cells accumulated alpha precursors bearing mostly Man8GlcNAc2 units and beta precursors bearing Man8GlcNAc2 units that represent the substrates of the rate-limiting step in the secretory pathway. In spite of the fact that their N-linked oligosaccharides were trimmed at different rates, free alpha, free beta, and alpha beta dimer were all secreted into the medium at the same rate, with a half-time of 35 min. The secreted hCG forms were stable in the chase medium between 4 and 8h, indicating that extracellular degradation, combination of free subunits to form dimer, or dissociation of dimer to form free subunits did not occur.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of cytokines in inflammatory synovitis. The coordinate regulation of intercellular adhesion molecule 1 and HLA class I and class II antigens in rheumatoid synovial fibroblasts.

This study was undertaken in an effort to understand the role of cytokines in T lymphocyte trafficking into inflamed synovium and in the potential enhancement of antigen presentation by human synovial fibroblasts. We found that interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and interferon-gamma (IFN gamma) each increased the cell surface expression of intercellular adhesion molecule 1 (ICAM-1) on human synovial fibroblasts in a dose- and time-dependent manner. Maximal ICAM-1 expression occurred within 8 hours of induction, with the following order of efficacy: IFN gamma greater than TNF alpha greater than IL-1 beta. The number of cells bearing the ICAM-1 antigen also increased, from a basal level of approximately 30% to more than 83% after cytokine induction (for all 3 cytokines). ICAM-1 expression rapidly decreased following cytokine removal. The expression of lymphocyte function-associated antigen 3 was also examined, but it was not changed by any of the 3 cytokines. Class I major histocompatibility complex antigen expression was increased modestly by all 3 cytokines, and expression was maximal by 24 hours after treatment. Only IFN gamma induced HLA class II antigen expression, and this expression persisted for up to 6 days following removal of the lymphokine. IL-6 and granulocyte-macrophage colony-stimulating factor had no effect on any of the parameters examined. Our data support an interactive role for inflammatory cytokines and the expression of adhesion ligands and HLA antigens by human synovial fibroblasts in the pathogenesis of synovial inflammation in rheumatoid arthritis.

Detection of a glycosylated, incompletely folded form of chorionic gonadotropin beta subunit that is a precursor of hormone assembly in trophoblastic cells.

The alpha and beta subunits of human chorionic gonadotropin are secreted both as a combined, noncovalently linked dimer form as well as uncombined, free forms by human trophoblastic cells. We have utilized the cultured choriocarcinoma cell line JAR to determine what regulates the combination of the two subunits. The human chorionic gonadotropin subunits produced by JAR cells were biosynthetically labeled with [35S] cysteine or [3H]mannose by a pulse-chase protocol, purified by immunoprecipitation with specific antisera that recognize free or combined subunits, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing or reducing conditions. Radioactively labeled bands were eluted from the gels and analyzed for total counts/minute incorporated, the ratio of free thiols to intramolecular cystine disulfides, and oligosaccharide composition. In some experiments, labeled gel bands were eluted with trypsin under nonreducing conditions, and the trypsin-released peptides were analyzed by high performance liquid chromatography. Using these procedures, the following results were obtained. The earliest, biosynthetically labeled form of the beta subunit detected in JAR cells contains high mannose N-linked oligosaccharides and has one-half of its incorporated cysteines present as free thiols. This form, termed pre-beta 1, has not yet combined with the alpha subunit even though the biosynthetically labeled alpha subunit is present in the cells at the same time. The pre-beta 1 form has a t1/2 of about 4 min and has a precursor-product relationship with a more completely disulfide-bonded form, termed pre-beta 2, which does combine with the alpha subunit to form a dimer. A subset of beta molecules produced in JAR cells does not attain the same disulfide bonding pattern as the pre-beta 2 form, does not combine with the alpha subunit, and is secreted as a free beta subunit into the culture medium. On the other hand, the earliest detectable form of the alpha subunit in JAR cells has all its thiols present as cystine disulfides, at a time when dimerization with the beta subunit has not yet taken place. These results strongly suggest that intramolecular disulfide bond formation in the beta subunit is the crucial and rate-limiting event in alpha beta dimer formation. The subset of beta molecules that remain free do not appear to form the appropriate intramolecular disulfides and thus do not achieve the correct conformation to combine with the alpha subunit.(ABSTRACT TRUNCATED AT 400 WORDS)

Biosynthesis and deposition of a noncovalent laminin-heparan sulfate proteoglycan complex and other basal lamina components by a human malignant cell line.

The basal lamina components laminin, heparan sulfate proteoglycan (HSPG), and type IV collagen were synthesized and codeposited in the extracellular matrix (ECM) by a cultured human cell line from gestational choriocarcinoma (JAR). Laminin and HSPG formed a noncovalent complex detected by the coimmunoprecipitation of HSPG with laminin from cell lysates and culture media. The complex was stable in the cell lysis buffer that contained detergents (1% Triton X-100, 0.5% deoxycholate, and 0.1% sodium dodecyl sulfate) and sodium chloride (from 0.15 to 1.0 M), but was dissociated by adding 8 M urea to the detergent lysates. Even though JAR cells produced roughly equal amounts of HSPG and chondroitin sulfate proteoglycan, only HSPG complexed with laminin, suggesting a specific interaction between these basal lamina components. The laminin-HSPG complex was deposited and retained in the ECM. This was shown biochemically by isolating an enriched fraction of ECM from JAR cells cultured on native type I collagen gels. At steady state, more than half (52%) of the laminin-HSPG in the culture was recovered in the ECM fraction, in contrast to 16% of the total laminin and 29% of the total type IV collagen, which were secreted to a greater extent than laminin-HSPG into the culture medium. The retention of the laminin-HSPG complex in the ECM suggests that it may participate in the assembly of the basal lamina-like extracellular matrix deposited by JAR cultures. Omission of ascorbate from the culture medium abolished the ECM deposition of type IV collagen but had little effect on the deposition of laminin or laminin-HSPG. This demonstrates that the stable deposition of laminin-HSPG and laminin in the collagen-based choriocarcinoma cultures is not dependent on an assembled network of type IV collagen.

T lymphocyte adhesion to human synovial fibroblasts. Role of cytokines and the interaction between intercellular adhesion molecule 1 and CD11a/CD18.

We studied the adhesion of human peripheral blood T lymphocytes to human synovial fibroblasts stimulated with interferon-gamma (IFN gamma), tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or combinations of these cytokines. T lymphocytes bound poorly to untreated human synovial fibroblasts. IFN gamma treatment resulted in the largest increase in adhesion, followed by TNF alpha and IL-1 beta. Combinations of IFN gamma + TNF alpha and IFN gamma + IL-1 beta had a synergistic effect on intercellular adhesion molecule 1 (ICAM-1) expression and adhesion. The increase in cellular adhesion induced by cytokines correlated with the up-regulation of the number of cells expressing ICAM-1 and the density of antigen/cell. There was no synergistic effect on leukocyte function-associated antigen 3 (LFA-3) or on HLA class I or class II antigen expression. Adhesion was only partially inhibited by anti-ICAM-1, anti-LFA-1, or anti-CD18. These findings suggest the existence of ICAM-1--independent and CD11/CD18-independent adhesion mechanisms. Anti-LFA-3 was completely ineffective as an inhibitor of adhesion. There was no additive or synergistic advantage of using combinations of antibodies to increase the level of inhibition, i.e., anti--ICAM-1 + anti-LFA-3, anti-ICAM-1 + anti-CD18, or anti-ICAM-1 + anti-LFA-1 (CD11a). Our data indicate that proinflammatory cytokines may play a prominent role in the formation and exacerbation of synovial hyperplasia, by regulating the recruitment and retention of T lymphocytes via the up-regulation of adhesion molecules on synovial fibroblasts.

The use of drugs to dissect the pathway for secretion of the glycoprotein hormone chorionic gonadotropin by cultured human trophoblastic cells.

Agents that affect intracellular cation and pH gradients and inhibit energy production have been tested for their ability to modulate the processing and secretion of the free alpha subunit and the alpha beta dimer of human chorionic gonadotropin (hCG) by cultured human trophoblastic cells (JAR). Incubation of JAR cells with monensin or nigericin, monovalent cation ionophores that produce equilibration of Na+ and K+ across cellular membranes, dicyclohexylcarbodiimide, an agent that inhibits intracellular membrane ATPases, and methylamine, which neutralizes intracellular pH gradients, produced similar effects on hCG processing and secretion. All these agents inhibited the processing of the asparagine-linked oligosaccharide chains of free alpha subunit and the alpha and beta subunits contained in the hCG dimer. Moreover, after treatment of JAR cells with these agents, there was an intracellular accumulation of precursor forms and an inhibition of secretion of "mature" forms of hCG. Monensin affected the processing and secretion of hCG subunits differently at different concentrations. At 5 X 10(-7) M, monensin inhibited the processing of the asparagine-linked oligosaccharides of hCG without altering the rate-limiting step in the secretory pathway or blocking hCG secretion. The intracellular hCG subunit precursors in both control and monensin-treated cells contained a similar array of high mannose oligosaccharides, predominantly of the Man8GlcNAc2 and Man9GlcNAc2 types. However, monensin-treated cells secreted hCG subunits that contained endo H-sensitive oligosaccharides of the high mannose (mostly Man5GlcNAc2) and hybrid types rather than the endo H-resistant complex chains synthesized by control cells. Nevertheless, a full complement of serine-linked oligosaccharides was added to the hCG-beta subunit in monensin-treated cells. These results indicate that the intracellular movement of hCG from the rough endoplasmic reticulum to the cell surface was not inhibited by monensin at a concentration that impaired Golgi-localized steps in the processing of asparagine-linked oligosaccharides. At 5 X 10(-6) M, monensin significantly inhibited secretion of hCG and created a new rate-limiting step in the processing pathway. hCG subunits bearing Man5GlcNAc2 units accumulated intracellularly, suggesting that the equilibration of intracellular Na+/K+ pools blocked oligosaccharide processing at an intra-Golgi point, perhaps by inhibiting movement of the glycoprotein hormone from the "cis" to the "trans" Golgi compartment. Since the other drugs mentioned above produced similar effects on hCG processing and secretion, it appears that maintenance of intracellular cation and pH gradients is necessary for the intra-Golgi transport of glycoprotein hormones.(ABSTRACT TRUNCATED AT 400 WORDS)

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells.

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.

Two-dimensional gel electrophoresis of cell proteins in childhood leukemia, with silver staining: a preliminary report.

We have investigated the potential of two-dimensional electrophoresis for identifying differences in protein patterns between acute lymphocytic (ALL) and acute myelogenous leukemia (AML). We studied blood cells directly isolated from seven children with non-T, non-B ALL and five with AML. Solubilized proteins were electrophoresed with the ISO-DALT system and made visible by silver staining. More than 800 spots were routinely detected in individual gels. Patterns for multiple runs of the same sample were indistinguishable. Most of the prominent spots were present in gels from all 12 of the individuals studied. However, substantial variability in spot intensity was observed, even among patients in the same group. Five spots were consistently present in gels of all the AML patient, and either completely absent or substantially decreased in ALL gels. Three other spots were present in ALL but not AML gels. We conclude that this may be a useful tool for characterizing leukemic cells.

Airway recruitment of leukocytes in mice is dependent on alpha4-integrins and vascular cell adhesion molecule-1.

The involvement of the alpha4-integrin very late activation antigen 4 and vascular cell adhesion molecule-1 (VCAM-1) in leukocyte trafficking into the airways of ovalbumin (OA)-sensitized and OA-challenged mice was investigated using in vivo administration of anti-alpha4 monoclonal antibodies (mAb) PS/2, R1-2, and M/K-2.7 (MK2), specific for VCAM-1. VCAM-1 was upregulated on endothelial cells in lung tissue after OA inhalation. PS/2, R1-2, or MK2 significantly inhibited the recruitment of eosinophils and lymphocytes into the bronchoalveolar lavage (BAL) fluid and decreased inflammation in the lung tissues. Escalating in vivo doses of PS/2 or MK2 increased circulating levels of rat immunoglobulin G in the plasma. The binding of phycoerytherin-labeled anti-alpha4 mAb to blood T cells from PS/2-treated mice was reduced, implying that alpha4 sites were already occupied. T cells and eosinophils in BAL fluid from mice treated with PS/2 or MK2 were phenotypically different from controls. Selective decreases of alpha4+ T cells in the BAL fluid after PS/2 or MK2 treatment were coupled with changes in CD8+, CD11a, and CD62L expression. The alpha4-integrin and VCAM-1 may have important roles in the antigen-induced recruitment of T cells and eosinophils during OA-induced airway inflammation. The data suggest that these adhesion molecules may be suitable targets for therapeutic intervention in certain conditions of pulmonary inflammation.