Platelet consumption is a prominent feature of disseminated intravascular coagulation. We investigated whether monocyte procoagulant activity (PCA) might play a role in platelet consumption associated with gram-negative septicemia. Human mononuclear cells exposed in vitro to lipopolysaccharide demonstrated parallel dose-dependent increases in PCA and ability to induce platelet aggregation. Induction of platelet aggregation required the generation of thrombin dependent on coagulation Factors VII, X, and II, and calcium. This is consistent with monocyte tissue factor initiating thrombin generation. A specific monoclonal antimonocyte antibody was used to identify monocytes via indirect immunofluorescence, and demonstrated that all monocytes were included in platelet aggregates. Mononuclear cells that did not express PCA did not induce platelet aggregation and monocytes were not surrounded by platelet clumps. These data suggest that monocytes induced to express tissue factor on their surface may be important mediators of endotoxin-induced platelet, as well as fibrinogen, consumption.
B S Schwartz, M C Monroe
Glomerular visceral epithelial cells are endowed with a sialic acid-rich surface coat (the "glomerular epithelial polyanion"), which in rat tissue contains the sialoprotein podocalyxin. We have identified a major membrane sialoprotein in human glomeruli that is similar to rat podocalyxin in its sialic acid-dependent binding of wheat germ agglutinin and in its localization on the surface of glomerular epithelial and endothelial cells, as shown by immunoelectron microscopy, using the monoclonal antibody PHM5. Differences in the sialoproteins of the two species are indicated by the discrepancy of their apparent molecular weights in sodium dodecyl sulfate gels, by the lack of cross reactivity of their specific antibodies, and by the lack of homology of their proteolytic peptide maps. It is therefore possible that the human glomerular sialoprotein and rat podocalyxin are evolutionarily distinct, but have similar functions.
D Kerjaschki, H Poczewski, G Dekan, R Horvat, E Balzar, N Kraft, R C Atkins
Bleomycin-induced lung disease is characterized by fibroblast proliferation and pulmonary fibrosis. In these studies, we demonstrate that fibroblasts are relatively resistant to clinically relevant amounts (below 10(-4) U/ml) of bleomycin and that these levels of bleomycin augment fibroblast proliferation in response to various fibroblast growth factors. These observations suggest that one mechanism by which bleomycin causes pulmonary fibrosis is augmentation of fibroblast proliferation.
P L Moseley, C Hemken, G W Hunninghake
In earlier model studies we demonstrated that artificially denatured hemoglobin binds to and clusters the protein, band 3, in the plane of the erythrocyte membrane. To determine whether denatured hemoglobin also clusters band 3 in vivo, we have compared the locations of denatured hemoglobin aggregates (Heinz bodies) with band 3 in sickle cells using phase contrast and immunofluorescence microscopy. We report that where Heinz bodies are found associated with the cytoplasmic surface of the membrane, clusters of band 3 are usually colocalized within the membrane. In contrast, normal erythrocyte membranes and regions of sickle cell membranes devoid of Heinz bodies display an uninterrupted staining of band 3. Similarly, ankyrin and glycophorin are periodically seen to aggregate at Heinz body sites, but the degree of colocalization is lower than for band 3. These data demonstrate that the binding of denatured hemoglobin to the membrane forces a redistribution of several major membrane components.
S M Waugh, B M Willardson, R Kannan, R J Labotka, P S Low
Parvalbumin (PA), one of the Ca2+-binding neuronal marker proteins, has been revealed to exist in the myelinated axons of the posterior root of the spinal cord and the peripheral nerve of rats. To investigate the role of PA for the genesis of diabetic neuropathy, the levels of PA in the sciatic nerve of normal and streptozotocin-induced diabetic rats were measured by radioimmunoassay (RIA) for PA. The immunohistochemical distribution of PA in the sciatic nerve from both groups was also studied. The RIA for PA revealed that the levels of PA in the sciatic nerve of diabetic rats were significantly decreased when compared with those of normal rats. However, the contents of S-100 protein, another type of Ca2+-binding glial marker protein, did not show any significant difference in the sciatic nerve from both groups. Immunohistochemically, the amount of PA containing myelinated axons of the diabetic nerve was markedly decreased when compared with nondiabetic subjects. These results suggest that the decreased level of PA in the peripheral nerve might contribute to the genesis of diabetic neuropathy.
T Endo, T Onaya
Sudden alteration in medium osmolality causes an osmometric change in proximal tubule cell size followed by restoration of cell volume toward normal in hypotonic but not in hypertonic medium. We determined the capability of isolated nonperfused proximal tubules to prevent a change in cell volume in anisotonic media. The external osmolality was gradually changed over a range from 110 to 480 mosM. At 1.5 mosM/min, cell volume remained constant between 167 +/- 9 and 361 +/- 7 mosM, a phenomenon termed isovolumetric regulation (IVR). Cells lost intracellular solutes in hypotonic and gained intracellular solutes in hypertonic media. Raffinose or choline chloride substitution showed that osmolality, rather than NaCl, signalled cell volume maintenance in hyperosmotic media. Cooling (7-10 degrees C) blocked IVR. IVR was maintained when osmolality was lowered at a rate of 27, but not at 42 mosM/min. IVR was not observed when the rate of osmolality increase exceeded 3 mosM/min. We conclude that proximal tubule cells sensitively regulate intracellular volume in an osmolality range of pathophysiologic interest by mechanisms dependent on the rate of net water movement across basolateral membranes and the absolute intracellular content of critical solutes.
J W Lohr, J J Grantham
To further our understanding of the molecular basis of DNA-autoantibody interactions, we have characterized the specificities of three IgG human myeloma proteins that bind DNA. We measured their binding to synthetic single- and double-stranded homopolynucleotides, random and alternating copolymers, oligonucleotides, and nucleotides or nucleosides conjugated to non-nucleic acid carriers. All three antibodies bound single-stranded nucleic acids, including both polyribonucleotides and polydeoxyribonucleotides. They varied in relative affinities for polynucleotides of varying base composition. Polymers containing the purines guanine or hypoxanthine and/or the pyrimidine thymine were most reactive with all three proteins. A myeloma protein that reacted with poly(G), poly(I), or poly(dT) also bound to the corresponding nucleosides or nucleotides conjugated to bovine serum albumin. None of the antibodies reacted with base-paired double-helical polynucleotides (double-stranded RNA, RNA-DNA hybrid or double-stranded DNA). The results indicate that base specificity is prominent in their reactions and that the accessible epitopes in single-stranded polynucleotides become masked upon base pairing in double-stranded helices. These findings suggest a model in which positions N1 and O6 of guanine and hypoxanthine and N3 and O4 of thymine interact with amino acids of the antibody-combining site.
M Zouali, B D Stollar
55 samples representing Hodgkin's and non-Hodgkin's lymphoma and other hyperplastic lesions of the lymph node were examined for rearrangement of the beta chain of the T cell antigen receptor (TcR) and Ig genes. In non-Hodgkin's lymphoma, rearrangement of TcR beta was found in all 14 T cell lymphomas and in two of the seven B cell lymphomas. Ig gene rearrangement was found in none of the 14 T cell lymphomas and in all seven B cell lymphomas. We also examined DNA from lymph nodes in which the lineage of the malignant cell is not clear. Rearrangement of TcR beta was found in all five lymphoepitheloid cell (Lennert's) lymphomas; four of eight Hodgkin's lymphomas; seven of ten Ki 1+ lymphomas; and all nine cases of angioimmunoblastic lymphoadenopathy (AIL). Ig gene rearrangement was found in none of five lymphoepitheloid cell lymphomas; none of eight Hodgkin's lymphomas; three of ten Ki 1+ lymphomas; and four of nine cases of AIL. These findings indicate that genetic studies of TcR and Ig genes are useful in identifying the presence of a clonal population in a lymph node, in determining the extent of the clonal population, and aid in identifying lineage. Of special interest was the finding that some cases of Hodgkin's lymphoma and AIL contain clonal rearrangement of the TcR genes, which suggests that in those cases the malignant cells may be of T cell origin.
H Griesser, A Feller, K Lennert, M Minden, T W Mak
Drug-induced triggered arrhythmias in heart muscle involve oscillations of membrane potential known as delayed or early afterdepolarizations (DADs or EADs). We examined the mechanism of DADs and EADs in ferret ventricular muscle. Membrane potential, tension and aequorin luminescence were measured during exposure to elevated [Ca2+]0, strophanthidin and/or isoproterenol (to induce DADs), or cesium chloride (to induce EADs). Ryanodine (10(-9)-10(-6) M), an inhibitor of Ca2+ release from the sarcoplasmic reticulum, rapidly suppressed DADs and triggered arrhythmias. When cytoplasmic Ca2+-buffering capacity was enhanced by loading cells with the Ca2+ chelators BAPTA or quin2, DADs were similarly inhibited, as were contractile force and aequorin luminescence. In contrast to DADs, EADs induced by Cs were not suppressed by ryanodine or by loading with intracellular Ca2+ chelators. The possibility that transsarcolemmal Ca2+ entry might produce EADs was evaluated with highly specific dihydropyridine Ca channel agonists and antagonists. Bay K8644 (100-300 nM) potentiated EADs, whereas nitrendipine (3-20 microM) abolished EADs. We conclude that DADs and DAD-related triggered arrhythmias are activated by an increase in intracellular free Ca2+ concentration, whereas EADs do not require elevated [Ca2+]i but rather arise as a direct consequence of Ca2+ entry through sarcolemmal slow Ca channels.
E Marban, S W Robinson, W G Wier
To test whether insulin secretion is self-regulatory, canine pancreata were isolated and perfused in vitro and were infused with 0.3, 0.6, or 1.2 mU/ml exogenous insulin. Basal and arginine-stimulated concentrations of C-peptide, glucagon, and somatostatin were measured. There were no significant differences between basal secretion nor the increment of arginine-stimulated secretion for each respective hormone at each exogenous insulin concentration. The second preparation studied was a vascularly isolated, yet innervated, in situ perfused pancreas. Exogenous insulin (1 mU/kg per min) was infused "systemically"; the pancreas received no insulin. Endogenous pancreatic insulin and C-peptide secretion was suppressed, while pancreatic glucagon secretion increased during systemic insulin infusion. No changes in pancreatic hormone secretion occurred after the sympathetic nerves were sectioned. These results suggest that exogenous insulin does not directly suppress the B cell, but can suppress insulin secretion through an indirect neurally mediated, insulin-dependent nerve mechanism.
J Stagner, E Samols, K Polonsky, W Pugh
Dietary protein restriction imposed before renal injury is established in the remnant kidney model in the rat reduces glomerular hypertension and hyperperfusion and renal injury. We demonstrate that dietary protein restriction (6% vs. 20%) imposed on a background of established renal injury in the remnant model leads to a greater preservation of renal function as measured by glomerular filtration rate and fractional clearances of albumin and IgG, despite the persistence of systemic hypertension. In similarly prepared rats, dietary protein restriction (6% vs. 20%) led to a lower glomerular capillary hydraulic pressure, a higher ultrafiltration coefficient, and similar single nephron filtration rates. In addition, less impairment of glomerular permselectivity was demonstrable after protein restriction. Our data demonstrate that the preservation of renal function with dietary protein restriction after established glomerular injury follows upon reduction of glomerular capillary hydraulic pressure, despite constancy of single nephron filtration rate and plasma flow and persistence of arterial hypertension.
K A Nath, S M Kren, T H Hostetter
A unique kindred with premature cardiovascular disease, tubo-eruptive xanthomas, and type III hyperlipoproteinemia (HLP) associated with familial apolipoprotein (apo) E deficiency was examined. Homozygotes (n = 4) had marked increases in cholesterol-rich very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), which could be effectively lowered with diet and medication (niacin, clofibrate). Homozygotes had only trace amounts of plasma apoE, and accumulations of apoB-48 and apoA-IV in VLDL, IDL, and low density lipoproteins. Radioiodinated VLDL apoB and apoE kinetic studies revealed that the homozygous proband had markedly retarded fractional catabolism of VLDL apoB-100, apoB-48 and plasma apoE, as well as an extremely low apoE synthesis rate as compared to normals. Obligate heterozygotes (n = 10) generally had normal plasma lipids and mean plasma apoE concentrations that were 42% of normal. The data indicate that homozygous familial apoE deficiency is a cause of type III HLP, is associated with markedly decreased apoE production, and that apoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.
E J Schaefer, R E Gregg, G Ghiselli, T M Forte, J M Ordovas, L A Zech, H B Brewer Jr
A purified recombinant human granulocyte-macrophage colony stimulating factor (rH GM-CSF) was a powerful stimulator of mature human eosinophils and neutrophils. The purified rH GM-CSF enhanced the cytotoxic activity of neutrophils and eosinophils against antibody-coated targets, stimulated phagocytosis of serum-opsonized yeast by both cell types in a dose-dependent manner, and stimulated neutrophil-mediated iodination in the presence of zymosan. In addition, rH GM-CSF enhanced N-formylmethionylleucylphenylalanine(FMLP)-stimulated degranulation of Cytochalasin B pretreated neutrophils and FMLP-stimulated superoxide production. In contrast, rH GM-CSF did not promote adherence of granulocytes to endothelial cells or plastic surfaces. rH GM-CSF selectively enhanced the surface expression of granulocyte functional antigens 1 and 2, and the Mo1 antigen. rH GM-CSF induced morphological changes and enhanced the survival of both neutrophils and eosinophils by 6 and 9 h, respectively. These experiments show that granulocyte-macrophage colony stimulating factor can selectively stimulate mature granulocyte function.
A F Lopez, D J Williamson, J R Gamble, C G Begley, J M Harlan, S J Klebanoff, A Waltersdorph, G Wong, S C Clark, M A Vadas
The T cell tropic retrovirus of macaque monkeys simian T lymphotropic virus type III (STLV-III) has morphologic, growth, and antigenic properties indicating that it is related to human T cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), the etiologic agent of the acquired immune deficiency syndrome (AIDS) of humans. STLV-III has recently been shown to induce an AIDS-like disease in macaque monkeys. In this study the humoral immune responses of six experimentally infected monkeys have been characterized to determine whether certain parameters of the antibody response to the virus might be predictive of the clinical outcome of this infection. Two distinct patterns of antibody responses were found. Four animals that died within 160 d of inoculation developed low titer anti-STLV-III antibody responses that recognized only the viral envelope protein, and progressive declines in total plasma IgG levels and absolute peripheral blood T4 lymphocyte numbers. The two animals that lived longer (one died at 352 d, the other remains alive at 430 d) developed high titer anti-STLV-III antibody responses that recognized both viral envelope and core proteins, increases in total plasma IgG, and a later decrease in number of peripheral blood T4 lymphocytes. Interestingly, the single animal that has remained clinically healthy after infection was the only one to develop detectable STLV-III neutralizing antibodies.
M Kannagi, M Kiyotaki, R C Desrosiers, K A Reimann, N W King, L M Waldron, N L Letvin
Excessive collagen deposition plays a critical role in the development of fibrosis, and early or active fibrosis may be more susceptible to therapeutic intervention than later stages of scarring. However, at present there is no simple method for assessing the collagen-synthesizing and secreting activity of fibroblasts in human tissues. Type I procollagen carboxyterminal domains are proteolytically removed during collagen secretion. Thus, antibodies to these domains should stain fibroblasts synthesizing type I collagen but not extracellular collagen fibrils which could mask the signal from the cells. We developed and characterized a monoclonal antibody (Anti-pC) specific for the carboxyterminal propeptide of type I procollagen. To determine the relationship between Anti-pC staining and collagen synthesis, we stained embryonic and adult chicken tendon. Embryonic chick tendon fibroblasts actively synthesizing type I collagen stained heavily with Anti-pC, while quiescent adult tendon fibroblasts did not stain with Anti-pC. Wounded adult tendons developed fibroblasts that stained with Anti-pC at the wound site. Thus, Anti-pC specifically visualized fibroblasts actively synthesizing collagen. Lung biopsies from patients with fibrotic lung disease were stained with Anti-pC. Interstitial and intraalveolar fibroblasts in biopsies from patients with active fibrosis stained intensely with Anti-pC, while normal human lung was unstained. The absence of staining in normal lung supports the hypothesis that fibrosis is associated with an altered collagen-synthesizing phenotype of tissue fibroblasts. Anti-pC may provide a useful clinical tool for assessing fibrogenic activity at sites of tissue injury.
J A McDonald, T J Broekelmann, M L Matheke, E Crouch, M Koo, C Kuhn 3rd
The transepithelial potential difference (PD) of cystic fibrosis (CF) airway epithelium is abnormally raised and the Cl- permeability is low. We studied the contribution of active Na+ absorption to the PD and attempted to increase the Cl- permeability of CF epithelia. Nasal epithelia from CF and control subjects were mounted in Ussing chambers and were short-circuited. The basal rate of Na+ absorption was raised in CF polyps compared with control tissues. Whereas beta agonists induced Cl- secretion in normal and atopic epithelia, beta agonists further increased the rate of Na+ absorption in CF epithelia without inducing Cl- secretion. This unusual effect is not due to an abnormal CF beta receptor because similar effects were induced by forskolin, and because cAMP production was similar in normal and CF epithelia. We conclude that CF airway epithelia absorb Na+ at an accelerated rate. The abnormal response to beta agonists may reflect a primary abnormality in a cAMP-modulated path, or a normal cAMP-modulated process in a Cl- impermeable epithelial cell.
R C Boucher, M J Stutts, M R Knowles, L Cantley, J T Gatzy
Stimuli of prostacyclin (PGI2) biosynthesis such as thrombin, bradykinin, histamine, and A23187 increase guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels in primary monolayer cultures of human umbilical vein endothelium by about twofold. This effect is dependent on the presence of extracellular Ca2+. Increases of about tenfold are observed when cyclic GMP phosphodiesterase activity is inhibited, which suggests that the observed increases in cyclic GMP involve the activation of guanylate cyclase. Activation of guanylate cyclase appears to involve an early event in the induction of PGI2 biosynthesis, as neither arachidonic acid nor its metabolites stimulate cyclic GMP accumulation. Although activators of guanylate cyclase such as atriopeptin III, sodium nitroprusside, and tert-butylhydroperoxide increase cyclic GMP levels by approximately 2-3-fold, they do not stimulate or modulate PGI2 production. We conclude that cyclic GMP does not play a primary role in mediating the induction or regulation of PGI2 biosynthesis in vascular endothelium.
A F Brotherton
The mechanism by which 2'-deoxyguanosine is toxic for lymphoid cells is relevant both to the severe cellular immune defect of inherited purine nucleoside phosphorylase (PNP) deficiency and to attempts to exploit PNP inhibitors therapeutically. We have studied the cell cycle and biochemical effects of 2'-deoxyguanosine in human lymphoblasts using the PNP inhibitor 8-aminoguanosine. We show that cytostatic 2'-deoxyguanosine concentrations cause G1-phase arrest in PNP-inhibited T lymphoblasts, regardless of their hypoxanthine guanine phosphoribosyltransferase status. This effect is identical to that produced by 2'-deoxyadenosine in adenosine deaminase-inhibited T cells. 2'-Deoxyguanosine elevates both the 2'-deoxyguanosine-5'-triphosphate (dGTP) and 2'-deoxyadenosine-5'-triphosphate (dATP) pools; subsequently pyrimidine deoxyribonucleotide pools are depleted. The time course of these biochemical changes indicates that the onset of G1-phase arrest is related to increase of the dATP rather than the dGTP pool. When dGTP elevation is dissociated from dATP elevation by coincubation with 2'-deoxycytidine, dGTP does not by itself interrupt transit from the G1 to the S phase. It is proposed that dATP can mediate both 2'-deoxyguanosine and 2'-deoxyadenosine toxicity in T lymphoblasts.
G J Mann, R M Fox
We have studied a woman with an apparent receptor-mediated resistance to cortisol on the basis of elevated 24-h mean plasma cortisol levels and increased urinary free cortisol. Plasma ACTH concentrations were normal but she was resistant to adrenal suppression by dexamethasone. No stigmata of Cushing's syndrome were seen. To study the proposed end-organ resistance to cortisol, we examined the glucocorticoid receptor (GR) in lymphocytes and in fibroblasts from this patient and from her son. Several molecular properties of the GR of lymphocytes from the patient were indistinguishable from that of normal control subjects. In thermolability assays, however, the patient's GR as well as her son's GR showed a striking heat sensitivity at 40 degrees and 45 degrees C when compared with GR from normal persons. In addition, data from the thermolability assays correlated well with the lack at 45 degrees C of dexamethasone-induced decrease in in vitro [3H]thymidine incorporation into lymphocytes derived from both patients.
M Brönnegård, S Werner, J A Gustafsson
With the exception of aldosterone, little is known about the hormonal regulation of distal nephron acidification. These experiments investigated the effects of prostaglandin E2, indomethacin, lysyl-bradykinin, 8-bromo-cyclic AMP, and forskolin on proton secretion in the major acidifying segment of the distal nephron, the medullary collecting duct from inner stripe of outer medulla. Using in vitro microperfusion and microcalorimetry, net bicarbonate reabsorption (proton secretion) was measured in rabbit medullary collecting ducts before, during, and after exposure to each test substance. PGE2 reduced proton secretion 12.2%, while the following substances stimulated proton secretion: indomethacin 14.2%; 8-bromo-cyclic AMP 34.5%; forskolin 39%. Lysyl-bradykinin was without effect. These studies demonstrate that distal nephron acidification, in addition to being stimulated by aldosterone, is significantly inhibited by the hormone PGE2. The stimulation of proton secretion by cAMP suggests that other hormones known to activate adenylate cyclase may also influence distal nephron acidification.
S Hays, J P Kokko, H R Jacobson
Previous data suggest that apolipoprotein (apo) CIII may inhibit both triglyceride hydrolysis by lipoprotein lipase (LPL) and apo E-mediated uptake of triglyceride-rich lipoproteins by the liver. We studied apo B metabolism in very low density (VLDL), intermediate density (IDL), and low density lipoproteins (LDL) in two sisters with apo CIII-apo AI deficiency. The subjects had reduced levels of VLDL triglyceride, normal LDL cholesterol, and near absence of high density lipoprotein (HDL) cholesterol. Compartmental analysis of the kinetics of apo B metabolism after injection of 125I-VLDL and 131I-LDL revealed fractional catabolic rates (FCR) for VLDL apo B that were six to seven times faster than normal. Simultaneous injection of [3H]glycerol demonstrated rapid catabolism of VLDL triglyceride. VLDL apo B was rapidly and efficiently converted to IDL and LDL. The FCR for LDL apo B was normal. In vitro experiments indicated that, although sera from the apo CIII-apo-AI deficient patients were able to normally activate purified LPL, increasing volumes of these sera did not result in the progressive inhibition of LPL activity demonstrable with normal sera. Addition of purified apo CIII to the deficient sera resulted in 20-50% reductions in maximal LPL activity compared with levels of activity attained with the same volumes of the native, deficient sera. These in vitro studies, together with the in vivo results, indicate that in normal subjects apo CIII can inhibit the catabolism of triglyceride-rich lipoproteins by lipoprotein lipase.
H N Ginsberg, N A Le, I J Goldberg, J C Gibson, A Rubinstein, P Wang-Iverson, R Norum, W V Brown
In vitro 1,25-dihydroxycholecalciferol (1,25(OH)2D3) decreased levels of preproparathyroid(preproPTH) hormone mRNA. We have now pursued these studies in vivo in the rat. Rats were administered vitamin D metabolites i.p. and the levels of preproPTH mRNA were determined in excised parathyroid-thyroid glands by blot hybridization. PreproPTH mRNA levels were less than 4% of basal at 48 h after 100 pmol 1,25(OH)2D3, with no increase in serum calcium. Gel blots showed that 1,25(OH)2D3 decreased preproPTH mRNA levels without any change in its size (833 basepair). Microdissected parathyroids after 1,25(OH)2D3 (100 pmol) showed mRNA levels for preproPTH were 40 +/- 8% of controls, but for beta-actin were 100% of controls. The relative potencies of vitamin D metabolites were: 1,25(OH)2D3 greater than 24,25(OH)2D3 greater than 25(OH)D3 greater than vitamin D3. In vitro nuclear transcription showed that 1,25(OH)2D3-treated (100 pmol) rats' PTH transcription was 10% of control, while beta-actin was 100%. These results show that 1,25(OH)2D3 regulates PTH gene transcription. PTH stimulates 1,25(OH)2D3 synthesis, which then inhibits PTH synthesis, thus completing an endocrinological feedback loop.
J Silver, T Naveh-Many, H Mayer, H J Schmelzer, M M Popovtzer
Human T lymphotropic virus-I (HTLV-I)-specific T cell lines were established and cloned. K5, an OKT8+ clone bearing multiple proviral integration sites, retained its HTLV-I-specific cytotoxicity and a normal dependence on interleukin 2 (IL-2), indicating that there is a finite number of transforming integration sites. R2, an OKT4+ HTLV-I-infected clone, initially mounted a proliferative response to HTLV-I; but then its IL-2-independent proliferation increased and the antigen specificity was lost. All HTLV-I-infected clones tested including K7, another OKT8+ transformed cytotoxic clone that had lost its reactivity, expressed comparable levels of T cell receptor beta-chain (TCR-beta) messenger (m)RNA. Although clones K5 and K7 had different functional properties, they had the same rearrangement of the TCR-beta gene, suggesting that they had the same clonal origin. These data indicate that HTLV-I-specific T cells retain their immune reactivity for variable periods of time following infection, but then usually lose it; in some cases, however, no alteration in function can be detected. The data also suggest that different consequences can take place in the same clone depending on the pattern of retroviral infection.
H Mitsuya, R F Jarrett, J Cossman, O J Cohen, C S Kao, H G Guo, M S Reitz, S Broder
Rat liver angiotensinogen cDNA (pRang 3) and mouse renin cDNA (pDD-1D2) were used to identify angiotensinogen and renin mRNA sequences in rat kidney cortex and medulla in rats on high and low salt diet. Angiotensinogen mRNA sequences were present in renal cortex and medulla in apparently equal proportions, whereas renin mRNA sequences were found primarily in renal cortex. Average relative signal of rat liver to whole kidney angiotensinogen mRNA was 100:3. Densitometric analysis of Northern blots demonstrated that renal cortical angiotensinogen mRNA concentrations increased 3.5-fold (P less than 0.001) and medulla, 1.5-fold (P less than 0.005) on low sodium compared with high sodium diet, whereas renal cortex renin mRNA levels increased 6.8-fold (P less than 0.0005). Dietary sodium did not significantly influence liver angiotensinogen mRNA levels. These findings provide evidence for sodium regulation of renal renin and angiotensinogen mRNA expressions, which supports potential existence of an intrarenally regulated RAS and suggest that different factors regulate renal and hepatic angiotensinogen.
J R Ingelfinger, R E Pratt, K Ellison, V J Dzau
Studies designed to characterize monocyte-derived recruiting activity (MRA) a monokine that stimulates endothelial cells to produce granulocyte macrophage-colony-stimulating activity (CSA) by endothelial cells, show that it is a thermolabile protein of from 12,000 to 24,000 D which, on chromatofocusing, shows three separate peaks of eluted activity from pH 7.5 to 5.0. Because these and many other properties of MRA are identical to those of interleukin 1 (IL-1), we tested the hypothesis that MRA and IL-1 are identical. We cultured vascular endothelial cells with various concentrations of purified native and recombinant IL-1 (pI 7 form), then tested the endothelial cell supernatants for GM-CSA. Purified native IL-1 and recombinant IL-1 stimulated endothelial cells to release CSA. The MRA of native IL-1, recombinant IL-1, and unfractionated monocyte conditioned medium was neutralized by a highly specific rabbit anti-human IL-1 antiserum. Chromatofocusing fractions that contained MRA contained immunoreactive IL-1 on immunoblotting and the bioactivity was neutralized completely by treatment with the antiserum. We conclude that IL-1 induces the release of CSA by vascular endothelial cells, that IL-1 is constitutively produced by monocytes in vitro, and that MRA and IL-1 are biologically, biophysically and, immunologically identical.
G C Bagby Jr, C A Dinarello, P Wallace, C Wagner, S Hefeneider, E McCall
The present studies examined the effect of acute in vitro acidosis on chloride reabsorption in the rabbit cortical thick ascending limb of Henle (cTALH). Four protocols were used: hypercapnic acidosis; "isocapnic" peritubular acidosis (bath bicarbonate reduction to 10 mM); isocapnic luminal acidosis (luminal bicarbonate reduction to 10 mM); isocapnic peritubular acidosis in the absence of luminal potassium. Transepithelial voltage (VT) decreased during hypercapnic acidosis and increased with recovery. Chloride reabsorption (pmol X mm-1 X min-1) decreased from 50.3 +/- 8.4 to 15.7 +/- 5.6, then increased to 45.6 +/- 11.1 with recovery. Likewise, VT was decreased reversibly during isocapnic peritubular acidosis, and chloride reabsorption decreased by 60%. Chloride reabsorption was greater (28.3 +/- 3.6) when tubules were perfused at normal luminal pH than at an acidotic luminal pH (11.4 +/- 4.5; P less than 0.05). Luminal potassium removal reduced chloride transport, and acidosis had no significant additional effect. Decreased chloride reabsorption in the cTALH during acidosis could contribute to the chloruresis associated with systemic acidosis. The symmetrical nature of this effect suggests that acidosis inhibits chloride reabsorption through an effect on cytosolic pH.
C S Wingo
Production of B cell growth factor (BCGF) from B-chronic lymphocytic leukemia (B-CLL) cells was demonstrated. Freshly isolated monoclonal B-CLL cells expressed surface mu, delta, B1, and Leu 1, but not Ba (an antigen expressed only on activated B cells). Upon stimulation with anti-IgM, they secreted BCGF, which could act on anti-IgM-stimulated autologous leukemic cells as well as anti-IgM-stimulated normal B cells. Cell lines established from these leukemic cells also constitutively secreted BCGF. The BCGF from B-CLL cells or established cell lines induced neither proliferation nor enhanced HLA-DR expression in resting B cells. These results show the presence of B cell-derived BCGF, which is distinct from BSF-1 and effective only on activated B cells. They also suggest that an autocrine mechanism may operate in the growth of B-CLL cells.
N Kawamura, A Muraguchi, A Hori, Y Horii, S Mutsuura, R R Hardy, H Kikutani, T Kishimoto
We have serially followed the function of intrahepatic canine islet autografts in 15 beagle dogs for up to 24 mo. Of these, only 20% sustained normal levels of fasting blood glucose for greater than 15 mo posttransplant. Failure of autograft function was accompanied by a preferential loss of well-granulated beta cells in the engrafted islets. The chronic stimulation of an initially marginal intrahepatic beta-cell mass ultimately resulted in metabolic deterioration and loss of beta cells below the minimal threshold required to maintain normal fasting blood glucose levels. It is possible that transplantation of a larger mass of islets would result in indefinite graft function in dogs. However, it remains to be demonstrated in larger mammals, including humans, whether an islet cell mass that is initially adequate in a heterotropic site such as the liver can remain functionally competent over a prolonged period.
R Alejandro, R G Cutfield, F L Shienvold, K S Polonsky, J Noel, L Olson, J Dillberger, J Miller, D H Mintz
The monokine, cachectin/tumor necrosis factor (TNF) differs from interleukin 1 (IL-1) in primary structure and in recognition by a distinct cellular receptor. It does, however, encode effector functions that are similar to those of IL-1 and characteristic of the host response to inflammation or tissue injury. Accordingly, we examined the possibility that recombinant-generated human TNF regulates hepatic acute-phase gene expression. In picomolar concentrations, TNF mediated reversible, dose- and time-dependent increases in biosynthesis of complement proteins factor B and C3, alpha 1 antichymotrypsin, and decreases in biosynthesis of albumin and transferrin in human hepatoma cell lines (Hep G2, Hep 3B). Biosynthesis of complement proteins C2 and C4, and alpha 1 proteinase inhibitor were not affected by TNF. TNF also increased factor B gene expression, but had no effect on C2 gene expression, in murine fibroblasts transfected with cosmid DNA bearing the human C2 and factor B genes. The effect of TNF on acute-phase protein expression (C3, factor B, albumin) was pre-translational as shown by changes in specific messenger RNA content.
D H Perlmutter, C A Dinarello, P I Punsal, H R Colten
To examine whether hyperinsulinemia associated with glucocorticoid treatment results solely from hypersecretion of insulin or also involves altered fractional hepatic extraction, oral glucose (1 g/kg body wt) was administered to dogs with or without dexamethasone treatment (2 mg/d for 2 d). Dexamethasone significantly increased basal glucose and insulin concentrations in the portal vein, hepatic vein, and femoral artery, reduced basal fractional hepatic extraction of insulin from 43 +/- 4% to 22 +/- 4%, and, after oral glucose, increased retention by the liver of net glucose released into the portal system from 27 +/- 4% to 53 +/- 13%. Intraportal insulin infusion (1 and 2 mU/kg per min) after 7 d of dexamethasone treatment (2 mg/d) caused less suppression of endogenous glucose production, and less exogenous glucose was required to maintain an euglycemic clamp than in control animals. Dexamethasone treatment is associated with: decreased basal fractional hepatic insulin extraction contributing to hyperinsulinemia; and less suppression of endogenous glucose production and increase in peripheral uptake in response to insulin, but no reduction in net hepatic glucose uptake after oral glucose.
Z Chap, R H Jones, J Chou, C J Hartley, M L Entman, J B Field
We investigated atrial natriuretic factor (ANF) in humans, measuring plasma immunoreactive (ir) ANF (in femtomoles per milliliter), and renal, hormonal, and hemodynamic responses to ANF infusion, in normal subjects (NL) and congestive heart failure patients (CHF). Plasma irANF was 11 +/- 0.9 fmol/ml in NL and 71 +/- 9.9 in CHF (P less than 0.01); the latter with twofold right ventricular increment (P less than 0.05). In NL, ANF infusion of 0.10 microgram/kg per min (40 pmol/kg per min) induced increases (P less than 0.05) of absolute (from 160 +/- 23 to 725 +/- 198 mueq/min) and fractional (1-4%) sodium excretion, urine flow rate (from 10 +/- 1.6 to 20 +/- 2.6 ml/min), osmolar (from 3.2 +/- 0.6 to 6.8 +/- 1.2 ml/min) and free water (from 6.8 +/- 1.6 to 13.6 +/- 1.6 ml/min) clearances, and filtration fraction (from 20 +/- 1 to 26 +/- 2%). Plasma renin and aldosterone decreased 33% and 40%, respectively (P less than 0.01). Systolic blood pressure fell (from 112 +/- 3 to 104 +/- 5 mmHg, P less than 0.05) in seated NL; but in supine NL, the only hemodynamic response was decreased pulmonary wedge pressure (from 11 +/- 1 to 7 +/- 1 mmHg, P less than 0.05). In CHF, ANF induced changes in aldosterone and pulmonary wedge pressure, cardiac index, and systemic vascular resistance (all P less than 0.05); however, responses of renin and renal excretion were attenuated. ANF infusion increased hematocrit and serum protein concentration by 5-7% in NL (P less than 0.05) but not in CHF.
R J Cody, S A Atlas, J H Laragh, S H Kubo, A B Covit, K S Ryman, A Shaknovich, K Pondolfino, M Clark, M J Camargo
The damage to pulmonary alveolar epithelial cells that occurs in many inflammatory conditions is thought to be caused in part by phagocytic neutrophils. To investigate this process, we exposed monolayers of purified rat alveolar epithelial cells to stimulated human neutrophils and measured cytotoxicity using a 51Cr-release assay. We found that stimulated neutrophils killed epithelial cells by a process that did not require neutrophil-generated reactive oxygen metabolites. Pretreatment of neutrophils with an antibody (anti-Mo1) that reduced neutrophil adherence to epithelial cells limited killing. Although a variety of serine protease inhibitors partially inhibited cytotoxicity, we found that neutrophil cytoplasts, neutrophil lysates, neutrophil-conditioned medium, purified azurophilic or specific granule contents, and purified human neutrophil elastase did not duplicate the injury. We conclude that stimulated neutrophils can kill alveolar epithelial cells in an oxygen metabolite-independent manner. Tight adherence of stimulated neutrophils to epithelial cell monolayers appears to promote epithelial cell killing.
R H Simon, P D DeHart, R F Todd 3rd
The involvement of the lymphocyte function-associated antigen-1 (LFA-1) membrane molecule in cytolytic T lymphocyte (CTL) interactions with lymphoid target cells was investigated using CTL clones derived from two patients with a heritable deficiency of LFA-1. LFA-1 surface expression on the CTL clones was 1% of the normal level of LFA-1, unchanged with prolonged culture, and identical on 14 different CTL clones. The function of the LFA-1 molecule was addressed using the LFA-1-deficient CTL clones and LFA-1-deficient lymphoid target cells. The lytic activity of the LFA-1-deficient CTL clones was 43% of control when tested against a target cell line expressing normal levels of LFA-1 and less than 10% of control when tested against an LFA-1-deficient target cell line. These results demonstrate a direct involvement of LFA-1 in CTL-mediated cytolysis and suggest a more general dependence on LFA-1 in lymphoid cell-cell interactions.
S J Mentzer, B E Bierer, D C Anderson, T A Springer, S J Burakoff
We surveyed 20 Philadelphia chromosome (Ph1) positive chronic myelogenous leukemia (CML) samples by Southern blot hybridization to determine the location of the breakpoints that occur on chromosomes 9 and 22 in the Ph1 translocation. Only 3 of 20 samples exhibited breakpoints on chromosome 9 within 18 kilobases (kb) of the v-abl homologous sequences. Mapping of these three chromosome 9 breakpoints indicates that each is at a separate location within this 18-kb region, indicating that there are no breakpoint "hot spots" in this area. In contrast, all 20 CML samples exhibited breaks on chromosome 22 within a 5.0-kb Bgl II fragment that lies within the previously described breakpoint cluster region (bcr). Several patients with CML blast crisis exhibiting multiple Ph1 chromosomes/metaphase exhibited amplified and rearranged c-abl-related fragments. These additional Ph1 chromosomes in blast crisis cells do not arise from a second, independent 9:22 translocation but rather result from a duplication of the preexisting Ph1 chromosome.
S J Collins
The presence of apolipoprotein (apo) B in liver and intestine from a patient with abetalipoproteinemia was evaluated by immunohistochemistry with a polyclonal and six monoclonal antibodies to different apo B-48 and B-100 epitopes. In normal liver, apo B was present inside and outside hepatocytes. The patients liver exhibited staining in the cytoplasm with the polyclonal and three monoclonal antibodies. By immunoelectron-microscopy, apo B was found to be present in the smooth endoplasmatic reticulum and the Golgi complex. Normal intestinal epithelium was labeled with polyclonal and all monoclonal antibodies, including those specific for apo B-100. The patients epithelium stained with polyclonal and six monoclonals, and apo B was present in the Golgi complex. Thus, normal intestinal mucosa expressed apo B-48 and B-100 epitopes, which indicates apo B-100 synthesis in the gut. The synthesis of the apo B molecule in the patient seems to be retained in both liver and gut, which suggests a posttranslational defect.
R P Dullaart, B Speelberg, H J Schuurman, R W Milne, L M Havekes, Y L Marcel, H J Geuze, M M Hulshof, D W Erkelens
Specific humoral substances produced and secreted by human tumors that cause hypercalcemia have not been identified. Certain growth factors (such as epidermal growth factor, platelet-derived growth factor, and transforming growth factors-alpha and -beta) have been shown to stimulate the resorption of bone in organ culture by both prostaglandin-dependent and prostaglandin-independent pathways. In this report we demonstrate that epidermal growth factor and recombinant human transforming growth factor-alpha induce a significant rise in plasma calcium concentration when administered repeatedly to intact mice for periods ranging from 24 h to 16 d. The elevation of plasma calcium is not dependent on dietary calcium and is not invariably accompanied by an increase in systemic levels of the prostaglandin E2 metabolite 13,14-dihydro-15-keto-prostaglandin E2. The in vivo calcium-mobilizing activity of epidermal growth factor and transforming growth factor-alpha indicate that these or related growth factors need be considered as potential mediators of tumor-induced hypercalcemia.
A H Tashjian Jr, E F Voelkel, W Lloyd, R Derynck, M E Winkler, L Levine
The causes of primary moderate hypercholesterolemia are not understood, but some patients have reduced fractional clearance rates (FCRs) for low density lipoproteins (LDL). This could be due to either decreased activity of LDL receptors or to a defect in structure (or composition) of LDL that reduces its affinity for receptors. To distinguish between these causes, simultaneous turnover rates of autologous and normal homologous LDL were determined in 15 patients with primary moderate hypercholesterolemia. In 10, turnover rates of both types of LDL were indistinguishable, which indicated that autologous LDL was cleared as efficiently as normal homologous LDL. In five others, FCRs for autologous LDL were significantly lower than for homologous LDL. Two of the latter five were treated with mevinolin, and although FCRs for both types of LDL rose during treatment, differences in FCRs between the two types of LDL persisted. In these five patients, autologous LDL appeared to be a poor ligand for LDL receptors.
G L Vega, S M Grundy
Graft vs. host disease (GVHD) remains one of the main problems associated with bone marrow transplantation. The current studies were undertaken to determine whether treatment of the donor inoculum with the anticytotoxic cell compound L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) would alter the development of GVHD in a murine model. Irradiated recipient mice transplanted with a mixture of control bone marrow and spleen cells from naive semiallogeneic donors died rapidly from GVHD, whereas the recipients of cells incubated with 250 microM Leu-Leu-OMe all survived. In addition, Leu-Leu-OMe treatment of cells obtained from donors immunized against host alloantigens resulted in significantly prolonged survival. Phenotypic characterization of spleen cells from the various groups of mice that had received Leu-Leu-OMe-treated cells and survived consistently revealed the donor phenotype. Treatment of marrow cells with 250 microM Leu-Leu-OMe appeared to have no adverse effects on stem cell function. Erythropoiesis was undiminished, as assayed by splenic 5-iodo-2'-deoxyuridine-125I uptake. Moreover, granulocytic and megakaryocytic regeneration were histologically equivalent in the spleens of recipients of control or Leu-Leu-OMe-treated cells. Treatment of the donor inoculum with Leu-Leu-OMe thus prevents GVHD in this murine strain combination with no apparent stem cell toxicity.
M Charley, D L Thiele, M Bennett, P E Lipsky