Human antiidiotypic antibodies to anti-DNA antibodies can be separated into at least two categories based on their binding to anti-DNA, antiidiotypic antibodies, and antigens. One type was found mainly in inactive stage of SLE. The antiidiotypic antibodies appear to be directed towards idiotype (Id) determinants in the antigen-binding sites of anti-DNA antibodies. Antibody from patient T.K. acted like a mirror image of anti-single-stranded DNA antibodies, O-81, as determined by a competitive inhibition RIA. Antibodies from patient S.U. also seemed to be Ab 2 beta and Ab 2 gamma to anti-double-stranded(ds) DNA antibodies, NE-1. Most of normal subjects, on the other hand, had antibodies that bound to the human monoclonal anti-ds DNA antibodies, NE-1, NE-13, 7F4, and O-81. The Id-anti-Id interaction was not inhibited by the addition of DNA. Thus, normal subjects had Ab2 alpha activity that recognizes Id determinants in the framework region common among anti-DNA antibodies, whereas antiidiotypic antibodies in most SLE sera appear to show Ab 2 beta and Ab 2 gamma activity. The results provide evidence that the Id network system regulates immunological tolerance to DNA in humans.
T Sasaki, T Muryoi, O Takai, E Tamate, H Saito, K Yoshinaga
The cell surface expression of alpha:beta heterodimer was studied using WT31 monoclonal antibody, in peripheral blood lymphocytes (PBL) from a patient who developed a prolonged immunodeficiency after allogeneic bone marrow transplantation. This patient, grafted for chronic myelogenous leukemia, received T cell depleted bone marrow from her HLA, A, B, D matched sibling. The late occurrence of opportunistic infection, led us to analyze the phenotype of patient PBL. 70% of PBL were CD3+ and 29% WT31+, indicating that the majority of CD3+ PBL did not express the alpha:beta heterodimer. Transcription of the genes encoding the alpha, beta, and gamma chains was assessed in cell lines derived from PBL, by Northern blot analysis. We showed that the CD3+ WT31- subset expressed a truncated, beta mRNA (1.0 kb) and also truncated alpha transcript (1.4 kb). To determine the CD3-associated structure on CD3+ WT31- cell line, immunoprecipitation assays were performed using monoclonal anti-CD3 and an hetero antiserum against gamma peptides. These CD3+ WT31- cells expressed a disulfide linked dimer, composed of products of gamma gene (37 kD, 40 kD) and of undefined delta chain (45 kD). Functional analyses were performed in PBL before and after sorting with WT31 and anti-CD3 antibody. These circulating CD3+ WT31- cells were unable to proliferate when triggered with anti-T3 beads and they seemed to mediate a suppressor activity on CD3+ WT31+ cells.
E Vilmer, P Guglielmi, V David, G Leca, C Rabian, L Degos, M Boiron, A Bensussan
Extracellular-superoxide dismutase (EC-SOD) is heterogenous in the vasculature with regard to heparin affinity and can be separated into three fractions: A, without affinity; B, with weak affinity; and C, with relatively strong heparin affinity. The plasma clearance of intravenously injected 125I-labeled and unlabeled human EC-SOD C was studied in rabbits. About 90% of injected 125I-EC-SOD C was eliminated from the blood within 5-10 min. Injection of heparin after 10 or 20 min led to an immediate release of all sequestered 125I-EC-SOD C back to the blood plasma. Later injections of heparin led to diminished release, although release could still be demonstrated after 72 h. A half-time of approximately 10 h could be calculated for heparin-releasable 125I-EC-SOD C. Unlabeled EC-SOD C, determined as enzymic activity and with ELISA, was likewise sequestered and released to the same degree as 125I-labeled EC-SOD C by heparin as tested at 20 min and 5 h. The immediacy of the heparin-induced release indicates that the sequestered enzyme had been bound to endothelial cell surfaces. The length of the half-time suggests that the putative cell surface binding has a physiological function and is not primarily a step in enzyme degradation. The distribution of sequestered 125I-labeled EC-SOD C to different organs was determined at times between 10 min and 24 h. Of the organs, the liver contained the most 125I-EC-SOD C, followed by kidney, spleen, heart, and lung. At all investigated times, the content in the analyzed organs was nearly as large as the amount that could be promptly released to plasma by intravenous heparin. This indicates that almost all 125I-EC-SOD C in the organs was present on endothelial cell surfaces and was not bound by other tissue cell surfaces, or was present within the cells.
K Karlsson, S L Marklund
To examine whether the hypothalamic corticotropin-releasing hormone (CRH) neuron is regulated by CRH, by products of the proopiomelanocortin (POMC) gene, and/or by glucocorticoids, we used a rat hypothalamic organ culture system in which rat CRH secretion from single explanted hypothalami was evaluated by an RIA (iCRH) specific for rat CRH. The effects of graded concentrations of ovine CRH (oCRH), adrenocorticotropin hormone (ACTH), beta-endorphin (beta-EP), alpha-melanocyte-stimulating hormone (alpha-MSH), corticotropin-like intermediate lobe peptide (CLIP), ovine beta-lipotropin (ovine beta-LPH), and dexamethasone (DEX) upon unstimulated and serotonin- (5HT), acetylcholine- (ACh), and norepinephrine-(NE) stimulated CRH secretion were determined. oCRH and DEX inhibited unstimulated iCRH secretion with ID50 at the 10(-8) M range. ACTH had no detectable suppressive effect at 10(-8) M. oCRH, ACTH, and DEX inhibited 5HT-, ACh-, and NE-stimulated iCRH secretion in a dose-dependent fashion. beta-EP, alpha-MSH, and CLIP also inhibited 5HT-induced iCRH secretion. Of the latter peptides, the strongest inhibitor was beta-EP and the weakest was CLIP. Ovine beta-LPH had only a weak inhibitory effect on 5HT-induced iCRH secretion. Generally, the concentrations required for 50% suppression of neurotransmitter-stimulated iCRH secretion were significantly lower than those required for a similar suppression of unstimulated iCRH secretion. In conclusion, these data suggest the presence of multiple negative feedback loops involved in the regulation of the hypothalamic CRH neuron: an ultrashort CRH-mediated loop, a short, hypothalamic POMC-derived peptide loop, and a long, glucocorticoid-mediated negative feedback loop. The potency of these negative feedback loops may be determined by the state of activation of the CRH neuron.
A E Calogero, W T Gallucci, P W Gold, G P Chrousos
Current concepts of the pathogenesis of sarcoidosis suggest that the expanded numbers of activated T-helper/inducer cells at sites of disease activity result, at least in part, from their proliferation in the local milieu. Normal clonal proliferation of T cells involves activation and expression of the IL 2 receptor (IL 2R) gene. Thus, knowing that IL 2R mRNA transcripts are relatively long lived, we hypothesized that sarcoid blood T cells may contain IL 2R mRNA transcripts and express functional surface IL 2R, although the cells are probably activated elsewhere. Northern analysis using a 32P-labeled cDNA probe for the IL 2R p55 protein demonstrated that blood T cells of patients with active sarcoidosis, but not of normal patients, express 3.5- and 1.5-kb IL 2R mRNA transcripts, the same as those observed in normal T cells activated in vitro. Consistent with this, using flow cytometry and an MAb directed against the IL 2R p55 protein (2A3), we observed detectable levels of IL 2R surface protein on increased numbers of blood T cells of active sarcoidosis patients (4.7 +/- 0.9%) compared with blood T cells of normal patients (0.9 +/- 0.2%). Importantly, when the sarcoid blood T cells were exposed to IL 2 in vitro, they proliferated at a rate greater than that of normal blood T cells under the same conditions, suggesting that the IL 2R spontaneously expressed by sarcoid blood T cells were functionally active. In the context of the known compartmentalization of spontaneous IL 2 production and T cell proliferation at sites of disease in active pulmonary sarcoidosis, these IL 2R positive blood T cells would probably have a proliferative advantage if they trafficked to sites of active sarcoidosis, such as the lower respiratory tract.
K Konishi, D R Moller, C Saltini, M Kirby, R G Crystal
This study examines the relationship between impaired fatty acid oxidation and the pathogenesis of Reye syndrome. We present a hypothesis proposing that many clinical signs of this childhood disease are caused by accumulation of unusual acyl CoA esters, precursors to deacylated metabolites found in the patients' blood and urine. A new method was developed to measure acyl CoA compounds in small human liver biopsy samples, offering several advantages over previous techniques. A major finding was an accumulation in Reye syndrome patients of short- and medium-chain acyl CoA intermediates of fatty acid and branched-chain amino acid oxidation. These metabolites included octanoyl, isovaleryl, butyryl, isobutyryl, propionyl, and methylmalonyl CoA esters. The findings were explained in a model of hepatic fatty acid oxidation involving three interrelated pathways: mitochondrial beta-oxidation, peroxisomal beta-oxidation, and omega-oxidation in the endoplasmic reticulum. The results suggest that pathogenesis in Reye syndrome stems from generalized mitochondrial damage resulting in accumulation of acyl CoA esters. High levels of these compounds lead to inhibition of mitochondrial pathways for ureogenesis, gluconeogenesis, and fatty acid oxidation. The inhibited pathways, in turn, could cause the hyperammonemia, hypoglycemia, and hypoketonemia observed in patients. The model also explains underlying biochemical differences between patients with Reye syndrome and medium-chain acyl CoA dehydrogenase deficiency, another disorder of fatty acid metabolism. Acetyl CoA levels, in the latter disease, were dramatically decreased, compared with both human controls and Reye syndrome patients.
B E Corkey, D E Hale, M C Glennon, R I Kelley, P M Coates, L Kilpatrick, C A Stanley
Antitumor activity of phorbol myristate acetate-(PMA) stimulated neutrophils was measured against CCRF-CEM cells. Neutrophils and tumor cells were incubated (a) as a suspension with continuous mixing to maximize the availability of oxygen or (b) after centrifugation as a pellet to maximize cell-cell contact. The cells were then incubated briefly as a suspension with [14C]glutamine under conditions that blocked further damage to the tumor cells. When cells were incubated as a suspension, inhibition of tumor-cell glutamine uptake was mediated by the myeloperoxidase/hydrogen peroxide/chloride system of stimulated neutrophils. Inhibition was blocked by adding catalase, an inhibitor of myeloperoxidase, or compounds that scavenge hypochlorous acid or chloramines. When cells were incubated as a pellet, a portion of the inhibition could not be blocked in this way, indicating that a nonoxidative mechanism contributed to inhibition. In both systems, inhibition of glutamine uptake was rapid and was obtained at effector-cell/target-cell ratios as low as 0.5:1. This inhibition was obtained under conditions that did not result in 51Cr release from cells labeled with [51Cr]-chromate, indicating that inhibition of glutamine uptake measured cytotoxicity rather than cytolysis. 51Cr release was observed only when cells were incubated together for an hour or more as a pellet at high E/T ratios. This cytolysis was mediated by the myeloperoxidase system, and a nonoxidative contribution to cytolysis was not observed. The results indicate that stimulated neutrophils are potent antitumor effectors cells when cytotoxicity rather than cytolysis is the measure of activity. Because glutamine is required for growth of many tumor cells, inhibition of glutamine uptake may represent a significant tumoristatic or tumoricidal effect.
D B Learn, E L Thomas
This study examines the potential influence of genetic variation on the metabolism of LDL. Restriction fragment length polymorphisms (RFLP) of the gene coding for apo B were identified using the endonucleases Xba I, Eco RI, and Msp I in a group of 19 subjects with moderate hyperlipidemia. There was a significant association between the Xba I polymorphism and the total fractional clearance rate (FCR) of LDL. The individuals with the X1X1 genotype had, on average, a 22% higher FCR (P less than 0.025) than those with the genotype X2X2 (X2 allele = presence of Xba I cutting site). This difference was attributable to increased clearance by the receptor-mediated pathway of LDL catabolism. In this group of subjects, there was no association of LDL kinetic parameters and RFLPs of the LDL receptor gene or the AI- CIII- AIV gene cluster. The data suggest that variation in apo B itself, presumably acting through variable binding to the LDL receptor, makes a significant contribution to the rate of catabolism of LDL.
T Demant, R S Houlston, M J Caslake, J J Series, J Shepherd, C J Packard, S E Humphries
Serum PGI2 stabilizing factor (PSF) was purified from human serum to a single protein with a molecular weight of 28,000 D by SDS-PAGE. Analyses of NH2-terminal sequence (32 residues), COOH-terminal sequence (3 residues) and the composition of amino acids disclosed its homology with human apolipoprotein A-I (Apo A-I), a major apolipoprotein of HDL. Apolipoprotein A-II, C-I, C-II, C-III, D and E, as well as LDL, and VLDL did not possess this activity. The alpha-helix structure of Apo A-I is necessary for the binding of PGI2. HDL and nascent HDL reconstituted from Apo A-I and phospholipid significantly prolonged the half-life of PGI2. PGI2 stabilization by HDL and Apo A-I may be an important protective action against the accumulation of platelet thrombi at sites of vascular damage. The beneficial effect of HDL in the prevention of coronary artery disease may be partly due to this action.
Y Yui, T Aoyama, H Morishita, M Takahashi, Y Takatsu, C Kawai
The gastrin-releasing peptide (GRP) is a neuropeptide hormone and growth factor produced normally by neural and neuroendocrine cells, as well as by human small-cell lung cancer (SCLC) tumors and derived cell lines. This study compares the structure of the human prepro-GRP gene in four SCLC cell lines that express variable levels of steady-state GRP mRNA. The regulation of GRP gene expression appears to be at the level of primary transcription based on nuclear run on studies. In the two SCLC cell lines expressing GRP we find a single transcription start site for GRP mRNA, and near this site we find four DNase I hypersensitive sites. These hypersensitive sites are absent in the two cell lines that do not express GRP. The presence of DNase hypersensitive sites in the promoter region of the GRP gene is the structural feature that best correlates with transcriptional activation. These four DNase hypersensitive sites are candidates for cis acting regulatory regions, which may be important in determining the level of transcription of the human prepro GRP gene.
S Markowitz, G Krystal, A M Lebacq-Verheyden, J Way, E A Sausville, J Battey
The majority of C1q-binding IgG in sera of some patients with systemic lupus erythematosus (SLE) cosediments with monomeric IgG. This study was undertaken to provide definitive proof that the low-molecular weight C1q-binding IgG consists of autoantibodies to C1q. Monomeric C1q-binding IgG was isolated from five SLE plasmas by C1q affinity chromatography and gel filtration. All C1q-binding IgG preparations and their F(ab')2 fragments bound to both C1q and the collagen-like region of C1q by an ELISA. To rule out the possibility that small DNA-antiDNA immune complexes caused this binding activity, Fab' fragments of the C1q-binding IgG preparations were digested with DNase I to degrade any DNA. The Fab' fragments continued to bind to C1q and its collagen-like region after this treatment. C1q-binding IgG was heterogenous on isoelectric focusing. Interaction of C1q-binding IgG with solid-phase C1q was retained in 1 M NaCl, whereas the binding of DNA or heat-aggregated IgG to solid-phase C1q was abrogated or markedly diminished. The association constant of C1q-binding IgG with solid-phase C1q was 2.7 X 10(7) M-1. We conclude that low-molecular weight C1q-binding IgG in the studied patients with SLE consists of autoantibodies to the collagen-like region of C1q.
S Uwatoko, M Mannik
Allergic contact dermatitis to Toxicodendron radicans (poison ivy) is mediated by the hapten urushiol. An urushiol-specific, interleukin 2 (IL-2)-dependent T cell clone (RLB9-7) was generated from the peripheral blood of a patient with a history of allergic contact dermatitis to T. radicans. This clone proliferated specifically to both leaf extract and pure urushiol. Although the clone had the phenotype CD3+CD4+CD8+, proliferation to antigen was blocked by anti-CD8 and anti-HLA-A, B, C, but not by anti-CD4, suggesting that CD4 was not functionally associated with the T cell receptor. Furthermore, studies with antigen-presenting cells from MHC-typed donors indicated that the clone was MHC class 1 restricted. RLB9-7 was WT31 positive, indicating it bears the alpha beta T cell receptor. The clone lacked significant natural killer cell activity and produced only low levels of IL-2 or gamma-interferon upon antigen stimulation. Addition of RLB9-7 to autologous peripheral blood mononuclear cells in the presence of urushiol inhibited the pokeweed mitogen-driven IgG synthesis. This suppression was resistant to irradiation (2,000 rad) and was not seen when RLB9-7 was added to allogeneic cells, even in the presence of irradiated autologous antigen-presenting cells, suggesting that suppression was MHC restricted and not mediated by nonspecific soluble factors. However, RLB9-7 cells in the presence of urushiol inhibited the synthesis of tetanus toxoid-specific IgG by autologous lymphocytes, indicating that the suppression, although triggered specifically by urushiol, was nonspecific.
R S Kalish, C Morimoto
Recent reports using anesthetized ventilator-dependent animal models, have suggested that in certain shock states, a disparity exists between arterial and mixed venous blood gases with regard to acid-base status and oxygenation. In a chronically instrumented unanesthetized canine model of acute cardiac tamponade breathing room air, we studied the effect of a graded decline in cardiac output on arterial and mixed venous pH, PCO2, and PO2. Cardiac tamponade resulted in a profound arterial respiratory alkalosis, whereas mixed venous pH, PCO2, and calculated serum bicarbonate levels remained relatively unchanged. As intrapericardial pressure increased and cardiac output declined, the difference between arterial and mixed venous PCO2 progressively increased. Further, whereas arterial oxygenation improved as cardiac output declined, mixed venous oxygenation steadily worsened. This disparity began early in cardiac tamponade (reductions in cardiac output of 20-40%) long before arterial blood pressure began to fall and progressively worsened as hemodynamic deterioration and lactic acidosis developed. Our findings are consistent with the hypothesis that a reduction in blood flow, resulting in decreased CO2 delivery to the lungs, is the primary mechanism responsible for the difference in pH and PCO2 observed between arterial and mixed venous blood. In this conscious, spontaneously breathing animal model, mixed venous blood gases thus are superior to arterial blood gases in assessing acid-base status and oxygenation, even early in acute cardiac tamponade when the decline in cardiac output is in the range of 20 to 40% and arterial blood pressure has not changed significantly.
D W Mathias, P S Clifford, H S Klopfenstein
To further our understanding of the functional role of catecholaminergic systems in regulating hypothalamic corticotropin-releasing hormone (CRH) secretion, we assessed the direct effects of a multiplicity of catecholamine agonists and antagonists on hypothalamic CRH secretion. To accomplish this, we used an in vitro rat hypothalamic organ culture system in which CRH secretion from single explants was evaluated by a specific RIA (IR-rCRH). Norepinephrine (NE) stimulated IR-rCRH secretion dose dependently, with peak effects in the nanomolar range. The effect of NE was antagonized by the mixed alpha antagonist phentolamine, the alpha 1 antagonist prazosin, and the alpha 2 antagonist yohimbine, but not by the beta blocker, L-propanolol. Compatible with these data were the findings that the alpha 1 agonist phenylephrine and the alpha 2 agonist clonidine both stimulated IR-rCRH secretion in a dose-dependent fashion. On the other hand, whereas the beta agonist, isoproterenol, caused a weak, non-dose-dependent increase in IR-rCRH secretion, this effect could not be antagonized by L-propanolol. Despite pretreatment with serotonin and acetylcholine antagonists, the effect of NE upon IR-rCRH secretion was undiminished, suggesting that NE-induced CRH secretion is not mediated by either neurotransmitter. On the other hand, pretreatment with gamma-aminobutyric acid (GABA) attenuated NE-induced IR-rCRH secretion. Whereas epinephrine (E) stimulated IR-rCRH secretion, this occurred only at higher concentrations, and was antagonized by phentolamine, but not by L-propanolol. Dopamine (DA) had a weak stimulatory effect that could be antagonized by the DA1 receptor antagonist, SCH 23390, but not by phentolamine. We conclude that NE and E stimulate hypothalamic IR-rCRH secretion via alpha 1 and alpha 2 receptors. The effect of NE upon IR-rCRH secretion is not apparently mediated by serotonergic or cholinergic interneurons, but is modulated by the inhibitory neurotransmitter, GABA. These data support the idea that the central catecholaminergic systems are excitatory rather than inhibitory upon CRH secretion when acting directly at the hypothalamic level.
A E Calogero, W T Gallucci, G P Chrousos, P W Gold
Heterozygous hypobetalipoproteinemia is characterized by reduced plasma concentrations of LDL cholesterol, total triglycerides, and apo B to less than 50% of normal values. The molecular basis of this disorder remains unknown. The phenotype cosegregates with a DNA haplotype of the apo B gene in an Idaho pedigree, with a maximum decimal logarithm of the ratio (LOD) score of 7.56 at a recombination rate of zero. Individuals carrying this haplotype had total cholesterol levels of 96 mg/dl, LDL cholesterol levels of 37 mg/dl, triglycerides levels of 51 mg/dl, and apo B levels of 38 mg/dl. This study strongly suggests that apo B mutations underlie hypobetalipoproteinemia, and demonstrates the power of the candidate gene approach in linkage analysis for unraveling genetic determinants in metabolic disorders of undefined etiology.
M Leppert, J L Breslow, L Wu, S Hasstedt, P O'Connell, M Lathrop, R R Williams, R White, J M Lalouel
We have investigated the genetic origin of autoantibody production in several strains of mice that spontaneously develop a systemic lupus erythematosus-like disease. Restriction fragment length polymorphism analyses of gene loci encoding kappa light chain variable regions (Igk-V) demonstrated, as shown previously for the Ig heavy chain locus, that autoantibody production and disease occur in different Igk-V haplotypes. Moreover, autoimmune mice with known genetic derivation inherited their Igk-V loci essentially unaltered from their nonautoimmune ancestors. New Zealand black lupus mice, with unknown genetic derivation, had a possibly recombinant Igk-V haplotype, composed of V kappa loci that were primarily indistinguishable from those of nonautoimmune strains from either of the two potential donor haplotypes. The heavy and light chain gene segments (variable, diversity, joining) encoding anti-DNA antibodies were diverse and often closely related, or even identical, to those found in antibodies to foreign antigens in normal mice. Only 1 of 11 sequenced variable region genes could not be assigned to existing variable region gene families; however, corresponding germline genes were present in the genome of normal mice as well. These data argue against abnormalities in the genes and mechanisms generating antibody diversity in lupus mice and suggest a remarkable genetic and structural diversity in the generation of anti-DNA binding sites.
R Kofler, R Strohal, R S Balderas, M E Johnson, D J Noonan, M A Duchosal, F J Dixon, A N Theofilopoulos
Serum osteocalcin was measured in patients with idiopathic hypoparathyroidism or pseudohypoparathyroidism, before or during the treatment with active vitamin D3 (1,25(OH)2D3 or 1 alpha OHD3). Serum osteocalcin and plasma 1,25(OH)2D were decreased in 11 patients with idiopathic hypoparathyroidism before treatment (2.8 +/- 1.27 ng/ml, P less than 0.001 and 14.3 +/- 4.27 pg/ml, P less than 0.001, respectively). In 24 patients with idiopathic hypoparathyroidism during the treatment, serum osteocalcin and plasma 1,25(OH)2D were within the normal range (4.5 +/- 0.74 ng/ml and 25.7 +/- 5.69 pg/ml, respectively). In five patients with pseudohypoparathyroidism before treatment, plasma 1,25(OH)2D was decreased (15.6 +/- 10.6 pg/ml, P less than 0.001) but serum osteocalcin was normal (7.8 +/- 1.66 ng/ml). In nine patients with pseudohypoparathyroidism during the treatment with active vitamin D3, serum osteocalcin and plasma 1,25(OH)2D were normal (6.8 +/- 1.47 ng/ml and 27.2 +/- 6.0 pg/ml, respectively). Serum PTH in pseudohypoparathyroidism was increased before treatment (0.70 +/- 0.34 ng/ml, P less than 0.05) and was normal during the treatment (0.50 +/- 0.13 ng/ml). In idiopathic hypoparathyroidism, the active vitamin D3 increased serum osteocalcin without PTH. In pseudohypoparathyroidism, PTH may increase serum osteocalcin or modulate the effect of active vitamin D3 on serum osteocalcin.
K Mizunashi, Y Furukawa, R Miura, S Yumita, H E Sohn, K Yoshinaga
To examine whether Cl- is transported via transcellular pathways in the thin ascending limb of Henle's loop (TAL), conventional microelectrode technique was applied in isolated TAL segments of hamsters perfused in vitro. The average basolateral membrane voltage (VB) was -24.5 +/- 1.5 mV (n = 18). Ouabain (10(-4) M) had no effect on VB. Sudden reduction of basolateral Cl- concentration from 165 to 5 mmol/liter caused a large depolarizing spike (+49.1 +/- 2.7 mV, n = 18), while the transepithelial potential (VT) showed lumen positive deflection by 33.4 +/- 1.2 mV, which indicates that a large Cl- conductance exists in the basolateral membrane. Reduction of luminal Cl- concentration caused sustained depolarization of luminal cell membrane from +24.5 +/- 2.1 to -9.7 +/- 3.4 mV (n = 6), which indicates that there is also a Cl- conductance in the luminal membrane. Since we have previously shown that acidification of ambient solution suppresses the transmural Cl- permeability, we tested whether acid pH also inhibits the Cl- conductance of the basolateral membrane. When pH of the bathing fluid was lowered to 5.8, the depolarizing spike of VB and the change of VT upon sudden reduction of basolateral Cl- were almost completely abolished. From these results we conclude: (a) both the luminal and the basolateral membrane of hamster TAL segments have Cl- conductances, and (b) Cl- transport in the TAL takes place, at least in part, via a transcellular route when a transepithelial Cl- gradient is present.
K Yoshitomi, Y Kondo, M Imai
The phagocytic receptor for unopsonized Pseudomonas aeruginosa was characterized functionally using human monocyte-derived macrophages. Freshly isolated human peripheral blood monocytes were unable to ingest unopsonized P. aeruginosa; ingestion did not occur until the cells had been in culture for 2 d and it became maximal after 4 d. Macrophages plated on coverslips derivatized with anti-BSA IgG or with human gamma-globulin lost the capacity to phagocytose unopsonized P. aeruginosa, unopsonized zymosan, and EIgG but bound C3bi-coated erythrocytes normally. Each of the four human IgG subclasses and Fc fragments of anti-BSA IgG inhibited phagocytosis of both unopsonized P. aeruginosa and EIgG. Phagocytosis of P. aeruginosa and zymosan was markedly impaired and EIgG minimally inhibited if macrophages were plated on coverslips derivatized with mannan or when mannan was added to the phagocytosis buffer. Phagocytosis of P. aeruginosa and zymosan, and binding of EC3bi was dependent on the presence of divalent cations, but phagocytosis of EIgG was not. The macrophage phagocytic receptor for unopsonized P. aeruginosa was inactivated by proteolytic enzymes. Phagocytosis of P. aeruginosa was inhibited by D-mannose, L-fucose, and alpha methyl mannoside, but not by L-mannose, D-fucose, or D-glucose. The same sugars inhibited phagocytosis of unopsonized zymosan. We conclude that phagocytosis of unopsonized P. aeruginosa by human monocyte-derived macrophages is facilitated by mannose receptors.
D P Speert, S D Wright, S C Silverstein, B Mah
The glycoprotein hormone inhibin is produced by the Sertoli cells of the testis under the influence of follicle-stimulating hormone (FSH) and is postulated in turn to inhibit FSH secretion. Luteinizing hormone (LH) is not recognized to have an important role in the control of inhibin secretion in any species. To determine the relative roles of FSH and LH in the control of inhibin secretion in man, we examined the effects of selective FSH and LH replacement on serum inhibin levels in normal men whose endogenous gonadotropins were suppressed by testosterone (T). After a 3-mo control period, nine men received 200 mg T enanthate i.m. weekly for 3-9 mo. During T treatment, serum LH and FSH levels were markedly suppressed and serum inhibin levels fell to 40% of control values. While continuing T, 3-5 mo of treatment with purified hFSH (n = 4) or hLH (n = 4) increased the respective serum gonadotropin level into the upper normal range and significantly increased inhibin levels back to 64 and 55% of control values, respectively. Supraphysiological LH replacement with high doses of human chorionic gonadotropin (n = 3) returned serum inhibin levels to 63% of control values. In no case did inhibin levels return fully to control levels. In conclusion, serum inhibin levels fell during gonadotropin suppression and were partially and approximately equally restored by either FSH or LH treatment. FSH presumably acts directly on the Sertoli cell to increase inhibin secretion whereas LH may act via increases in intratesticular T levels and/or other factor(s).
R I McLachlan, A M Matsumoto, H G Burger, D M de Kretser, W J Bremner
The role of central alpha-1 adrenergic receptors in cataplexy was investigated in genetically narcoleptic Doberman pinschers. Treatment of narcoleptic dogs with 25-600 micrograms/kg prazosin, a selective alpha-1 adrenergic receptor blocker, exacerbated cataplexy, whereas treatment with the alpha-1 agonist, methoxamine, ameliorated it. Subsequent studies showed that the beneficial effects of classical treatments of human narcolepsy (amphetamines and tricyclic antidepressants) are antagonized by prazosin, suggesting that these drugs are active through an indirect alpha-1 stimulation (via an increase of norepinephrine in the synaptic cleft). Other studies confirmed that the observed effects were not due to peripheral alpha-1 cardiovascular involvement. Atropine, a central anticholinergic agent, but not methylatropine, a peripheral one, completely suppressed the prazosin effect, which suggests that adrenergic and cholinergic systems act sequentially and not independently to generate cataplexy. Little is known about the physiological role of central alpha-1 adrenoceptors. This series of experiments implicates these receptors in narcolepsy-cataplexy.
E Mignot, C Guilleminault, S Bowersox, A Rappaport, W C Dement
31P nuclear magnetic resonance (NMR) spectroscopy in vivo and fluorometry were used to measure muscle ATP, total creatine, pH, and Mg2+ in vivo; and to calculate creatine phosphate (CrP), the ratios of CrP/inorganic phosphate (Pi), CrP/ATP, free ADP levels, and the free-energy change in ATP hydrolysis so nutritional effects could be ascertained. These parameters were determined in vivo in resting control, 2-d-fasted, and hypocalorically fed rats and in animals similarly hypocalorically fed and then refed. The ATP, Pi, and intracellular Mg2+ levels were comparable in the four groups. When the fasted and underfed animals were compared with the control and refed animals, there were falls in the ratios of CrP/Pi and CrP/ATP, in the calculated CrP, and the free-energy change of ATP hydrolysis, but a rise in the calculated free ADP. In the hypocaloric group, intracellular pH fell significantly and a large peak was noted in the phosphodiester region. The data are consistent with the hypothesis that ATP levels are maintained at the cost of CrP, suggesting that ATP production is disturbed by aerobic and anaerobic mechanisms.
C Pichard, C Vaughan, R Struk, R L Armstrong, K N Jeejeebhoy
Ecto-5'-nucleotidase (ecto-5'-NT) is believed to be a maturation marker for human B lymphocytes because its expression increases during normal development and is reduced in many patients with B cell immunodeficiencies. To determine whether this enzyme defines functional subsets of B lymphocytes, human peripheral blood B cells, separated into ecto-5'-NT positive and negative populations by using goat anti-5'-NT antibodies and the fluorescence-activated cell sorter, were compared for their ability to secrete polyclonal immunoglobulin. Both populations synthesized equivalent quantities of IgM in response to a T cell-dependent (PWM) or T cell-independent (EBV) stimulator of polyclonal immunoglobulin biosynthesis. However, ecto-5'-NT+ B lymphocytes synthesized 8- to 26-fold more IgG per cell than ecto-5'-NT- B cells. These data provide the first direct evidence that ecto-5'-NT is a marker for the functional maturation of human B cells and support the hypothesis that ecto-5'-NT deficiency in patients with hypogammaglobulinemia results from a block in B lymphocyte maturation.
L F Thompson, J M Ruedi
Polymorphic minor transplantation antigens probably play an important role in immune mediated graft rejections of bone marrow transplants. Mapping of these antigens on hematopoietic progenitor cells (HPC) is important since these antigenic determinants may serve as target structures in the rejection process, and it ultimately opens the possibility to match for these antigens. Using a cell-mediated cytotoxicity assay with H-Y-specific cytotoxic T lymphocytes as effector cells, a dose-dependent growth inhibition up to 100% of myeloid (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) HPC of male donors was obtained, indicating expression of the H-Y antigen on these progenitor cells. In contrast, inhibition of relatively mature erythroid and myeloid progenitor cells was only 40-50%, indicating that the recognition of the H-Y antigen diminished during maturation of erythroid and myeloid HPC. Our results show that the H-Y antigen can be recognized on HPC as a target for cytotoxic T cell responses. This may be important in graft rejection of male donor bone marrow grafts by female recipients.
P J Voogt, E Goulmy, W E Fibbe, W F Veenhof, A Brand, J H Falkenburg
By preventing macrophages from destroying phagocytized microorganisms, glucocorticoids lower natural resistance of the host against many pathogens. gamma-Interferon has an opposite effect and restores activity of dexamethasone-treated mononuclear phagocytes against some but not all microorganisms. In the present studies we show that dexamethasone impairs activity of human blood monocytes kept in culture for 36 h against Listeria monocytogenes, without impairing H2O2 or O2- secretion. Likewise dexamethasone does not interfere with activation of systems generating oxygen metabolites by lymphokines. Thus gamma-interferon increases three- to fivefold the capacity of dexamethasone-treated monocytes to secrete O2- or H2O2 upon stimulation by opsonized zymosan, live bacteria, or phorbol myristate acetate. Concurrently gamma-interferon restores listericidal activity of dexamethasone-treated monocytes. After gradual activation by exposure to gamma-interferon for progressive time periods, listericidal activity becomes tightly correlated (r = 0.922-0.994) with the amount of H2O2 or O2- secreted by dexamethasone-treated monocytes. Activation of oxidative systems by the lymphokine is, however, not correlated with the restoration of activity against Aspergillus spores, lost during dexamethasone treatment, which does not depend on antimicrobial oxygen metabolites. Taken together, these observations lend to the hypothesis that glucocorticoids impair nonoxidative defense mechanisms of "resting" macrophages, while gamma-interferon restores macrophage function impaired by glucocorticoids by activating alternate killing systems, which are at least partly of oxidative nature.
A Schaffner, P Rellstab
Cellular calcium overload figures prominently in the pathogenesis of the contractile dysfunction observed after brief periods of ischemia (myocardial stunning). Because acidosis is known to antagonize Ca influx and the intracellular binding of Ca, we reasoned that acidosis during reperfusion might prevent Ca overload and ameliorate functional recovery. We measured developed pressure (DP) and 31P-nuclear magnetic resonance spectra in 26 isovolumic Langendorff-perfused ferret hearts. After 15 min of global ischemia, hearts were reperfused either with normal solution (2 mM [Ca]o, Hepes-buffered, pH 7.4 bubbled with 100% O2; n = 6) or with acidic solutions (pH 6.6 during 0-3 min, pH 7.0 during 4-6 min) before returning to the normal perfusate (n = 7). Ventricular function after 30 min of reperfusion was much greater in the acidic group (105 +/- 5 mmHg at 2 mM [Ca]o) than in the unmodified reperfusion group (79 +/- 7 mmHg, P less than 0.001); similar differences in DP were found over a broad range of [Ca]o (0.5-5 mM, P less than 0.001) and during maximal Ca2+ activation (P less than 0.001). Intramyocardial pH (pHi) was lower in the acidic group than in the unmodified group during early reperfusion, but not at steady state. Phosphate compounds were comparable in both groups. To clarify whether the protective effect of acidosis is due to intracellular or extracellular pH, we produced selective intracellular acidosis during early reperfusion by exposure to 10 mM NH4Cl for 6 min just before ischemia (n = 6). For the first 12 min of reperfusion with NH4Cl-free solution (pH = 7.4), pHi was decreased relative to the unmodified group. Recovery of DP was practically complete, and maximal Ca2+-activated pressure was comparable to that in a nonischemic control group (n = 5). These results indicate that transient intracellular acidosis can prevent myocardial stunning, presumably owing to a reduction of Ca influx into cells and/or competition of H+ for intracellular Ca2+ binding sites during early reperfusion.
M Kitakaze, M L Weisfeldt, E Marban
Uptake of [3H]oleate by canine or rat cardiac myocytes is saturable, displays the countertransport phenomenon, and is inhibited by phloretin and trypsin. Cardiac myocytes contain a basic (pI approximately 9.1) 40-kD plasma membrane fatty acid binding protein (FABPPM) analogous to those recently isolated from liver, adipose tissue, and gut, unrelated to the 12-14-kD cytosolic FABP in these same tissues. An antibody to rat liver FABPPM selectively inhibits specific uptake of [3H]oleate by rat heart myocytes at 37 degrees C, but has no influence on nonspecific [3H]oleate uptake at 4 degrees C or on specific uptake of [3H]glucose. Uptake of long-chain free fatty acids by cardiac muscle cells, liver, and adipose tissue and absorption by gut epithelial cells is a facilitated process mediated by identical or closely related plasma membrane FABPs.
D Sorrentino, D Stump, B J Potter, R B Robinson, R White, C L Kiang, P D Berk
The possibility of Fc-dependent uptake of IgG immune complexes was examined in subcultured rat mesangial cells free of monocytes. 195Au-labeled colloidal gold particles were coated either with BSA only or with BSA followed by rabbit anti-BSA-IgG or the F(ab')2 fragment of the IgG. Mesangial cells preferentially took up 195Au particles covered with BSA-anti-BSA-IgG over those covered with BSA or the F(ab')2 fragment. This uptake was a time-dependent and saturable process inhibitable by sodium azide or cytochalasin B. Using phase-contrast microscopy in the light reflectance mode, it was established that essentially all mesangial cells took up IgG-coated gold particles. By electron microscopy the process was shown to consist of vesicular uptake with delivery to endosomes. Mesangial binding-uptake of the IgG-covered particles was associated with stimulation of PGE2 synthesis and production of platelet-activating factor, a lipid mediator of inflammation. To characterize the potential Fc receptor for IgG we used the rosetting technique with sheep red blood cells coated with IgG subclass-specific mouse monoclonal antibodies. 50% of mesangial cells exhibited rosetting with red cells coated with mouse IgG2a, whereas negligible rosetting was observed with IgG2b or IgG1. Competition experiments confirmed the specificity of IgG2a binding. We conclude that cultured rat mesangial cells exhibit specific receptors for IgG and that occupancy of Fc receptors results in endocytosis and is associated with generation of PGE2 and platelet-activating factor. These observations may be of significance for immune-mediated glomerular diseases.
R Neuwirth, P Singhal, B Diamond, R M Hays, L Lobmeyer, K Clay, D Schlondorff
A possible role for C1q in antibody-dependent granulocyte-mediated killing of nonphagocytosable targets was investigated utilizing IgG-dependent granulocyte cytotoxicity directed against microfilariae of Dirofilaria immitis. Granulocyte-mediated killing of microfilariae is enhanced by addition of fresh serum. Lack of C4 did not significantly reduce the observed increase in cytotoxicity. The addition of highly purified monomeric human Clq (0.2 microgram/ml) in the presence of immune IgG resulted in a two- to fivefold enhancement of killing (P less than 0.025). C1q enhancement of killing occurred in the absence of fluid-phase IgG, but killing was significantly less than when both fluid-phase IgG and C1q were present. The effect of C1q was inhibited by the addition of solubilized type I collagen (44-92% inhibition of killing, P less than 0.05). Significant 125I-Clq binding to microfilariae occurred only in the presence of immune IgG. In addition, C1q in concentrations ranging from 0.5 to 2.0 micrograms/ml resulted in a dose-dependent increase in binding of 125I-immune IgG to microfilariae. Finally, when purified C1q was added to preopsonized, washed microfilariae, granulocyte production of superoxide was increased from 0.25 +/- 0.07 to 0.68 +/- 0.07 nm/10(6) cells.10 min (P less than 0.01). These results describe a novel functional role for C1q in enhancement of antibody-dependent cellular cytotoxicity towards nonphagocytosable targets.
A Hamada, J Young, R A Chmielewski, B M Greene
Growth factor receptors for T lymphocytes, such as interleukin 2 and insulin, are present on activated but not resting T lymphocytes. We sought to determine if insulin-like growth factor I (IGF-I) could act as a growth factor for human T cells and to characterize its receptor on resting and activated cells. Recombinant IGF-I induced two separate functions. It was chemotactic for and increased incorporation of tritiated thymidine into both unactivated (resting) and mitogen-activated T cells. High-affinity 125I-IGF-I binding to human T cells was saturable with an apparent Kd of 1.2 +/- .6 X 10(-10) M for binding to activated T cells and 1.2 +/- .9 X 10(-10) for unactivated T cells. The calculated binding for activated cells was 330 +/- 90 and for resting cells 45 +/- 9 high-affinity receptor sites per cell. Affinity cross-linking of 125I-IGF-I to resting or activated T cells revealed a radioligand-receptor complex of 360,000 mol wt when analyzed by SDS-PAGE without reduction and complexes of 270,000 and 135,000 mol wt upon reduction; prior incubation with excess unlabeled IGF-I prevented formation of the 125I-IGF-I receptor complex. Our data suggest that both resting and activated T lymphocytes bear functional IGF-I receptors similar to those found in other tissues. These receptors may mediate T cell growth and chemotaxis.
V F Tapson, M Boni-Schnetzler, P F Pilch, D M Center, J S Berman
A young man with a long history of abnormal bleeding was seen in January 1985. Coagulation tests showed dysfibrinogenemia and an antifibrinogen autoantibody was demonstrable in his serum. This antibody, when purified, was capable of inhibiting the polymerization of normal fibrin monomers, apparently through binding to the alpha fibrinogen chain. 6 mo later the patient was asymptomatic, coagulation tests were normal, and the antifibrinogen autoantibody was barely detectable. At this time, affinity-purified autologous and rabbit antifibrinogen antibodies were capable of absorbing an IgG kappa antibody from the patient's serum, which reacted indistinctly with both autologous and xenogeneic antifibrinogen antibodies in enzyme immunoassays. It has been concluded that the patient's dysfibrinogenemia was the result of an antifibrinogen autoantibody, and that later on an anti-idiotype antibody, which binds an interspecies cross-reactive idiotype expressed on anti-human fibrinogen antibodies, inhibited the production of the antifibrinogen autoantibody which led to the remission of the disorder.
Lipid A is the toxic principle of lipopolysaccharide of gram-negative bacteria, which causes a spectrum of changes in blood cells and vascular cells. We now report that human platelets are directly stimulated by endotoxic lipid A that activates protein kinase C. Rapid phosphorylation of a human platelet protein of Mr 47,000, a marker of protein kinase C activation, accompanies secretion of [14C]serotonin and aggregation triggered by endotoxic lipid A. These events are time and concentration dependent, with phosphorylation reaching maximum in 2 min and the concentration of lipid A causing a 50% effect (EC50) between 12 and 15 microM. Phospholipase C activation in lipid A-stimulated platelets was not observed as judged by a lack of generation of [3H]diacylglycerol in [3H]arachidonic acid-labeled platelets and a lack of generation of [32P]-phosphatidic acid in 32PO4-labeled platelets. Lipid A did not induce formation of TXA2 as measured by radioimmunoassay for TXB2. The stimulation of human platelets and activation of protein kinase C by endotoxic lipid A was blocked by lipid X, a structural precursor of lipid A. Lipid X also blocked the stimulation of human platelets by phorbol 12-myristate 13-acetate, suggesting that lipid A, lipid X and phorbol ester share reactive site(s) on the human platelet membrane. Although lipid X inhibited thrombin-induced phosphorylation of P47 it did not suppress secretion of [14C]serotonin, indicating the role of protein kinase C-independent pathways in platelet stimulation by thrombin. The inhibitory effect of lipid X did not involve generation of cyclic AMP in human platelet membrane preparations. These results indicate that human platelets are stimulated by endotoxic lipid A, a naturally occurring biologic modifier of protein kinase C. Due to the widespread presence of this enzyme in blood cells, vascular cells, and neurons, its modulation by lipid A may represent a significant mechanism underlying hematologic and circulatory derangements observed in endotoxic shock in humans.
J Grabarek, S Timmons, J Hawiger
Using a new method for long-term recording of monophasic action potentials from the human heart, we studied in 17 patients the effects on ventricular action potential duration (APD) of three clinically pertinent cycle length perturbations: (1) single extrastimuli, (2) abrupt sustained rate acceleration and deceleration, and (3) different steady-state cycle lengths. Results were: (a) APD after single extrastimuli at progressively longer cycle lengths were related to the extrastimulus cycle length with a biphasic electrical restitution curve which after an initial steep rise and a subsequent transient descent rose again more gradually to a plateau at cycle lengths above 800-1,000 ms. (b) After a sustained step decrease in cycle length, the first APD shortened abruptly while final steady-state adaptation required up to several minutes. The transition between the rapid and slow phase of APD change was characterized by a variable alternans of APD which correlated inversely with the preceding diastolic interval. (c) In the steady state, APD correlated linearly with cycle length, increasing an average of 23 ms per 100 ms cycle length increase (r = 0.995). The divergence between steady-state and non-steady-state APD, and the slowness of steady-state adaptation, are important factors to be considered in clinical electrophysiologic studies and in rate correction algorithms of APD or QT intervals, respectively.
M R Franz, C D Swerdlow, L B Liem, J Schaefer
The effect of pH, PO2, and inorganic phosphate on the uptake and metabolism of hypoxanthine by erythrocytes has been studied. Uptake of hypoxanthine and accumulation of inosine 5'-monophosphate (IMP) were markedly increased at acid pH, high external phosphate concentrations, and low PO2. Release of accumulated IMP as hypoxanthine occurred at alkaline pH values and low external phosphate concentrations. Conditions favoring IMP accumulation gave rise, in the absence of hypoxanthine, to a corresponding increase in 5'-phosphoribosyl-1-pyrophosphate. Intracellular phosphate concentrations were markedly pH dependent and a model is presented whereby hypoxanthine uptake and release are controlled by intracellular concentrations of inorganic phosphate and 2,3-bisphosphoglycerate. These allosteric effectors influence, in opposing ways, two enzymes governing IMP accumulation, namely 5'-phosphoribosyl-1-pyrophosphate synthetase and 5'-nucleotidase. These metabolic properties suggest that the erythrocyte could play a role in the removal of hypoxanthine from anoxic tissue.
P A Berman, D A Black, L Human, E H Harley
The HLA class I molecules identified serologically as HLA-B27 are highly associated with ankylosing spondylitis and related human disorders. All known HLA-B27 amino acid sequences contain a cysteine residue at position 67; no other published HLA class I sequence contains a cysteine within the hypervariable region of the alpha 1 domain, which extends from amino acid residues 63-84. To investigate the role of this cysteine residue in the antigenic structure of HLA-B27, we isolated a genomic clone encoding a molecule of the HLA-B27.1 subtype and performed oligonucleotide-directed mutagenesis to convert the cysteine at position 67 to a tyrosine. When transfected into mouse L cells, both the wild-type and Cys67----Tyr67 mutant B27 genes directed the synthesis and surface expression of molecules reactive with the monomorphic anti-HLA class I antibody W6/32. However, only the L cells transfected with the wild-type B27 gene reacted with the anti-B27 antibody ME1; L cells transfected with the mutant B27 were completely unreactive with this antibody. Experiments with hybrid exons created from the HLA-B27 and HLA-A2 genes yielded results consistent with the mapping of the ME1 epitope to the B27 alpha 1 domain. A second anti-B27 antibody, GS145.2, also showed markedly reduced binding to the Cys67----Tyr67 mutant. These studies document the importance of the unique Cys67 residue in the antigenic structure of HLA-B27.
J D Taurog, F A el-Zaatari
We studied the role of diminished sympathetic nervous system (SNS) activity and endogenous opiate activation in the hypotensive action of taurine, a sulfur amino acid, in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Supplementation of taurine could prevent the development of DOCA-salt hypertension in rats, but failed to change blood pressure in vehicle-treated control rats. Cardiac NE turnover, which was determined from the rate of decline of tissue NE concentration after the administration of alpha-methyl-p-tyrosine, was markedly accelerated in DOCA-salt rats, but 1% taurine supplement restored it to normal. Moreover, naloxone (2 mg/kg), the specific opiate antagonist, increased blood pressure in taurine-treated DOCA-salt rats, restoring it to levels similar to those in the DOCA-salt rats. In contrast, taurine did not decrease cardiac NE turnover in the control rats, nor did naloxone increase blood pressure in the taurine-treated control rats. Moreover, supplementation of taurine increased both beta-endorphin-like immunoreactive material and taurine contents in the hypothalamus of DOCA-salt rats, whereas it did not increase beta-endorphin in that of control rats despite increased taurine contents. Thus, taurine not only normalized the increased cardiac SNS activity but also elicited an opiate-mediated vasodepressor response only in DOCA-salt rats. It is suggested, therefore, that endogenous opiate activation, which is intimately related to SNS suppression, may contribute to the antihypertensive effect of taurine in sodium chloride hypertension.
T Fujita, Y Sato
The pathogenesis of renal fibrosis in crescentic nephritis is incompletely understood. To improve our understanding of this process, crescentic nephritis was induced in New Zealand White rabbits by administration of guinea pig antiglomerular basement membrane IgG after sensitization with guinea pig IgG, and their kidneys were analyzed for the development of fibrosis. Collagen synthesis in renal cortical tissue was significantly elevated by day 3, peaked at days 7-15, and returned towards baseline by day 21. Collagen content of both glomeruli and cortex were increased starting on days 14-16, and remained constant in cortex thereafter. Light microscopic analysis was much less sensitive, revealing fibrosis only after day 21. Immunofluorescence revealed that type IV collagen was distributed primarily in the glomerulus, while types I and III were increased in the glomerulus and interstitium. Thus, in this model of crescentic nephritis, fibrosis, as assessed biochemically, developed early at time points when morphologic analysis failed to detect such a development. Hence early therapeutic intervention, before morphologic evidence of fibrosis is evident, may be more successful in arresting the progression of this disease before it reaches irreversible terminal stages.
G Downer, S H Phan, R C Wiggins
Renin has been identified biochemically and immunohistochemically in the adrenal gland. We examined the subcellular distribution and behavior of adrenal renin. By differential centrifugation of adrenal capsules, we found renin mainly in mitochondrial fractions. By Percoll density gradient centrifugation of this fraction, dense granules were separated from mitochondria and microsomes. The renin activity in the dense granules from the capsules of nephrectomized rats was 15 times greater than that of the intact rat. Immunohistochemical studies revealed that the dense granules increased in number after bilateral nephrectomy. Immunogold staining of these granules showed unequivocally the presence of renin in these granules. Adrenal capsules in organ culture were found to release renin at a steady rate. Renin release from bilaterally nephrectomized rat adrenals was 46 times faster than from the organs of intact animals. The mechanism of the control of renin secretion from the adrenal gland was different from the kidney in that the secretion was stimulated by potassium chloride (10 mM) or angiotensin II (10(-9)-10(-7) M) but not by ACTH (10(-9)-10(-7) M), suggesting stimulation by intracellular calcium. These results provide evidence that the adrenal synthesizes renin, stores it in specific secretory granules and secretes it in a regulated manner.
K Mizuno, L H Hoffman, J C McKenzie, T Inagami
A hematopoietic cell (CFU-B1) capable of producing blast cell containing colonies in vitro was detected using a semisolid culture system. The CFU-B1 has the capacity for self-renewal and commitment to a number of hematopoietic lineages. Monoclonal antibody to the human progenitor cell antigen-1 (HPCA-1) and a monoclonal antibody against the major histocompatibility class II antigen (HLA-DR) were used with fluorescence activated cell sorting to phenotype the CFU-B1. The CFU-B1 was found to express My10 but not HLA-DR antigen; experiments using complement-dependent cytotoxicity to eliminate DR positive cells confirmed this finding. Pretreatment of marrow cells with two chemotherapeutic agents, 5-fluorouracil and 4-hydroperoxycyclophosphamide facilitated detection of CFU-B1 derived colonies, while diminishing or totally inhibiting colony formation by other hematopoietic progenitor cells. CFU-B1-derived colony formation was dependent upon the addition of exogenous hematopoietic growth factors. Media conditioned either by the human bladder carcinoma cell line 5637 or lectin stimulated leukocytes, as well as recombinant granulocyte-macrophage colony stimulating factor, interleukin 3 or interleukin 1 alpha promoted blast cell colony formation. By contrast, neither recombinant erythropoietin, recombinant interleukin 4, purified macrophage colony stimulating factor or recombinant granulocyte colony-stimulating factor alone promoted blast cell colony formation.
J Brandt, N Baird, L Lu, E Srour, R Hoffman
Epidermal growth factor (EGF), an endogenous mitogenic peptide, has recently been shown to be a potent vasoconstrictor of vascular smooth muscle. In view of its potential role in proliferative and inflammatory renal glomerular diseases, we examined the effects of EGF both on cultured rat mesangial cells and on in vivo glomerular hemodynamics. Mesangial cells possess specific, saturable EGF receptors of differing affinities, with Kd's of 0.1 and 1.7 nM, respectively. EGF produced a rapid increase in intracellular pH of 0.12 +/- 0.01 pH U, which was sodium dependent and amiloride inhibitable. The addition of EGF to mesangial cells cultured on either glass or dimethylpolysiloxane substratum induced reproducible cell contraction. Intrarenal EGF infusion did not affect systemic blood pressure or hematocrit but reversibly decreased GFR and renal blood flow from 4.19 +/- 0.33 to 3.33 +/- 0.26 and from 1.17 +/- 0.09 to 0.69 +/- 0.07 ml/min, respectively. Glomerular micropuncture confirmed decreases in single nephron plasma flow and in single nephron GFR (from 142 +/- 9 to 98 +/- 8 and from 51.6 +/- 11.7 to 28.5 +/- 3.5 nl/min, respectively) which were due to significant increases in both pre- and postglomerular arteriolar resistances (from 1.97 +/- 0.31 to 2.65 +/- 0.36 and from 1.19 +/- 0.11 to 2.00 +/- 0.15 10(10) dyn.s.cm-5 respectively) and to a significant decrease in the ultrafiltration coefficient, Kf, which fell from 0.100 +/- 0.019 to 0.031 +/- 0.007 nl/(s.mmHg). These studies demonstrate that mesangial cells possess specific receptors for EGF, and exposure of these cells to physiologic concentrations of EGF results in an in vitro functional response characterized by activation of Na+/H+ exchange and by resultant intracellular alkalinization, as well as by cell contraction. EGF administration in vivo significantly reduces the glomerular capillary ultrafiltration coefficient, Kf, which, in combination with EGF-induced constriction of both preglomerular and postglomerular arterioles, results in acute major reductions in the rates of glomerular filtration and perfusion.
R C Harris, R L Hoover, H R Jacobson, K F Badr
In this study we examined the leukocytic oxidant species that induce oxidant damage of DNA in whole cells. H2O2 added extracellularly in micromolar concentrations (10-100 microM) induced DNA strand breaks in various target cells. The sensitivity of a specific target cell was inversely correlated to its catalase content and the rate of removal of H2O2 by the target cell. Oxidant species produced by xanthine oxidase/purine or phorbol myristate acetate-stimulated monocytes induced DNA breakage of target cells in proportion to the amount of H2O2 generated. These DNA strand breaks were prevented by extracellular catalase, but not by superoxide dismutase. Cytotoxic doses of HOCl, added to target cells, did not induce DNA strand breakage, and myeloperoxidase added extracellularly in the presence of an H2O2-generating system, prevented the formation of DNA strand breaks in proportion to its H2O2 degrading capacity. The studies also indicated that H2O2 formed hydroxyl radical (.OH) intracellularly, which appeared to be the most likely free radical responsible for DNA damage: .OH was detected in cells exposed to H2O2; the DNA base, deoxyguanosine, was hydroxylated in cells exposed to H2O2; and intracellular iron was essential for induction of DNA strand breaks.
I Schraufstätter, P A Hyslop, J H Jackson, C G Cochrane
We studied Heinz body-containing erythrocytes with three different unstable hemoglobins: Nottingham, Brockton, and unclassified. We demonstrated two classes of membrane protein defects in unstable hemoglobin-containing cells (UH-RBCs), a defect of the spectrin-depleted inside-out vesicle (UH-IOV), and a defect of spectrin (UH-spectrin) itself. The composition of UH-IOVs is the same as control with respect to quantity of ankyrin and proportion inside-out. However, UH-IOVs bind even less spectrin than IOVs derived from sickle erythrocytes (SS-IOVs), suggesting a severe functional defect in the ankyrin of UH-RBCs (UH-ankyrin). Further evidence that UH-ankyrin is abnormal is demonstrated by the virtual absence of ankyrin in isotonic membrane shells of UH-RBCs (UH-shells), and abnormal mobility and decreased binding of the 72-kD (spectrin-binding) alpha-chymotryptic fragment of UH-ankyrin (UH-72-kD) to control spectrin. All UH-RBC membranes were spectrin-deficient (60% of control). In addition, spectrin isolated from UH-RBCs (UH-spectrin) was abnormal in two respects: (a) presence of a fast-moving band on nondenaturing polyacrylamide gels of both 0 degree C and 37 degrees C extracts, and (b) decreased binding to actin in the presence of protein 4.1. UH-spectrin did exhibit normal self-association, binding to IOVs and binding to actin in the absence of protein 4.1. This pattern of normal and abnormal spectrin functions has been described for spectrin subjected to mild diamide oxidation, suggesting the role of oxidation is the pathogenesis of membrane defect(s) of erythrocytes with abnormal hemoglobins.
O S Platt, J F Falcone
Gastric acid exerts a feedback inhibition on the secretion of gastrin from antral G cells. This study examines whether gastrin gene expression is also regulated by changes in gastric pH. Achlorhydria was induced in rats by the gastric H+/K+ ATPase inhibitor, omeprazole (100 mumol/kg). This resulted in fourfold increases in both serum gastrin (within 2 h) and gastrin mRNA levels (after 24 h). Antral somatostatin D cells probably act as chemoreceptors for gastric acid to mediate a paracrine inhibition on gastrin secretion from adjacent G cells. Omeprazole-induced achlorhydria reduced D-cell activity as shown by a threefold decrease in antral somatostatin mRNA levels that began after 24 h. Exogenous administration of the somatostatin analogue SMS 201-995 (10 micrograms/kg) prevented both the hypergastrinemia and the increase in gastrin mRNA levels caused by omeprazole-induced achlorhydria. Exogenous somatostatin, however, did not influence the decrease in antral somatostatin mRNA levels seen with achlorhydria. These data, therefore, support the hypothesis that antral D cells act as chemoreceptors for changes in gastric pH, and modulates somatostatin secretion and synthesis to mediate a paracrine inhibition on gastrin gene expression in adjacent G cells.
S J Brand, D Stone
The inner medullary collecting duct (IMCD) effects net sodium reabsorption under the control of volume regulatory hormones, including atrial natriuretic peptides (ANP). These studies examined the mechanisms of sodium transport and its regulation by ANP in fresh suspensions of IMCD cells. Sodium uptake was inhibited by amiloride but insensitive to furosemide, bu-metanide, and hydrochlorthiazide. These results are consistent with uptake mediated by a sodium channel or Na+/H+ exchange. To determine the role of sodium channels, cells were hyperpolarized by preincubation in high potassium medium followed by dilution into potassium-free medium. Membrane potential measurements using the cyanine dye, Di(S)-C3-5 verified a striking hyperpolarization of IMCD cells using this protocol. Hyperpolarization increased the apparent initial rate of sodium uptake fourfold. Amiloride and ANP inhibited potential-stimulated sodium uptake 73% and 65%, respectively; the two agents together were not additive. Addition of 5 mM sodium to hyperpolarized cells resulted in a significant amiloride-sensitive depolarization. Half-maximal inhibition of potential-driven sodium uptake occurred at 3 X 10(-7) M amiloride, and 5 X 10(-11) M ANP. We conclude that sodium enters IMCD cells via a conductive, amiloride-sensitive sodium channel, which is regulated by ANP. ANP inhibition of luminal sodium entry in the IMCD appears to contribute to the marked natriuretic effect of this hormone in vivo.
M L Zeidel, D Kikeri, P Silva, M Burrowes, B M Brenner
Angiotropin is a differentiation factor for microvascular endothelial cells isolated from serum-free cultures of lectin-activated, porcine monocytes. We used an ear lobe model in rabbits, single intradermal injection of angiotropin to induce phenotypical changes of the endothelial cells in capillaries and postcapillary venules, vascular engorgement, and subsequent angiogenesis in dose-dependent manner. The vascular changes are associated with epidermal and stromal cell proliferation. Angiogenesis and tissue proliferation occur in the absence of tissue necrosis and do not lead to scar formation. Angiotropin-induced angiogenesis is not inhibited by local dexamethasone although it involves a defined turnover of inflammatory cells. Proliferation is transient and regressive events follow. The overall tissue reaction resembles changes found in the undamaged skin margin of a primary healing wound during the inflammatory/proliferative phase. From these observations we conclude that angiotropin is an important secretory product of activated peripheral macrophages that triggers inflammatory and proliferative reactions in wound healing by activating microvascular endothelial cells.
M Höckel, W Jung, P Vaupel, H Rabes, C Khaledpour, J H Wissler
Brush-border vesicles purified from rabbit ileal villus cells were used to evaluate how Ca++/calmodulin (CaM) regulates the neutral linked NaCl absorptive process, part of which is a Na+/H+ exchanger. After freezing and thawing to allow incorporation of macromolecules into the vesicles, the effect of Ca++/CaM on brush-border Na+ uptake with an acid inside pH gradient, and on Na+/H+ exchange was determined. Freezing and thawing vesicles with 0.85 microM free Ca++ plus 5 microM exogenous CaM failed to alter Na+/H+ exchange as did the addition of exogenous ATP plus an ATP regenerating system, which was sufficient to elevate intravesicular ATP to 47 microM from a basal level of 0.4 microM. However, the combination of Ca++/CaM plus ATP inhibited Na+ uptake in the presence of an acid inside pH gradient and inhibited Na+/H+ exchange, while Na+ uptake in the absence of a pH gradient was not altered. This effect required a hydrolyzable form of ATP, and did not occur when the nonhydrolyzable ATP analogue, AMP-PNP, replaced ATP. Under the identical intravesicular conditions used for the transport studies, Ca++ (0.85 microM) plus exogenous CaM (5 microM), in the presence of magnesium plus ATP, increased phosphorylation of five brush-border peptides. These data are consistent with Ca++/CaM acting via phosphorylation to regulate the ileal brush-border Na+/H+ exchanger.
R P Rood, E Emmer, J Wesolek, J McCullen, Z Husain, M E Cohen, R S Braithwaite, H Murer, G W Sharp, M Donowitz
The pathogenesis of ischemic renal tubular cell injury involves a complex interaction of different processes, including membrane phospholipid alterations and depletion of high-energy phosphate stores. To assess the role of membrane phospholipid changes due to activation of phospholipases in renal tubule cell injury, suspensions enriched in rabbit renal proximal tubule segments were incubated with exogenous phospholipase A2 (PLA2). Exogenous PLA2 did not produce any significant change in various metabolic parameters reflective of cell injury in control nonhypoxic preparations despite a significant decrease in phosphatidylethanolamine (PE) and moderate increases in lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). In contrast, exogenous PLA2 treatment of hypoxic tubules resulted in a severe degree of cell injury, as demonstrated by marked declines in tubule K+ and ATP contents and significant decreases in tubule uncoupled respiratory rates, and was associated with significant phospholipid alterations, including marked declines in phosphatidylcholine (PC) and PE and significant rises in LPC, LPE, and free fatty acids (FFA). The injurious metabolic effects of exogenous PLA2 on hypoxic tubules were reversed by addition of ATP-MgCl2 to the tubules. The protective effect of ATP-MgCl2 was associated with increases in tubule PC and PE contents and declines in LPC, LPE, and FFA contents. These experiments thus indicate that an increase in exogenous PLA2 activity produces renal proximal tubule cell injury when cell ATP levels decline, at which point phospholipid resynthesis cannot keep pace with phospholipid degradation with resulting depletion of phospholipids and accumulation of lipid by-products. High-energy phosphate store depletion appears to be an important condition for exogenous PLA2 activity to induce renal tubule cell injury.
V D Nguyen, D A Cieslinski, H D Humes
In obese women (n = 16) at their weight, fasting adipose tissue lipoprotein lipase (LPL) activity, obtained by elution with serum and heparin at 4 degrees and 37 degrees C, was inversely correlated to plasma estradiol levels (r = -0.724; P = 0.002) and (r = -0.641; P = 0.010), respectively. Furthermore, fasting postheparin plasma LPL activity during a heparin infusion, showed an even stronger inverse correlation to plasma estradiol when measured at 60 min (r = -0.815; P less than 0.001). None of the above parameters was correlated to the body mass index. Postprandial LPL activity in postheparin plasma, measured 10 min after a heparin injection, showed a strong positive correlation with plasma free testosterone (r = 0.780; P = 0.001). Neither of these parameters was correlated with the body mass index. The origin of this LPL activity is presently unknown but could conceivably represent a pool of LPL from skeletal muscle. Since it has been shown convincingly that estrogen decreases adipose tissue LPL activity in the rat, the present studies strongly suggest that estradiol is a major negative regulator of fasting adipose tissue LPL activity in women.
P H Iverius, J D Brunzell
Radiolabeled UDPGlc incubated with rough endoplasmic reticulum (RER)-derived microsomes from rat liver became associated with the vesicles. This microsomal uptake of nucleotide sugar was time and temperature dependent. Analysis of the molecular species containing radiolabel revealed that initial uptake represented entry of predominantly intact UDPGlc in the microsomes. Conclusive evidence for proper translocation of UDPGlc across the microsomal membrane into the intravesicular space was obtained by demonstrating that UDPGlc was transported into an osmotically sensitive compartment. Microsomal uptake of UDPGlc exhibited features characteristic of carrier-mediated transport including saturation, specificity, and countertransport. Inhibition and trans-stimulation studies showed that other uridine-containing nucleotide sugars and 5'-UMP were substrates of the postulated microsomal carrier system for UDPGlc, while cytosine- or guanosine-containing nucleotides and non-5'-uridine monophosphates were, at best, very poor substrates. UDPGlc translocation activities were lower in smooth microsomal fractions than in the RER-derived vesicles, indicating that contamination with Golgi membranes could not be responsible for microsomal transport of UDPGlc. Our findings suggest that rat liver endoplasmic reticulum possesses a carrier system mediating proper translocation of UDPGlc and 5'-uridine-substituted structural analogues across the membrane.
F Vanstapel, N Blanckaert
Aberrant expression of MHC class II molecules on endocrine cells has been proposed to induce autoimmune reactions in thyroid and endocrine pancreas. The present study examines whether MHC class II positive insulin-containing islet cells occur at the onset of diabetes in rats, in analogy to the findings in man. At the onset of diabetes, both streptozotocin-treated and diabetes-prone BB rats exhibited numerous class II positive islet cells that presented ultrastructural features of monocytes and were surrounded by class II negative islet B cells. These class II positive cells were characterized by vacuoles that contained insulin immunoreactive granules and disrupted membranes. Similar cells also appeared positive for the monocyte marker OX-42. The presence of class II positive monocytes with insulin-containing vacuoles may indicate a removal of damage B cells by infiltrating leukocytes. A similar electron microscopical study in man will be necessary to distinguish the putative endocrine pancreatic B cells with aberrant class II expression from infiltrating nonendocrine class II positive cells with insulin-containing phagosomes.
P A In't Veld, D G Pipeleers
The transgenic mouse system provides an in vivo setting in which to examine the effects on mouse red cells of hemoglobin genes that have been genetically introduced into the animals' genome. In this report, we have analyzed the cellular properties of red cells from homozygous beta-thalassemic mice (Hbbth-1/Hbbth-1), homozygous beta-thalassemic transgenic mice containing a human beta-sickle (beta(s)) gene (Hbb(th-1)/Hbb(th-1) + beta(s)), and normal animals. The presence of human beta(s)-globin chains in red cells from the Hbbth-1/Hbb(th-1) + beta(s) transgenic animals was noted to have a significant effect on cellular deformability and density distribution, as well as on the degree of anemia in these animals. We conclude from these studies that red cell deformability and density distribution is a sensitive means for assessing at the cellular level the effects of globin genes genetically introduced into whole organisms. In addition, these studies suggest that small decreases in the amount of excess alpha-globin chains can significantly ameliorate the severity of anemia in the beta-thalassemic mouse.
E M Rubin, Y W Kan, N Mohandas
Platelet-derived growth factor (PDGF) mRNA, and mRNA for its receptor, have been localized to specific cell types within the human atherosclerotic plaque, using in situ hybridization. The predominant cell types found to express PDGF A and B chain mRNA are mesenchymal-appearing intimal cells and endothelial cells, respectively, with little or no expression detected in macrophages. The distribution of PDGF receptor mRNA containing cells was also examined and found to be localized predominantly in the plaque intima.
J N Wilcox, K M Smith, L T Williams, S M Schwartz, D Gordon
Thyroid-stimulating hormone (TSH) and insulin-like growth factor-1 (IGF-1) synergistically stimulate DNA synthesis in thyroid cells. In this report, a novel mechanism for mediation of this synergistic interaction is described in rat thyroid (FRTL-5) cells. Because phorbol myristate acetate stimulates DNA synthesis, the effects of TSH, IGF-1 and insulin on FRTL-5 cell content of 1,2-diacylglycerol (1,2-DG), the endogenous activator of protein kinase C, were measured. After 6 d, TSH, IGF-1 and insulin caused increases in cellular 1,2-DG (mean +/- SE) to 180 +/- 10%, 540 +/- 50%, and 360 +/- 40% of control, respectively, whereas TSH plus IGF-1 and TSH plus insulin synergistically increased 1,2-DG to 1,890 +/- 310% and 1,690 +/- 230%, respectively. In the absence of insulin, the effect of TSH to elevate 1,2-DG exhibited an EC50 of approximately 2,000 microU/ml. The synergistic interaction of insulin and TSH was found to increase the potency of TSH by 300-fold (EC50 was approximately 7 microU/ml) in addition to increasing the efficacy of TSH. The effect of TSH appeared to be mediated by TSH-stimulated increases in cyclic AMP (cAMP). Forskolin and 8-bromo-cAMP, like TSH, caused modest increases in 1,2-DG and DNA synthesis, whereas forskolin plus insulin and 8-bromo-cAMP plus insulin markedly elevated 1,2-DG content and stimulated DNA synthesis. Under all conditions, increases in 1,2-DG content correlated with stimulation of DNA synthesis. These findings suggest that the synergistic stimulation of DNA synthesis in thyroid cells by TSH, via cAMP, and IGF-1 is mediated by 1,2-DG. Moreover, they implicate a novel interaction between the lipid and adenylyl cyclase signaling systems for the regulation of cell proliferation.
L Brenner-Gati, K A Berg, M C Gershengorn