H W Harris Jr, K Strange, M L Zeidel
In vitro studies indicate that oxidatively modified low density lipoprotein (oxLDL) promotes leukocyte adhesion to the vascular endothelium, a constant feature of early atherogenesis. Using intravital fluorescence microscopy in the dorsal skinfold chamber model in awake Syrian golden hamsters, we studied whether (a) oxLDL elicits leukocyte/endothelium interaction in vivo, and whether (b) leukotrienes play a mediator role in this event. Leukocyte/endothelium interaction was assessed in the time course after intravenous injection of native human LDL (4 mg/kg body wt) and of oxLDL (7.5 microM Cu++, 6 h, 37 degrees C) into control hamsters and into hamsters, pretreated with the selective leukotriene biosynthesis inhibitor MK-886 (20 mumol/kg, i.v.). While no effect was seen after injection of native LDL, oxLDL elicited an immediate induction of leukocyte adhesion to the endothelium of arterioles and postcapillary venules. Total and differential leukocyte counts suggest that all leukocyte subsets were likewise affected by oxLDL with no specific preference for monocytes. Stimulation of leukocyte adhesion was entirely prevented in inhibitor-treated animals, suggesting the important mediator role of leukotrienes in oxLDL-induced leukocyte/endothelium interaction.
H A Lehr, C Hübner, B Finckh, S Angermüller, D Nolte, U Beisiegel, A Kohlschütter, K Messmer
Sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was examined in microsomes prepared from the left ventricular myocardium of eight heart transplant recipients with end-stage idiopathic dilated cardiomyopathy and six unmatched organ donors with normal cardiac function. At cAMP concentrations less than or equal to 1.0 microM, sarcoplasmic reticulum-associated cAMP phosphodiesterase activity was functionally homogeneous. cAMP phosphodiesterase activity was inhibited competitively by cGMP (Ki = 0.031 +/- 0.008 microM) and the cilostamide derivative OPC 3911 (Ki = 0.018 +/- 0.004 microM), but was essentially insensitive to rolipram. Vmax and Km were 781.7 +/- 109.2 nmol/mg per min and 0.188 +/- 0.031 microM, respectively, in microsomes prepared from nonfailing hearts and 793.9 +/- 68.9 nmol/mg per min and 0.150 +/- 0.027 microM in microsomes prepared from failing hearts. Microsomes prepared from nonfailing and failing hearts did not differ with respect to either the ratio of cAMP phosphodiesterase activity to ATP-dependent Ca2+ accumulation activity or the sensitivity of cAMP phosphodiesterase activity to inhibition by OPC 3911. These data suggest that the diminished inotropic efficacy of phosphodiesterase inhibitors in failing human hearts does not result from changes in the level, kinetic properties, or pharmacologic sensitivity of sarcoplasmic reticulum-associated cAMP phosphodiesterase activity.
M A Movsesian, C J Smith, J Krall, M R Bristow, V C Manganiello
We have examined the mechanisms involved in the adherence of normal peripheral blood eosinophils to cultured human umbilical vein endothelial cells (HEC) under three conditions: (a) adherence in the absence of treatment of HEC or eosinophils with activating agents (basal adherence); (b) adherence induced by stimulation of eosinophils with phorbol ester (eosinophil-dependent adherence); and (c) adherence induced by pretreatment of HEC with LPS, tumor necrosis factor (TNF), or IL-1 (endothelial-dependent adherence). A mechanism was identified that was equally active in basal, eosinophil-dependent, and endothelial-dependent adherence. This mechanism was optimally active in the presence of both Ca++ and Mg++, and reduced in the presence of Ca++ only or Mg++ only. Furthermore, like the other mechanisms of eosinophil adherence, it was active at 37 degrees C but not at 4 degrees C. A second mechanism of adherence was involved in eosinophil- and in endothelial-dependent adherence. This mechanism was dependent on the CD11/CD18 adhesion complex of eosinophils (i.e., inhibited by anti-CD18 MAb) and it was active in the presence of Ca++ and Mg++ or Mg++ only, but not Ca++ only. The third mechanism of adherence was specific for endothelial-dependent adherence. It involved the endothelial ligand vascular cell adhesion molecule-1 (VCAM-1) and the eosinophil receptor very late activation antigen-4 (VLA-4, CD49d/CD29, i.e., inhibited by anti-VCAM-1 MAb or anti-VLA-4 MAb). This mechanism was active in the presence of Ca++ and Mg++ but not of Ca++ only or Mg++ only, and was not up- or downregulated when eosinophils were stimulated with phorbol ester. In contrast, the endothelial leukocyte adhesion molecule-1 (ELAM-1), that binds neutrophils and monocytes, was not involved in eosinophil adherence to LPS-, TNF-, or IL-1-stimulated HEC (i.e., not inhibited by anti-ELAM-1 MAb). We conclude that eosinophils, like monocytes and lymphocytes, bind to the cytokine-induced endothelial ligand VCAM-1 via the integrin receptor VLA-4.
A Dobrina, R Menegazzi, T M Carlos, E Nardon, R Cramer, T Zacchi, J M Harlan, P Patriarca
To investigate the mechanism(s) of insulin-induced suppression of plasma amino acid concentration and release, we studied forearm as well as whole-body leucine and phenylalanine uptake and release during a peripheral insulin infusion in postabsorptive normal subjects using isotope-dilution methods. Before insulin, leucine and phenylalanine release exceeded uptake (P less than 0.01 and P less than 0.07, respectively). A net output of alpha-ketoisocaproate (KIC) was also observed. During insulin, arterial plasma leucine, KIC and phenylalanine concentrations decreased (P less than 0.05 or less vs. basal), despite ongoing net output of these substrates by the forearm, that persisted after correction for the mean transit time spent through the extracellular muscular space. By the end of insulin, whole-body leucine and phenylalanine concentrations and rate of appearance were decreased (P less than 0.01 vs. basal). However, release and uptake of both amino acids by the forearm were not significantly decreased vs. the preinsulin values. These data indicate that systemic hyperinsulinemia acutely decreases plasma amino acid concentrations by acting primarily at sites other than skeletal muscle.
P Tessari, S Inchiostro, G Biolo, E Vincenti, L Sabadin
Tumor necrosis factor (TNF) is a peptide secreted by macrophages in response to endotoxin that can produce many of the changes seen in septic shock. After cecal ligation and puncture (CLP) rats gradually develop tachycardia, hypotension, tachypnea, and hypothermia. At 5 h post-CLP, rats have a peak in serum levels of endotoxin and 60% of rats have blood cultures that grow Gram-negative rods (Escherichia coli and Klebsiella pneumonia). At 20 h post-CLP all rats develop positive blood cultures. Serum levels of TNF are not reproducibly measurable in rats following CLP. Rats undergoing CLP have a 50-80% mortality with deaths usually occurring 24-72 h postinjury. Repetitive (twice daily x 6 d) i.p. injection of sublethal doses of recombinant human TNF-alpha (100 micrograms/kg) to rats undergoing CLP 1 d after the treatment period resulted in a significant reduction in mortality compared to control rats previously unexposed to rTNF (P less than 0.03). Animals treated with rTNF had no hypotension or hypothermia after CLP and regained normal food intake faster than control rats. 12 h after CLP the gene expression for manganous superoxide dismutase (MnSOD), an inducible mitochondrial metalloenzyme responsible for cellular resistance to injury from toxic reactive oxygen species, was higher in livers of rats treated with rTNF suggesting that the TNF treatment augmented expression of this protective enzyme. Unlike MnSOD, expression of the gene for copper-zinc SOD was not affected by CLP or rTNF treatment. The results suggest that prior treatment with recombinant TNF can ameliorate the lethality, hypotension, hypothermia, and anorexia of Gram-negative sepsis in rats and that the mechanism may be related to enhanced hepatic expression of the gene for MnSOD. Repeated administration of recombinant TNF may be a strategy to minimize mortality and morbidity of Gram-negative sepsis.
H R Alexander ... M C Stovroff, J A Norton
Glucocorticoids inhibit plasma extravasation induced in the rat tracheal mucosa by substance P and other tachykinins released from sensory nerves. This study was performed to determine whether this antiinflammatory effect of glucocorticoids is mediated by the tachykinin-degrading enzymes neutral endopeptidase (NEP) and kininase II (angiotensin converting enzyme, ACE). In addition, we studied the effect of dexamethasone on a nonpeptide inflammatory mediator, platelet-activating factor (PAF), which is not degraded by NEP or ACE. Adult male pathogen-free F344 rats were treated for 2 d with dexamethasone (0.5 mg/kg per d i.p.), or with the vehicle used to dissolve the steroid. The magnitude of plasma extravasation produced by an intravenous injection of substance P (5 micrograms/kg) or PAF (10 micrograms/kg) was then assessed by using Monastral blue pigment as an intravascular tracer. The role of NEP and ACE activities in the changes produced by dexamethasone was investigated by examining the effect of the selective inhibitors of these enzymes, phosphoramidon and captopril. Dexamethasone reduced the substance P-induced extravasation by 57% but did not affect the PAF-induced extravasation. The suppressive effect of dexamethasone on substance P-induced extravasation was completely reversed by simultaneously inhibiting NEP and ACE activities, but the inhibition of these enzymes had no effect on PAF-induced extravasation, regardless of whether the rats were pretreated with dexamethasone or not. These results suggest that NEP and ACE mediate a selective inhibitory effect of glucocorticoids on neurogenic plasma extravasation.
G Piedimonte, D M McDonald, J A Nadel
We have compared the abilities of extracellular ATP (acting via P2-purinergic receptors) and formylated peptides (FMLP) to stimulate both phospholipase D (PLD)-based signal transduction and primary granule (azurophilic) secretion in HL-60 cells induced to differentiate along the granulocytic pathway. In undifferentiated HL-60 cells, neither ATP nor FMLP elicited significant PLD activation or increased secretion despite the previously documented ability of ATP to stimulate large increases in polyphosphoinositide hydrolysis and Ca2+ mobilization. Conversely, within 1 d after induction of granulocytic differentiation by dibutyryl cAMP, both ATP and FMLP induced large increases in azurophilic secretion and corresponding increases in PLD activity. ATP-activated PLD activity was near-maximal within 1 d after dibutyryl cAMP treatment, while the FMLP-induced activity increased continuously over 4 d, with a maximal level twice that stimulated by ATP. Additional experiments characterized the activation of PLD by receptor-independent pathways at different stages of differentiation; these included studies of phorbol ester action in intact cells and GTP gamma S action in electropermeabilized cells. An apparent role for guanine nucleotide-binding regulatory proteins in PLD regulation was also indicated by the significant reduction in FMLP- and ATP-stimulated PLD activity observed in cells pretreated with pertussis toxin. At all stages of differentiation, there was good correlation between the relative efficacies of ATP versus FMLP in stimulating both secretion and PLD activity. These data indicate: (a) that the receptor-regulated phospholipase D signaling pathway is induced during differentiation of myeloid progenitor cells; and (b) that differential activation of this signaling system by various Ca(2+)-mobilizing receptor agonists may underlie the differential regulation of secretion and other phagocyte functions by such agents.
M S Xie, L S Jacobs, G R Dubyak
Ischemia may cause increased or decreased distensibility of the left ventricle, but the cellular mechanisms involved have not been clarified. We examined the possible contributions of changes in intracellular inorganic phosphate, pH, and Ca2+ concentrations to altered diastolic function in cultured myocytes subjected to partial metabolic inhibition. Paced cultured embryonic chick and adult rabbit ventricular myocytes superfused with 20 mM 2-deoxyglucose (2DG) exhibited an increase in end-diastolic intracellular free calcium concentration ([Ca2+]i) and an upward shift in end-diastolic cell position. These results indicate that glycolytic blockade increases diastolic and systolic calcium in paced ventricular myocytes, and that this elevated diastolic calcium influences the extent of diastolic relaxation. In contrast, paced ventricular myocytes superfused with 1 mM cyanide (CN) exhibited a similar increase in end-diastolic [Ca2+]i but a decrease in end-diastolic cell position and amplitude of motion. Although changes in ATP contents were similar in both groups (2DG, -29.9%; CN, -40.1%), alterations of intracellular pH and inorganic phosphate concentrations were different. In 2DG-treated cells, pHi did not decrease significantly (7.18 +/- 0.04 to 7.12 +/- 0.11, n = 14) but in the CN group it decreased markedly within 6 min (7.18 +/- 0.04 to 6.76 +/- 0.11, n = 11, P less than 0.01). Intracellular inorganic phosphate decreased slightly in the 2DG group (-14.8%, NS) but increased in cells exposed to CN (45.7%, P less than 0.02). We conclude that while a prominent increase in diastolic [Ca2+]i occurs in rapidly paced ventricular myocytes exposed to either inhibitors of glycolysis or oxidative phosphorylation, the effects of this increase in [Ca2+]i on diastolic distensibility may be influenced by intracellular accumulation of metabolites that decrease the sensitivity of myofilament to [Ca2+]i.
H Ikenouchi, O Kohmoto, M McMillan, W H Barry
Considering the special relation of human multiple myeloma (MM) to bones, it is of importance to clarify the early steps of bone involvement in this disease. In this work, using bone histomorphometry (including histoenzymologic and kinetic studies for the first time), we have evaluated the bone remodeling (i.e., bone resorption and bone formation rates) of 16 individuals with early MM in comparison with that of 10 with benign monoclonal gammopathy (BMG) and that of 17 patients with previously untreated overt MM. A significantly increased osteoblastic recruitment was observed in the individuals with early MM when compared with those with BMG (P less than 0.01). A significant (P less than 0.01) increased bone resorption (i.e., eroded surfaces, osteoclast numbers and surfaces) was observed from the early stage of MM in comparison with the BMG status where bone resorption remained within the normal range. At the tissue level, there was no difference in terms of bone resorption between early and overt MM. On the other hand, osteoblast activity was significantly reduced in patients with overt MM (P less than 0.05 by comparison with those with early MM). A significant enhancement of osteoblastic recruitment with an increased generation of new osteoclasts is an early critical event in the pathogenesis of human MM. Of particular importance is the early stimulation of osteoblasts, since these cells produce high amounts of IL-6, a potent myeloma cell growth factor and a critical cytokine for the formation of osteoclasts in the bone marrow.
R Bataille, D Chappard, C Marcelli, P Dessauw, P Baldet, J Sany, C Alexandre
Resting natural killer (NK) cells express the p75 chain of the IL-2 receptor (IL-2R beta) and most NK cells express the CD2 (erythrocyte rosette) receptor. The cell adhesion molecule, LFA-3, is a natural co-ligand for CD2. Tac antigen (IL-2R alpha), a p55 IL-2R subunit, can be expressed after NK activation and may play a role in IL-2-induced NK proliferation. Little is known of the molecular mechanisms underlying cytokine production in NK cells. We investigated the roles of IL-2R alpha, IL-2R beta, and CD2/LFA-3 in the molecular regulation of NK cell granulocyte/macrophage-colony-stimulating factor (GM-CSF) production. Enriched populations of peripheral blood NK cells were separated into CD16-positive and CD16-negative fractions by flow cytometry; positively selected cells were greater than 97% positive for CD16 (the FcIII receptor for IgG which is present on almost all NK cells), less than 1% positive for the T cell antigen CD3, and did not demonstrate rearrangement of the T cell receptor beta chain gene by Southern blot. NK cell supernatants were harvested after 3-4 d of incubation with 0-100 U/ml IL-2, or after incubation with anti-CD2 (T11(3] MAb and sheep red blood cells (SRBC are a homologue for LFA-3). Parallel cell aliquots were harvested at 3-16 h for transcriptional run-on assays, S1 nuclease assays, and actinomycin D mRNA t1/2 determinations. IL-2-activated NK supernatants contained large amounts of GM-CSF (178 +/- 35 pg/ml) by ELISA as did supernatants from CD2-activated NK cells (T11(3) MAb + SRBC: 212 +/- 42) vs. less than 20 pg/ml for NK cells incubated alone or with either SRBC or T11(3) MAb alone. Sepharose-linked anti-CD3 MAb did not induce GM-CSF release from NK cells. By S1 analysis, both IL-2 and CD2 stimulation markedly augmented GM-CSF mRNA expression but with very different latencies of onset. IL-2R beta MAb inhibited greater than 85% of GM-CSF release from IL-2-activated NK cells and markedly suppressed IL-2-induced GM-CSF mRNA expression, whereas IL-2R alpha MAb even at 2,000-fold molar excess of IL-2 had little effect (less than 10%) on either GM-CSF release or mRNA expression. Run-on assays showed that GM-CSF is constitutively transcribed in NK cells and that IL-2 and CD2-activated cells had a three- to fourfold increased rate of GM-CSF transcription compared to nonstimulated cells. The t1/2 of GM-CSF mRNA in IL-2-activated NK cells was identical to that of unstimulated NK cells (15 min), whereas GM-CSF mRNA t1/2 in CD2-activated NK cells was increased 2.5-fold. We conclude that GM-CSF production in NK cells is regulated by both the IL-2Rbeta and the CD2 receptor but not by IL-2Ralpha, that both transcriptional and posttranscriptional signals act together to modulate the level of GM-CSF mRNA in NK cells, and that the molecular mechanisms underlying NK cell GM-CSF production are dependent in part on differential surface receptor activation.
L J Levitt, A Nagler, F Lee, J Abrams, M Shatsky, D Thompson
The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.
M Garbarz, W T Tse, P G Gallagher, C Picat, M C Lecomte, F Galibert, D Dhermy, B G Forget
A nonmotile mutant of Borrelia burgdorferi, the etiologic agent of Lyme disease, was isolated and characterized. The mutant was compared with the wild-type predecessor as well as with a motile back-revertant of the same genetic background. The mutant lacked, by morphologic, biochemical, and immunologic criteria, the major structural protein of flagella, flagellin. This mutation was not associated with major DNA rearrangements or with failure of transcription. An apparent consequence of a loss of flagella was reduced ability to penetrate human endothelial cell layers in vitro. In another assessment of functional significance, the flagella-less mutant was equal if not superior to flagella-bearing, isogenic isolates when examined in an enzyme-linked immunosorbent assay for anti-B. burgdorferi antibodies in the sera of Lyme disease patients. These studies of a mutant, the first among pathogenic Borrelia spp. to be characterized, indicate that the flagellum and motility it confers play a role in B. burgdorferi's invasion of human tissues. A flagella-less B. burgdorferi may be useful as the basis of a more specific immunoassay and a vaccine for protection against Lyme disease.
A Sadziene, D D Thomas, V G Bundoc, S C Holt, A G Barbour
Defensins induce ion channels in model lipid bilayers and permeabilize the membranes of Escherichia coli. We investigated whether similar membrane-active events occur during defensin-mediated cytolysis of tumor cells. Although defensin-treated K562 targets did not release chromium-labeled cytoplasmic components for 5-6 h, they experienced a rapid collapse (within minutes) of the membrane potential, efflux of rubidium, and influx of trypan blue. Defensin treatment also blunted the subsequent acidification response induced by nigericin, thereby further supporting the notion of enhanced transmembrane ion flow during exposure. These initial effects on the plasma membrane were not sufficient for subsequent lysis; a second phase of injury was required which involved the continued presence of defensin. The rapid membrane permeabilization phase was inhibited by azide/2-deoxyglucose, cytochalasin B, and increased concentrations of extracellular potassium and was unaffected by actinomycin-D, cycloheximide, and varying the calcium concentration. In contrast, the second phase was unaffected by cytochalasin B, inhibited by azide/2-deoxyglucose, enhanced by actinomycin D and cycloheximide, and varied with calcium concentration. These results indicate the initial adverse effect of defensins on mammalian cells occurs at the cell membrane. It is possible that the second phase of injury is mediated intracellularly by defensin that has been internalized through this leaky membrane.
In the present micropuncture study, the autoregulation of glomerular capillary hydrostatic pressure (PG) in Munich-Wistar rats 24 h after 75% nephrectomy (Nx) or sham operation (Sh) was investigated. The effect of varying renal perfusion pressure (RPP) on paired determinations of directly measured PG was evaluated in glomeruli of nephrons in which distal fluid delivery was present (unblocked). Autoregulation of PG in Sh glomeruli with unblocked tubules occurred at RPP values between 99.5 +/- 1.0 and 132.1 +/- 1.0 mmHg. In contrast, in Nx glomeruli with unblocked tubules PG increased by 0.32 +/- 0.07 mmHg/mmHg increase in RPP over this same range of RPP (P less than 0.0001). To determine whether enhanced prostaglandins synthesis was responsible for the altered regulation of PG in Nx glomeruli, we repeated the micropuncture measurements in a setting of prostaglandin synthesis inhibition. Although prostaglandins synthesis inhibition did not affect the autoregulation of PG in Sh glomeruli, it did normalize the autoregulatory capacity for PG of Nx glomeruli with unblocked tubules. Thus, acute Nx is associated with a significant loss of the autoregulatory capacity for PG and this impairment appears to be related to a prostaglandin-mediated alteration of the responsiveness of the vascular effector site for autoregulation.
J C Pelayo, J Y Westcott
To assess the importance of the role of thyroidal iodine in the pathogenesis of thyroiditis in the obese strain (OS) chicken, a model of spontaneous and severe disease, we studied the effect of antithyroid drugs that reduce thyroidal iodine or prevent its metabolism. Reduction of thyroidal iodine was achieved with KClO4, an inhibitor of iodine transport and mononitrotyrosine (MNT), a drug that promotes loss of thyroidal iodine as iodotyrosines. A regimen consisting of KClO4 and MNT administration beginning in ovo and continuing after hatching reduced thyroidal infiltration to 2% of control values and decreased thyroglobulin antibody (TgAb) production for as long as 9 wk. Untreated birds had severe disease by 5 wk of age. The suppression of disease was independent of TSH, not mediated by generalized immunosuppression and reversed by excess dietary iodine. Two drugs that inhibit the metabolism of iodine, propylthiouracil (PTU) and aminotriazole, reduced thyroidal infiltration and TgAb levels, although to a lesser extent. When splenocytes from OS chickens with thyroiditis were transferred to Cornell strain (CS) chickens, a related strain that develops late onset mild disease, only the recipients that were iodine supplemented developed thyroiditis. In conclusion, autoimmune thyroiditis in an animal model can be prevented by reducing thyroidal iodine or its metabolism and optimal effects require intervention at the embryonic stage.
T R Brown, R S Sundick, A Dhar, D Sheth, N Bagchi
This study was initiated to characterize nonadrenergic-noncholinergic (NANC) inhibitory neurotransmission in penile corpus cavernosum. Using organ baths, isometric tension measurements were made in strips of human and rabbit corpus cavernosum. In examining endothelium-mediated responses, cumulative additions of exogenous acetylcholine elicited dose-dependent relaxations which were significantly reduced or completely inhibited in tissues treated with NG-monomethyl L-arginine (L-NMMA; an inhibitor of nitric oxide synthesis), oxyhemoglobin (a nitric oxide scavenger), or methylene blue (a guanylate cyclase blocker). Tissues exposed to hypoxic conditions (PO2 = 5-10 mmHg) also did not respond to exogenous acetylcholine. Mechanical removal of the endothelium in human corporal strips or in situ treatment of rabbit corpora with detergent blocked the relaxation to acetylcholine. Transmural electrical stimulation of corporal tissue strips denuded of functional endothelium, in the presence of adrenergic blockade with bretylium and muscarinic receptor blockade with atropine, caused frequency-dependent relaxation. This neurogenic relaxation was reduced or prevented by L-NMMA, oxyhemoglobin, methylene blue, and hypoxia. The effects of L-NMMA were reversed by L-arginine and the effects of hypoxia were readily reversed by normoxic conditions. Authentic, exogenous nitric oxide relaxed corporal strips which were contracted with adrenergic agonists and this effect was significantly inhibited by oxyhemoglobin. It is concluded that (a) endothelium-mediated responses of corpus cavernosum smooth muscle are mediated by a diffusible nitric oxide-like substance; (b) NANC neurogenic inhibitory responses do not require functional endothelium, and (c) nitric oxide, or a closely related substance, may act as an inhibitory neurotransmitter in penile corpus cavernosum smooth muscle.
N Kim, K M Azadzoi, I Goldstein, I Saenz de Tejada
The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.
C Pothoulakis, J T LaMont, R Eglow, N Gao, J B Rubins, T C Theoharides, B F Dickey
Renal hydrogen ion excretion increases with chronic acid loads and decreases with alkali loads. We examined the mechanism of adaptation by analyzing vacuolar proton-translocating adenosine triphosphatase (H+ ATPase) 31-kD subunit protein and mRNA levels, and immunocytochemical distribution in kidneys from rats subjected to acid or alkali loads for 1, 3, 5, 7, and 14 d. Acid- and alkali-loaded rats exhibited adaptive responses in acid excretion, but showed no significant changes in H+ ATPase protein or mRNA levels in either cortex or medulla. In contrast, there were profound adaptive changes in the immunocytochemical distribution of H+ ATPase in collecting duct intercalated cells. In the medulla, H+ ATPase staining in acid-loaded rats shifted from cytoplasmic vesicles to plasma membrane, whereas in alkali-loaded rats, cytoplasmic vesicle staining was enhanced, and staining of plasma membrane disappeared. In the cortical collecting tubule, acid loading increased the number of intercalated cells showing enhanced apical H+ ATPase staining and decreased the number of cells with basolateral or poorly polarized apical staining. The results indicate that both medulla and cortex participate in the adaptive response to acid and alkali loading by changing the steady-state distribution of H+ ATPase, employing mechanisms that do not necessitate postulating interconversion of intercalated cells with opposing polarities.
B Bastani, H Purcell, P Hemken, D Trigg, S Gluck
We have shown that urea transport across the terminal inner medullary collecting duct (terminal IMCD) is mediated by a vasopressin-stimulated, facilitated diffusion process exhibiting properties consistent with a transporter. To investigate whether hypertonic NaCl, as exists in vivo in the inner medulla, affects urea permeability, we studied isolated perfused rat terminal IMCD segments. Perfusate and bath osmolality were varied symmetrically by adding or removing NaCl or mannitol. Urea permeability rose progressively when osmolality was increased with NaCl or mannitol from 290 to 690 mOsm/kg H2O in the absence of vasopressin; there was no further increase at 890 mOsm/kg H2O. In the presence of 10(-8) M arginine vasopressin, urea permeability increased when NaCl was added to raise osmolality from 290 to 490 mOsm/kg H2O but there was no further increase at 690 mOsm/kg H2O. When 1 mM 8-bromo cyclic AMP was added to the bath, raising NaCl still increased urea permeability. These results suggest that urea transport across the rat terminal IMCD is regulated both by vasopressin and by osmolality at values present in the renal inner medulla. Osmolality seems to activate urea transport across the rat terminal IMCD by mechanisms distinct from those of vasopressin or cyclic AMP.
J M Sands, D C Schrader
We have examined secretory antibody and cell-mediated immune responses to oral cholera vaccine in the human gastrointestinal mucosa. Freshly isolated peripheral blood lymphocytes and intestinal lymphocytes obtained by enzymatic dispersion of duodenal biopsies were assayed for numbers of total and vaccine specific immunoglobulin-secreting cells by enzyme-linked immunospot assay (ELISPOT) techniques; the frequency of cells secreting interferon-gamma (IFN-gamma) was also examined by a new modification of the ELISPOT technique. After booster immunizations with oral cholera vaccine, large numbers of cholera toxin-specific antibody-secreting cells (ASC) appeared in the small intestine. The responses were dominated by IgA ASC. A single immunization, performed 5 mo after the initial vaccinations, gave rise to an ASC response similar to that seen after the first booster immunization, with respect to both magnitude and isotype distribution. Each of the immunizations also evoked an ASC response in blood which was of lower magnitude than that seen in the small intestine, and comprised similar proportions of IgA and IgG ASC. A booster immunization also resulted in increased frequencies of IFN-gamma-secreting cells, but this increase was confined to the duodenal mucosa. This study establishes the feasibility of studying, at the single-cell level, intestinal immune reactivity in humans. Furthermore, it indicates that the small intestinal mucosa is an enriched source of IFN-gamma. It also demonstrates marked differences between intestinal and peripheral blood immune responses after enteric immunization, and confirms the notion that the mucosal immune system in humans displays immunological memory.
M Quiding, I Nordström, A Kilander, G Andersson, L A Hanson, J Holmgren, C Czerkinsky
Although glucocorticoids are widely used in the treatment of immunohematologic disease, their relative efficacy is uncertain. We used an animal model, which has helped to elucidate the role of splenic macrophage Fc gamma receptors in the clearance of IgG-coated cells, to investigate whether each Fc gamma receptor is modulated by glucocorticoids to the same extent and to examine the relative potency of three commonly used glucocorticoids. Cortisol, prednisone, and dexamethasone all impaired the clearance of IgG-coated erythrocytes. However, dexamethasone was more effective than either prednisone or cortisol (P less than 0.001). Furthermore, splenic macrophages isolated from glucocorticoid-treated animals expressed impaired Fc gamma receptor function. This effect was greater in macrophages isolated from dexamethasone-treated animals, as compared to either cortisol- or prednisone-treated animals (P less than 0.001). To assess the effect of glucocorticoids on the two types of guinea pig splenic macrophage Fc gamma receptors, Fc gamma R1,2 and Fc gamma R2, specific immunoglobulin isotypes were used to measure macrophage binding of IgG-sensitized erythrocytes. Cortisol and prednisone primarily affected Fc gamma R2, whereas dexamethasone inhibited the function of both guinea pig Fc gamma receptors. Furthermore, dexamethasone was more effective (P less than 0.01) than either prednisone or cortisol in inhibiting the ability of both receptors to bind IgG-sensitized cells. Fluorescence-activated cell sorter analysis and fluorescence microscopy with monoclonal antibodies specific for each of these two receptors demonstrated that essentially all splenic macrophages expressed both receptors, and that these glucocorticoids decreased the level of each Fc gamma receptor protein expressed, rather than altering receptor mobility and clustering in the macrophage membrane. The effect on both Fc gamma receptors was greatest with dexamethasone and least with cortisol. These studies demonstrate the significant role of guinea pig splenic macrophage Fc gamma R2 in immune clearance and in the binding of IgG-coated cells. They demonstrate a differential effect of glucocorticoid hormones on Fc gamma receptor function and on surface receptor protein. Furthermore, they suggest that dexamethasone may be a more effective glucocorticoid than either prednisone or cortisol in inhibiting the clearance of IgG-coated cells by its effect on splenic macrophage Fc gamma receptors.
P Ruiz, F Gomez, M King, R Lopez, C Darby, A D Schreiber
Although the importance of the hepatic glucose load in the regulation of liver glucose uptake has been clearly demonstrated in in vitro systems, the relationship between the hepatic glucose load and hepatic glucose uptake has yet to be defined in vivo. Likewise, the effects of the route of glucose delivery (peripheral or portal) on this relationship have not been explored. The aims of the present study were to determine the relationship between net hepatic glucose uptake (NHGU) and the hepatic glucose load in vivo and to examine the effects of the route of glucose delivery on this relationship. NHGU was evaluated at three different hepatic glucose loads in 42-h fasted, conscious dogs in both the absence (n = 7) and the presence (n = 6) of intraportal glucose delivery. In the absence of intraportal glucose delivery and in the presence of hepatic glucose loads of 50.5 +/- 5.9, 76.5 +/- 10.0, and 93.6 +/- 10.0 mg/kg/min and arterial insulin levels of approximately 33 microU/ml, NHGU was 1.16 +/- 0.37, 2.78 +/- 0.82, and 5.07 +/- 1.20 mg/kg/min, respectively. When a portion of the glucose load was infused into the portal vein and similar arterial insulin levels (approximately 36 microU/ml) and hepatic glucose loads (52.5 +/- 4.5, 70.4 +/- 5.6, and 103.6 +/- 18.4 mg/kg/min) were maintained, NHGU was twice that seen in the absence of portal loading (3.77 +/- 0.40, 4.80 +/- 0.59, and 9.62 +/- 1.43 mg/kg/min, respectively). Thus, net hepatic glucose uptake demonstrated a direct dependence on the hepatic glucose load that did not reach saturation even at elevations in the hepatic glucose load of greater than three times basal. In addition, the presence of intraportal glucose delivery increased net hepatic glucose uptake apparently by lowering the threshold at which the liver switched from net glucose output to net glucose uptake.
S R Myers, D W Biggers, D W Neal, A D Cherrington
Insulin resistance is commonly associated with obesity and noninsulin-dependent diabetes. Whereas it predicts the development of diabetes, its effect on body weight change is unknown. We measured glucose disposal rates at submaximally- and maximally-stimulating insulin concentrations in 192 nondiabetic Pima Indians and followed their weight change over 3.5 +/- 1.8 y (mean +/- SD). Results: (a) Insulin-resistant subjects gained less weight than insulin-sensitive subjects (3.1 vs. 7.6 kg, P less than 0.0001). (b) The percent weight change per year correlated with glucose disposal at submaximally-(r = 0.19, P less than 0.01) and maximally-stimulating (r = 0.34, P less than 0.0001) insulin concentrations independent of sex, age, initial weight, and 24-h energy expenditure; the correlations were stronger for glucose oxidation than for glucose storage. (c) Weight gain was associated with an increase in insulin resistance more than four times that predicted from the cross-sectional data. We conclude that insulin resistance is associated with a reduced risk of weight gain in nondiabetic Pima Indians.
B A Swinburn, B L Nyomba, M F Saad, F Zurlo, I Raz, W C Knowler, S Lillioja, C Bogardus, E Ravussin
Persons with HIV infection sometimes develop aggressive psoriasis or Kaposi's sarcoma (KS) not usually seen in other immunosuppressed patients. However, a specific and direct pathophysiological role for HIV-1 in these AIDS-associated disorders remains unclear since HIV has not been easily detected in these skin lesions. By combining in situ hybridization with the sensitive detection technique of confocal laser scanning microscopy, we have demonstrated HIV RNA transcripts in 5 of 15 lesional skin biopsies from HIV-infected psoriasis patients, and in 3 of 8 Kaposi's sarcoma biopsies from HIV-infected patients. HIV transcripts were not detected in normal appearing skin from HIV-infected patients or in psoriatic and normal skin biopsies from uninfected individuals (P = 0.006). Although previous attempts to demonstrate viral sequences in psoriasis and KS lesions have been unsuccessful, in situ hybridization with confocal microscopy has shown the presence of HIV RNA transcripts predominantly within CD4+, Factor XIIIa positive dermal dendrocytes. HIV or cytokines produced by infected cells in skin lesions may therefore play a direct role in the pathogenesis of HIV-associated psoriasis and KS.
S E Mahoney, M Duvic, B J Nickoloff, M Minshall, L C Smith, C E Griffiths, S W Paddock, D E Lewis
Anti-idiotypic MAb were raised in syngeneic mice against a mouse MAb recognizing GD3 ganglioside (MAb R24). Two anti-idiotypic MAb, designated BEC2 and BEC3, recognized distinct determinants on MAb R24 that mapped near or within the GD3-binding site. New Zealand white rabbits, which express GD3 on normal tissues, were immunized with either BEC2, BEC3, or control MAb FLOPC-21. All rabbits developed high and equivalent titers of antibodies against mouse immunoglobulins. Immunization with BEC2 and BEC3 induced rabbit antibodies expressing R24 idiotype as demonstrated by their ability to inhibit BEC2 binding to R24. Antibodies (IgG and IgM) reacting with GD3 developed in five of eight rabbits immunized with BEC2 but not in rabbits immunized with BEC3 or with control MAb. Serum antibodies against GD3 did not cross-react with other gangliosides. These results show that MAb BEC2 can mimic GD3 ganglioside and can induce antibodies against GD3 ganglioside despite expression of GD3 on normal rabbit tissue.
P B Chapman, A N Houghton
Recently, combined serological and molecular studies of autoantibodies have revealed that these antibodies play an important role in the normal function of the immune system and in the development of the B cell repertoire. Accordingly, we hypothesized that a homozygous deletion of a critical autoantibody-associated Ig variable (V) gene may alter the immune system and thus predispose the host to autoimmune disorders. Initial experiments revealed several restriction fragment length polymorphisms (RFLP) of the Humhv3005 gene, that is likely to encode heavy chains of rheumatoid factors, and the closely related 1.9III gene. By probing EcoR1-digested DNA with the Humhv3005/P1 probe, we found that one of the four major hybridizing bands was missing in approximately 20% of patients with either rheumatoid arthritis or systemic lupus erythematosus, but only 2% of normal subjects. To delineate the genetic basis of this polymorphism, we have now employed the PCR to amplify and analyze hv3005, 1.9III, and homologous genes in individuals with characteristic RFLP genotypes. Our results indicate that the human Vh gene repertoire contains several hv3005- and 1.9III-like genes, and that a complete deletion of the hv3005-like genes is relatively restricted to a subset of autoimmune patients. These findings provide initial evidence for deletion of developmentally regulated autoreactive V genes in autoimmune diseases.
T Olee, P M Yang, K A Siminovitch, N J Olsen, J Hillson, J Wu, F Kozin, D A Carson, P P Chen
We explored the effects of two components of ischemia, hypoxia and glucose deprivation, on the beta-adrenergic receptor (beta AR)-adenylate cyclase system in a model of hypoxic injury in cultured neonatal rat ventricular myocytes. After 2 h of hypoxia in the presence of 5 mM glucose, cell surface beta AR density (3H-CGP-12177) decreased from 54.8 +/- 8.4 to 39 +/- 6.3 (SE) fmol/mg protein (n = 10, P less than 0.025), while cytosolic beta AR density (125I-iodocyanopindolol [ICYP]) increased by 74% (n = 5, P less than 0.05). Upon reexposure to oxygen cell surface beta AR density returned toward control levels. Cells exposed to hypoxia and reoxygenation without glucose exhibited similar alterations in beta AR density. In hypoxic cells incubated with 5 mM glucose, the addition of 1 microM (-)-norepinephrine (NE) increased cAMP generation from 29.3 +/- 10.6 to 54.2 +/- 16.1 pmol/35 mm plate (n = 5, P less than 0.025); upon reoxygenation cAMP levels remained elevated above control (n = 5, P less than 0.05). In contrast, NE-stimulated cAMP content in glucose-deprived hypoxic myocytes fell by 31% (n = 5, P less than 0.05) and did not return to control levels with reoxygenation. beta AR-agonist affinity assessed by (-)-isoproterenol displacement curves was unaltered after 2 h of hypoxia irrespective of glucose content. Addition of forskolin (100 microM) to glucose-supplemented hypoxic cells increased cAMP generation by 60% (n = 5; P less than 0.05), but in the absence of glucose this effect was not seen. In cells incubated in glucose-containing medium, the decline in intracellular ATP levels was attenuated after 2 h of hypoxia (21 vs. 40%, P less than 0.05). Similarly, glucose supplementation prevented LDH release in hypoxic myocytes. We conclude that (a) oxygen and glucose independently regulate beta AR density and agonist-stimulated cAMP accumulation; (b) hypoxia has no effect on beta AR-agonist or antagonist affinity; (c) 5 mM glucose attenuates the rate of decline in cellular ATP levels during both hypoxia and reoxygenation; and (d) glucose prevents hypoxia-induced LDH release, a marker of cell injury.
K J Rocha-Singh, N Y Honbo, J S Karliner
Several lines of experimental evidence suggest that inclusion of core sequences in the hepatitis B vaccine may represent a feasible strategy to increase the efficacy of the vaccination. In order to identify immunodominant core epitopes, peripheral blood T cells purified from 23 patients with acute hepatitis B and different HLA haplotypes were tested with a panel of 18 short synthetic peptides (15 to 20 amino acids [AA]) covering the entire core region. All patients except one showed a strong T cell proliferative response to a single immunodominant 20 amino acid sequence located within the aminoterminal half of the core molecule. Two additional important sequences were also identified at the aminoterminal end and within the carboxyterminal half of the core molecule. These sequences were able to induce significant levels of T cell proliferation in 69 and 73% of the patients studied, respectively. T cell response to these epitopes was HLA class II restricted. The observations that (a) polyclonal T cell lines produced by PBMC stimulation with native HBcAg were specifically reactive with the relevant peptides and that (b) polyclonal T cell lines produced with synthetic peptides could be restimulated with native HBcAg, provide evidence that AA sequences contained within the synthetic peptides represent real products of the intracellular processing of the native core molecule. In conclusion, the identification of immunodominant T cell epitopes within the core molecule provides the molecular basis for the design of alternative and hopefully more immunogenic vaccines.
C Ferrari ... T Giuberti, F V Chisari
We studied the effects of IL-4 on the spontaneous proliferation of chronic myelomonocytic leukemia (CMMoL) cells in vitro. IL-4 (100 U/ml) suppressed the spontaneous DNA synthesis by approximately 50% in 5 of 8 cases examined. IL-4 (100 U/ml) also inhibited the spontaneous colony formation by CMMoL cells in a methylcellulose culture by 50-97% in all of the 10 cases in which spontaneous colonies were formed. This IL-4-mediated suppression of the growth of CMMoL cells was completely abolished by the addition of anti-IL-4 neutralizing antibodies. The spontaneous CMMoL colonies were substantially suppressed by the addition of either anti-IL-6 or anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) antibodies to the colony assay system: the addition of both anti-IL-6 and anti-GM-CSF antibodies resulted in greater than 80% inhibition of the colony formation by CMMoL cells. On the other hand, none of anti-IL-1-beta, anti-granulocyte-CSF, anti-macrophage-CSF, or anti-tumor necrosis factor-alpha antibodies affected the CMMoL colony formation. In the supernatants from 24-h cultures of CMMoL cells, high levels of IL-6 and GM-CSF were demonstrated in 9 of 9 and 2 of 9 cases examined, respectively. IL-4 (100 U/ml) almost completely inhibited the secretion of IL-6 and GM-CSF by CMMoL cells. These observations suggest that IL-4 suppresses the spontaneous proliferation of CMMoL cells by inhibiting their production of IL-6 and/or GM-CSF, both of which could act in vitro as an autocrine growth factor for CMMoL cells.
K Akashi, T Shibuya, M Harada, Y Takamatsu, N Uike, T Eto, Y Niho
To help define the immunoregulatory defects in patients with onchocerciasis, flow cytometric analysis of circulating lymphocyte subpopulations was performed in parallel with functional assays. No significant differences in CD4/CD8 ratios were seen when microfilariae-positive individuals from Guatemala were compared with Guatemalan controls. However, the infected individuals had significantly increased numbers of circulating CD4+CD45RA+ lymphocytes (mean 38.3%) when compared with controls (mean 16.0%). Coexpression of the activation marker HLA-DR was significantly increased on CD4+ cells from infected individuals. In contrast, no up-regulation of HLA-DR was seen on CD8+ or CD19+ cells. At 1 year after initiation of treatment with semiannual doses of the microfilaricide ivermectin, there were significant increases (P less than 0.05) in the percentage of CD4+CD45RA- cells, the percentage of CD4+HLA-DR+ cells, and mitogen-induced lymphokine production (IL-2, IL-4). Despite these changes, parasite-specific IL-2 and IL-4 production which had been undetectable before treatment did not manifest itself even by the 2-yr follow-up. Defects in the T-cell activation pathway in Onchocerca volvulus-infected individuals may thus exist at several independent points; a state of parasite antigen-specific tolerance appears to remain even after the relative reversal of other generalized immunoregulatory defects.
D O Freedman, A Lujan-Trangay, C Steel, C Gonzalez-Peralta, T B Nutman
Expression of the human IL-6 gene in EBV-immortalized normal human B lymphocytes following retroviral-mediated transduction rendered these cells highly tumorigenic in athymic mice. The tumors were lymphomas composed of the originally inoculated human lymphoblastoid cells. Co-injection of IL-6 expressing EBV-immortalized cells with IL-6 nonexpressing control cells resulted in increased tumorigenicity of the IL-6 nonexpressing cells. The lymphoblastoid cells expressing IL-6 were indistinguishable from parental cell lines in morphology and in a variety of cell surface characteristics, and did not exhibit growth advantage over parental cell lines in vitro, such that increased tumorigenicity is unlikely to depend upon a direct oncogenic effect of IL-6 on the B cells. Rather, at high concentrations, IL-6 markedly inhibits human lymphoblastoid cell killing by IL-2-activated murine splenocytes in vitro, suggesting that IL-6-related tumorigenicity might depend upon IL-6 inhibiting cytotoxicity at the tumor site. Thus, production of IL-6 by tumor cells that results in natural killer cell dysfunctions illustrates a novel mechanism of tumor cell escape from immune surveillance.
J Tanner, G Tosato
Past studies have shown that freshly isolated human B cells from peripheral blood and tonsils do not express IL-6 receptors (IL-6R); however, mitogen or antigen activation of these B cells induces IL-6R and responsiveness to IL-6. In this study, we have shown that a high proportion of B cells enzymatically dissociated from human appendix, a gut-associated lymphoreticular tissue (GALT), expresses the IL-6R, and that recombinant human IL-6 induces significant increases in the number of Ig-producing cells. The recombinant human IL-6-induced increase in Ig-producing cells is restricted to the IgA isotype. Further, IgA2 is the major subclass; however, significant numbers of IgA1 producing cells are also seen. In contrast, human tonsillar and peripheral blood B cells express low levels of IL-6R, and exogenous IL-6 does not increase numbers of Ig-producing cells. When PBMC or tonsillar cells are stimulated with PWM, the former display an equal distribution of IgA1 and IgA2 secreting cells, while tonsillar B cells are mainly of the IgA1 subclass. The distribution of surface Ig-positive (sIg+) B cells in the appendix B cell population is sIgA+ greater than sIgG+ greater than sIgM+, and the sIgA+ B cells express higher levels of IL-6R when compared with sIgG+ or sIgM+ B cells. These studies show that human appendix contains B cell subsets that constitutively express IL-6R, and that a high proportion of these cells are committed to the IgA isotype. Furthermore, higher numbers of IL-6 responsive IgA2 B cells are present in the human appendix as compared to tonsils or PBMC.
K Fujihashi, J R McGhee, C Lue, K W Beagley, T Taga, T Hirano, T Kishimoto, J Mestecky, H Kiyono
We have previously shown that von Willebrand factor (vWF), a glycoprotein which plays a critical role in the adhesion of platelets to injured blood vessels, is present within vascular subendothelium. We investigated the identity of the subendothelial binding site(s) for vWF by examining vWF binding to subendothelial constituents and solubilized a 150-kD protein with SDS-urea that bound vWF. This protein had an amino-acid composition similar to that of the type VI collagen alpha-1/alpha-2 chains, was recognized by specific polyclonal antibodies against type VI collagen, and had a similar acidic isoelectric point. Furthermore, we found that purified type VI collagen also bound vWF. Thus, we have identified the extracted 150-kD protein as type VI collagen. This protein may play a significant role in the binding of vWF to vascular subendothelium in vivo.
J H Rand, N D Patel, E Schwartz, S L Zhou, B J Potter
Our present work characterized the role of hormone-mediated signal transduction pathways in regulating hepatic reduced glutathione (GSH) synthesis. Cholera toxin, dibutyryl cAMP (DBcAMP), and glucagon inhibited GSH synthesis in cultured hepatocytes by 25-43%. Cellular cAMP levels exhibited a lower threshold for stimulation of the GSH efflux than inhibition of its synthesis. The effect of DBcAMP was independent of the type of sulfur amino acid precursor and cellular ATP levels and unassociated with increased GSH mixed disulfide formation or altered GSH/oxidized glutathione ratio. In liver cytosols, addition of DBcAMP and cAMP-dependent protein kinase (A-kinase) inhibited GSH synthesis from substrates (cysteine, ATP, glutamate, and glycine) by approximately 20% which was prevented by the A-kinase inhibitor. However, if only substrates of the second step in GSH synthesis were used (gamma-glutamylcysteine, glycine, and ATP), DBcAMP and A-kinase exerted no inhibitory effect. Phenylephrine, vasopressin, and phorbol ester also inhibited GSH synthesis in cultured cells by approximately 20%, and depleted cell GSH independent of the type of sulfur amino acid precursor. Cellular cysteine level was unchanged despite the significant fall in GSH after glucagon or phenylephrine treatment. Pretreatment with either staurosporine, C-kinase inhibitor, or calmidazolium, a calmodulin inhibitor, partially prevented but, together, completely prevented the inhibitory effect of phenylephrine. The same combination had no effect on the inhibitory effect of glucagon. The effects of hormones were confirmed in both the intact perfused liver and after in vivo administration. Thus, two classes of hormones acting through distinct signal transduction pathways may down-regulate hepatic GSH synthesis by phosphorylation of gamma-glutamylcysteine synthetase.
S C Lu, J Kuhlenkamp, C Garcia-Ruiz, N Kaplowitz
Apolipoprotein (apo) E and the two B apolipoproteins, apoB48 and apoB100, are important proteins in human lipoprotein metabolism. Commonly occurring polymorphisms in the genes for apoE and apoB result in amino acid substitutions that produce readily detectable phenotypic differences in these proteins. We studied changes in apoE and apoB phenotypes before and after liver transplantation to gain new insights into apolipoprotein physiology. In all 29 patients that we studied, the postoperative serum apoE phenotype of the recipient, as assessed by isoelectric focusing, converted virtually completely to that of the donor, providing evidence that greater than 90% of the apoE in the plasma is synthesized by the liver. In contrast, the cerebrospinal fluid apoE phenotype did not change to the donor's phenotype after liver transplantation, indicating that most of the apoE in CSF cannot be derived from the plasma pool and therefore must be synthesized locally. The apoB100 phenotype (assessed with immunoassays using monoclonal antibody MB19, an antibody that detects a two-allele polymorphism in apoB) invariably converted to the phenotype of the donor. In four normolipidemic patients, we determined the MB19 phenotype of both the apoB100 and apoB48 in the "chylomicron fraction" isolated from plasma 3 h after a fat-rich meal. Interestingly, the apoB100 in the chylomicron fraction invariably had the phenotype of the donor, indicating that the vast majority of the large, triglyceride-rich apoB100-containing lipoproteins that appear in the plasma after a fat-rich meal are actually VLDL of hepatic origin. The MB19 phenotype of the apoB48 in the plasma chylomicron fraction did not change after liver transplantation, indicating that almost all of the apoB48 in plasma chylomicrons is derived from the intestine. These results were consistent with our immunocytochemical studies on intestinal biopsy specimens of organ donors; using apoB-specific monoclonal antibodies, we found evidence for apoB48, but not apoB100, in donor intestinal biopsy specimens.
M F Linton, R Gish, S T Hubl, E Bütler, C Esquivel, W I Bry, J K Boyles, M R Wardell, S G Young
Induction of HLA class I antigens on cultured melanoma cells FO-1 after transfection with a human or a mouse B2m gene was associated with a statistically significant reduction in their susceptibility to natural killer (NK) cell-mediated lysis. These results indicate that the structural differences between human and mouse beta 2-mu do not abolish the ability of the HLA class I molecular complex to modulate NK cell-mediated lysis of melanoma cells FO-1. The role of HLA class I antigens in the phenomenon is corroborated by the ability of anti-HLA class I MAb to enhance, although to a different extent, the susceptibility of transfected FO-1 cells to NK cell-mediated lysis. Gamma interferon (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) significantly reduced the susceptibility to NK cell-mediated lysis of transfected FO-1 cells. Surprisingly, TNF-alpha reduced the extent of lysis more than IFN-gamma, although the latter cytokine enhanced HLA class I antigen expression more than the former one. This finding, in conjunction with a reduction in the susceptibility to NK cell-mediated lysis of untransfected FO-1 cells incubated with IFN-gamma or TNF-alpha, suggests that the two cytokines reduce NK cell-mediated lysis of transfected cells by modulating not only the expression of HLA class I antigens, but also that of other structures. Induction of HLA class I antigens and their modulation with IFN-gamma did not affect the susceptibility to lymphokine-activated killer (LAK) cell-mediated lysis of transfected FO-1 cells. Characterization of the molecular mechanism(s) underlying abnormalities in HLA class I antigen expression by melanoma cells and of the role of these molecules in the interactions of melanoma cells with various types of effector cells may suggest novel immunotherapeutic approaches to melanoma.
M Maio, M Altomonte, R Tatake, R A Zeff, S Ferrone
Antibody inhibitors against human thrombin are rare and have remained poorly characterized. We report the case of a 40-yr-old patient who developed a potent thrombin inhibitor revealed by mild bleeding symptoms and marked prolongation of most laboratory clotting times. After two years of evolution, he died from cerebral hemorrhage. The inhibitor, a polyclonal IgG, was associated with hematological and immunological criteria of autoimmune disorder. Antithrombin IgG was isolated from the patient's plasma by protein A- and thrombin-affinity chromatography. Fab fragments inhibited amidolytic activity of alpha thrombin, and thrombin-thrombomodulin catalyzed protein C activation with a Ki of approximately 10(-8) M in a noncompetitive manner. Alpha to gamma conversion of thrombin resulted in a moderate loss of affinity for the inhibitor. Upon complex formation of thrombin with staphylocoagulase or alpha 2-macroglobulin (alpha 2M), inhibition was decreased by two orders of magnitude and acquired an apparent competitive character. In Western blot experiments, the antibody reacted with active alpha-thrombin, did not react with chloromethylketone-inhibited thrombin and reacted with a lower affinity with iPr2P-thrombin. The inhibitor did not block thrombin binding to benzamidine-, heparin-, or fibrin-Sepharose, but displaced proflavin from its complex with thrombin. Taken together, these results indicate that the patient's autoantibody recognized a conformational structure which includes, at least in part, the apolar binding site adjacent to the catalytic site of thrombin.
P Sié ... M C Guillin, B Boneu
In the context of the central role of the alveolar macrophage in host defense of the respiratory epithelial surface, and the ability of IFN-gamma to activate mononuclear phagocytes, we have evaluated strategies to use rIFN-gamma to activate human alveolar macrophages in vivo. To accomplish this, rIFN-gamma was administered to nonsmoking normals, the amounts of IFN-gamma quantified in serum and respiratory epithelial lining fluid (ELF) and the status of IFN-gamma related activation of blood monocytes and alveolar macrophages was evaluated by quantifying the expression of mRNA transcripts of IP-10, a gene induced specifically by IFN-gamma. Systemic administration (subcutaneous) of maximally tolerated amounts of rIFN-gamma (250 micrograms) was followed by detectable levels of IFN-gamma in serum but not ELF, the expression of IP-10 transcripts in blood monocytes but not alveolar macrophages, and multiple systemic adverse effects. To circumvent the inability of systemic administration to reach respiratory ELF and activate alveolar macrophages, rIFN-gamma (250-1,000 micrograms) was inhaled as an aerosol once daily for 3 d. Strikingly, while IFN-gamma was not detected in serum it was detectable in respiratory ELF in a dose-dependent fashion. Further, alveolar macrophages, but not blood monocytes, expressed IP-10 mRNA transcripts and, importantly, inhalation of aerosolized rIFN-gamma was not associated with local or systemic adverse effects. Thus, it is feasible to use rIFN-gamma to activate alveolar macrophages by targeting the cytokine directly to the lung. These data suggest a potential strategy for targeted cytokine therapy, without systemic side effects, to augment respiratory tract defenses in individuals at risk for or with lung infection.
H A Jaffe ... S Sherwin, R G Crystal
Osmotic injury induced by rapid correction of severe chronic hyponatremia has been implicated in the development of central pontine myelinolysis. Organic osmolytes known previously as "idiogenic osmoles" accumulate intracellularly to protect cells from osmotic injury. We investigated the changes of these organic osmolytes as well as electrolytes in the brain during the induction and correction of chronic hyponatremia. Using 1H-nuclear magnetic resonance spectroscopy and HPLC, we found that in rats with chronic hyponatremia (3 d, serum sodium = 109 +/- 3 meq/liter), brain concentrations of myoinositol (41%), glycerophosphorylcholine (45%), phosphocreatine/creatine (60%), glutamate (53%), glutamine (45%), and taurine (37%) were all significantly decreased compared with control values (percentage control value shown, all P less than 0.01). The contribution of measured organic osmolytes and electrolytes to the total brain osmolality change was 23 and 72%, respectively. With rapid correction by 5% NaCl infusion, significant brain dehydration and elevation of brain Na and Cl levels above the normal range occurred at 24 h. These changes were not seen with slow correction by water deprivation. Reaccumulation of most organic osmolytes except glycerophosphorylcholine is delayed during the correction of hyponatremia and is independent of the correction rate of serum sodium. It is concluded that: most of the change of brain osmolality in chronic hyponatremia can be accounted by the changes in organic osmolytes and brain electrolytes; and rapid correction of hyponatremia is associated with an overshoot of brain sodium and chloride levels along with a low organic osmolyte level. The high cerebral ion concentrations in the absence of adequate concentrations of organic osmolytes may be relevant to the development of central pontine myelinolysis.
Y H Lien, J I Shapiro, L Chan
Endothelin (ET), a peptide synthesized by endothelial cells (EC), causes a decreased renal blood flow and glomerular filtration rate and an increased mean arterial pressure when infused in animals. In tissue culture, ET causes smooth muscle cell (SMC) proliferation and contraction by influx of extracellular calcium, which is inhibited by calcium channel antagonists. Infusion of cyclosporine (CSA) hemodynamically parallels ET action, and knowing that CSA effects EC, we hypothesize that the vasoconstrictive effects of CSA are a result of ET synthesis by EC. Varying concentrations of CSA were incubated with EC resulting in ET present in the supernatants in a dose-dependent manner peaking at 75% above basal activity. Coincubation of either cremophor alone or cycloheximide with CSA resulted in minimal ET present in the EC supernatants (P less than 0.01 each). Incubation of conditioned media from CSA-treated EC with SMC caused proliferation at 114% above basal activity, which did not occur in the presence of CSA alone (P less than 0.01). This activity is specifically inhibited in the presence of an anti-ET antibody or nonspecifically in the presence of calcium channel antagonists (P less than 0.01 each). Therefore, CSA stimulates EC synthesis of ET which in turn causes SMC proliferation. This action is inhibited by the coincubation of a specific antibody to ET or a calcium channel antagonist. These findings may help in the understanding of CSA-induced hypertension and vasculopathy.
T E Bunchman, C A Brookshire
Using affinity-purified calmodulin-binding proteins from human epidermis we have developed a monoclonal IgM antibody, ROC 129.1, to a human desmosomal calcmodulin-binding protein. This antibody reacts with a submembranous 250-kD protein from human keratinocytes and stains human epidermis in a "cell-surface pattern". Permeability studies indicated that the epitope with which this monoclonal reacts is on the inner surface of the cell membrane. Immunoelectronmicroscopy localized the antigen to the desmosome. The epitope is restricted to stratified squamous epithelia and arises between 8-12 wk of fetal development. This desmosomal calmodulin-binding protein, which we have termed keratocalmin, may be involved in the calcium-regulated assembly of desmosomes.
J A Fairley, G A Scott, K D Jensen, L A Goldsmith, L A Diaz
Expression of the two sarcomeric actins, alpha-skeletal and alpha-cardiac, is regulated in the rodent heart in response to developmental, hormonal, and hemodynamic stimuli. Little is known in man, except that both isogenes were found to be coexpressed in three adult ventricles. In this report, we investigated the isoactin mRNA composition in ventricles from 21 control patients (4 fetal, 5 juvenile, 12 adult) and from 15 patients undergoing cardiac transplantation (5 idiopathic dilated cardiomyopathies, 5 ischemic myopathies with myocardial infarcts, 5 diverse etiologies) by two different and complementary techniques: RNA dot blot analysis with specific cDNA probes, and primer extensions with an oligonucleotide common to alpha-cardiac and alpha-skeletal actins. In the case of dot blot analysis, quantification of each isoform was performed by using as standards RNA transcripts obtained from cloned human alpha-actin sequences, and the total amount of sarcomeric actin mRNA was evaluated as a function of total poly(A+)RNA. We found that both isogenes are always coexpressed, and that the isoactin pattern changes during development. In utero and in neonatal hearts, alpha-skeletal actin mRNA represents less than or equal to 20% of sarcomeric actins, it increases to 48 +/- 6% during the first decade after birth and becomes the predominant isoform of adult hearts (60.4 +/- 8.5%). The 15 adult failing hearts exhibited the same isoactin pattern as the control ones (62.84 +/- 11.06%), and there was no difference in expression between patients with dilated cardiomyopathy or ischemic heart disease. These observations demonstrate that cardiac development in man, in contrast to rodent heart, is characterized by an up-regulation of the skeletal actin gene, the expression of which does not change in hypertrophied and failing hearts, and suggest that the actin and myosin heavy chain families are independently regulated in human heart.
K R Boheler, L Carrier, D de la Bastie, P D Allen, M Komajda, J J Mercadier, K Schwartz
Recent studies have demonstrated the existence of two members of a novel family of calcium-independent plasmalogen-selective phospholipases A2 in mammalian myocardium (Wolf, R. A., and R. W. Gross. 1985. J. Biol. Chem. 260:7295-7303; and Hazen, S. L., D. A. Ford, and R. W. Gross. 1991. J. Biol. Chem. 266:5629-5633). To examine the potential role of these calcium-independent phospholipases A2 in mediating membrane dysfunction during early myocardial ischemia, the temporal course of alterations in phospholipase A2 activity during global ischemia in Langendorf perfused rabbit hearts was quantified and compared with traditionally accepted markers of myocytic ischemic injury and anaerobic metabolism. We now report that membrane-associated calcium-independent plasmalogen-selective phospholipase A2 activity increased over 400% during 2 min of global ischemia (P less than 0.01), was near maximally activated (greater than 10-fold) after only 5 min of ischemia, and remained activated throughout the entire ischemic interval examined (2-60 min). Activation of membrane-associated plasmalogen-selective phospholipase A2 after 5 min of myocardial ischemia was rapidly reversible during reperfusion of ischemic tissue. Both the activation of phospholipase A2 and its reversibility during reperfusion were temporally correlated to alterations in myocytic anaerobic metabolism. Furthermore, activation of membrane-associated phospholipase A2 was essentially complete before electron microscopic evidence of cellular damage. Collectively, these results identify dynamic alterations in calcium-independent plasmalogen-selective phospholipase A2 activity during myocardial ischemia which precede irreversible cellular injury and demonstrate that activation of plasmalogen-selective phospholipase A2 is amongst the earliest biochemical alterations in ischemic myocardium.
D A Ford, S L Hazen, J E Saffitz, R W Gross
We examined the relative effects of thyrotropin (TSH) and TSH receptor autoantibodies in the sera of patients with autoimmune thyroid disease on three TSH-lutropin/chorionic gonadotropin (LH/CG) receptor extracellular domain chimeras. Each chimera binds TSH with high affinity. Only the chimera with TSH receptor extracellular domains ABC (amino acids 1-260) had a functional (cAMP) response to thyroid stimulatory IgG. The chimeras with TSH receptor domains CD (amino acids 171-360) and DE (amino acids 261-418) were unresponsive. The lack of response of the chimera with TSH receptor domains DE was anticipated because it fails to transduce a signal with TSH stimulation, unlike the other two chimeras. A different spectrum of responses occurred when the TSH-LH/CG chimeras were examined in terms of autoantibody competition for TSH binding. IgG with TSH binding-inhibitory activity when tested with the wild-type TSH receptor also inhibited TSH binding to the chimera with TSH receptor domains DE. Dramatically, however, these IgG did not inhibit TSH binding to the chimera with TSH receptor domains CD, and had weak or absent activity with the chimera with TSH receptor domains ABC. Chimeras with TSH receptor domains ABC and DE were equally effective in affinity-purifying IgG with thyroid-stimulatory and TSH binding-inhibitory activities. Nonstimulatory IgG with TSH binding-inhibitory activity inhibited the action of stimulatory IgG on the wild-type TSH receptor, but not with the chimera containing TSH receptor domains ABC. In summary, TSH receptor autoantibodies and TSH bind to regions in both domains ABC and DE of the TSH receptor extracellular region. Stimulatory and inhibitory TSH receptor autoantibodies, as well as TSH, appear to bind to different sites in domains ABC, but similar sites in domains DE, of the receptor. Alternatively, TSH and the different TSH receptor antibodies bind with differing affinities to the same site in the ABC region.
Y Nagayama, H L Wadsworth, D Russo, G D Chazenbalk, B Rapoport
The gene encoding a novel protein designated nm23-H1, which was recently identified as identical to the A subunit of nucleotide diphosphate kinase from human erythrocytes, has been proposed to play a role in tumor metastasis suppression. We report that untreated neuroblastoma tumors contain a cellular polypeptide (Mr = 19,000) designated p19, identified in two-dimensional electrophoretic gels, which occurs at significantly higher levels (P = 0.0001) in primary tumors containing amplified N-myc gene. The partial amino acid sequence obtained for p19 is identical to the sequence of the human nm23-H1 protein. An antibody to the A subunit of erythrocyte nucleotide diphosphate kinase reacted exclusively with p19. In this study, significantly higher levels of p19/nm23 occurred in primary neuroblastoma tumors from patients with advanced stages (III and IV) relative to tumors from patients with limited stages (I and II) of the disease. Even among patients with a single copy of the N-myc gene, tumors from patients with stages III and IV had statistically significantly higher levels of p19/nm23 than tumors from patients with stages I and II. Our findings indicate that, in contrast to a proposed role for nm23-H1 as a tumor metastasis suppressor, increased p19/nm23 protein in neuroblastoma is correlated with features of the disease that are associated with aggressive tumors. Therefore, nm23-H1 may have distinct if not opposite roles in different tumors.
N Hailat ... F Lottspeich, J R Strahler
A large series of T cell clones (TCC) specific for purified protein derivative (PPD) of Mycobacterium tuberculosis (total 60) or Toxocara canis excretory/secretory (TES) antigen (total 69) were established from the peripheral blood of two healthy individuals and analyzed for their profile of cytokine production in response to stimulation with either the specific antigen or the polyclonal activator phorbol myristate acetate plus anti-CD3 antibody. Under both these experimental conditions, the great majority of PPD-specific TCC secreted IL-2 and IFN-gamma but not, or limited amounts of, IL-4 and IL-5. In contrast, most TES-specific TCC secreted IL-4 and IL-5 but not, or limited amounts of, IL-2 and IFN-gamma. PPD-specific TCC that failed to secrete IL-4 and IL-5, and TES-specific TCC that failed to secrete IL-2 and IFN-gamma, were found to lack transcripts for IL-4 and IL-5, or for IL-2 and IFN-gamma, respectively. During the course of the study, over a 6-mo period, the functional phenotype of both TES- and PPD-specific TCC was repeatedly assessed and remained constant. These data demonstrate that T cells with stable Th1 or Th2 functional pattern exist not only in mice but also in humans and suggest that in the course of natural immunization certain infectious agents preferentially expand T cell subsets with stable and definite profile of cytokine production.
G F Del Prete, M De Carli, C Mastromauro, R Biagiotti, D Macchia, P Falagiani, M Ricci, S Romagnani
Chloroquine and ammonium chloride, by virtue of their basic properties, have been shown to raise endocytic and lysosomal pH and thereby interfere with normal iron metabolism in a variety of cell types, including mononuclear phagocytes. Cellular iron metabolism is of critical importance to Legionella pneumophila, an intracellular bacterial pathogen whose capacity to multiply in human mononuclear phagocytes is dependent upon the availability of intracellular iron. In view of this, we have studied the effects of chloroquine and ammonium chloride on L. pneumophila intracellular multiplication in human monocytes. Chloroquine, at a concentration of 20 microM, and ammonium chloride, at a concentration of 20 mM, inhibited L. pneumophila intracellular multiplication by 1.4 +/- 0.2 (SEM) logs and 1.5 +/- 0.2 logs, respectively. Chloroquine- and ammonium chloride-induced inhibition of L. pneumophila intracellular multiplication was completely reversed by iron nitrilotriacetate, an iron compound which is soluble in the neutral to alkaline pH range, but not by iron transferrin, which depends upon acidic intracellular conditions to release iron. Chloroquine had no major direct effect on L. pneumophila multiplication in artificial media except at extremely high concentrations (15,000-fold that which inhibited L. pneumophila multiplication in mononuclear phagocytes), and inhibition at such concentrations was not reversed by iron nitrilotriacetate. This study demonstrates that chloroquine and ammonium chloride inhibit the intracellular multiplication of L. pneumophila by limiting the availability of iron to the bacterium. It is possible that such a mechanism of action underlies chloroquine's antimicrobial effect against other intracellular pathogens, such as the agents of malaria and tuberculosis.
T F Byrd, M A Horwitz
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