Alveolar macrophages from normal individuals and patients with interstitial lung diseases spontaneously expressed a 4.2-kilobase mRNA complementary to the c-sis gene, a proto-oncogene coding for one of the chains of platelet-derived growth factor (PDGF). Concomitantly, these cells released a mediator with the properties of PDGF, including: chemotactic factor for smooth muscle cells whose activity was resistant to heat and acid, but sensitive to reduction; mitogenic (competence) activity for fibroblasts; ability to compete with PDGF for its receptor; and precipitated by an anti-PDGF antibody. While blood monocytes did not contain c-sis mRNA transcripts, monocytes matured in vitro expressed c-sis, consistent with the concept that expression of c-sis occurs during the differentiation of monocytes into alveolar macrophages. Together with the known actions of PDGF, these observations suggest that the c-sis proto-oncogene and its PDGF product are part of the armamentarium available to the alveolar macrophages for normal lung defense and participation in lung inflammation.
J F Mornex, Y Martinet, K Yamauchi, P B Bitterman, G R Grotendorst, A Chytil-Weir, G R Martin, R G Crystal
Certain hormonal and nonhormonal binding systems such as the leukoagglutinin-lymphocyte model exhibit complex receptor-ligand interactions that result in nonlinear Scatchard plots. Such plots are interpreted as indicating either homogeneous negatively interacting binding sites or heterogeneous sites with different and fixed affinity. We assessed the validity of these interpretations in our system by conjugating the ligand to a photoactivated heterobifunctional agent and cross-linking the conjugate to a subset of receptors before studying the binding interactions of non-cross-linked sites. Conjugation did not qualitatively or quantitatively affect the binding properties of the ligand. Cross-linking was specific, efficient, and stable and had no effect on irrelevant surface receptors. Cross-linking of only 3% of the total receptors resulted in 50% decreased ligand binding to high affinity sites consistent with a calculated inactivation of 85% and 2% of high and low affinity sites, respectively. Such preferential inactivation of high affinity sites in an unequivocal demonstration of binding sites heterogeneity in this system and shows a clear rejection of the homogeneous cooperation model.
G B Faguet, D Beebe
Studies have been performed on the biochemical mechanism of platelet activation induced by the fibrinolytic protease plasmin. In washed human platelets, greater than or equal to 1.0 caseinolytic units (CU/ml plasmin induced aggregation. Platelet [14C]serotonin release was stimulated by 1.0 CU/ml plasmin to an extent comparable to that induced by 1.0 U/ml thrombin. A dose- and time-dependent phosphorylation of the platelet 47,000- and 20,000-kD proteins was noted in 32PO4-labeled platelets incubated with plasmin; phosphorylation was not affected by extracellular Ca2+, but was completely inhibited by an increase in platelet cyclic AMP. Phosphorylation of these platelet proteins suggested that plasmin may act on platelets by stimulating a rise in cytosolic calcium concentration ([Cai2+]) and activating inositol phospholipid-dependent phospholipase C and protein kinase C. Using both quin2 fluorescence and aequorin luminescence as indicators, plasmin was found to elevate platelet [Cai2+] in the presence or absence of extracellular Ca2+. Phospholipase C activation was shown by the generation of [3H]diglyceride in [3H]arachidonic acid-labeled platelets and [32P]phosphatidic acid in 32PO4 labeled platelets exposed to plasmin. Plasmin did not induce formation of thromboxane A2 (TXA2). Only small amounts of this eicosanoid were detected late in the time course after plasmin stimulation. Our results indicate that plasmin causes platelet aggregation and secretion associated with phosphorylation of the 47,000- and 20,000-kD proteins, Ca2+ mobilization, and phospholipase C and protein kinase C activation.
A I Schafer, A K Maas, J A Ware, P C Johnson, S E Rittenhouse, E W Salzman
Protein 4.1, a principal component of the erythrocyte membrane skeleton, is thought to be important in regulating membrane stability through its interaction with spectrin and actin. A key role for protein 4.1 has been indicated in studies in which deficiency of this protein was shown to result in marked instability of the membrane. In order to obtain direct evidence for the functional role of protein 4.1, we reconstituted protein 4.1-deficient membranes with purified protein 4.1 and showed restoration of membrane stability. Erythrocyte membranes totally and partially deficient in protein 4.1 were reconstituted by exchange hemolysis with various concentrations of purified protein 4.1, and their stability measured using an ektacytometer. Native erythrocyte membranes totally deficient in protein 4.1 were markedly unstable, while those partially deficient had intermediate reductions in membrane stability. Reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability. Near-normal membrane stability could be restored to both totally and partially protein 4.1-deficient membranes. In contrast, the addition of protein 4.1 to resealed membranes did not improve membrane stability. This implies that the added protein 4.1 must have access to the cell interior in order to affect membrane stability. Furthermore, in control experiments, the addition of protein 4.1 to normal membranes did not increase their stability. Also, the addition of purified spectrin and human serum albumin during resealing did not improve stability of protein 4.1-deficient membranes. These results provide direct evidence for the crucial role of protein 4.1 in regulating erythrocyte membrane stability.
Y Takakuwa, G Tchernia, M Rossi, M Benabadji, N Mohandas
The regional cerebral metabolic rate for glucose was measured in normal and portacaval shunted rats and the effects of unilateral carotid infusions of "threshold" amounts of ammonia were assessed. 8 wk after shunting the glucose metabolic rate was increased in all 20 brain regions sampled. Effects on subcortical and phylogenetically older regions of the brain were most pronounced with a 74% increase observed in the reticular formation at the collicular level. Increases in the cerebral cortex ranged from 12 to 18%. Unilateral infusions of ammonia did not affect behavior but altered the electroencephalogram and selectively increased the glucose metabolic rate in the thalamus, hypothalamus, and substantia nigra in half of the animals, a pattern similar to that seen after a portacaval shunt, suggesting hyperammonemia as the cause of postshunt increases in glucose metabolism. Visual inspection of autoradiograms, computed correlation coefficients relating interregional metabolism, and principal component analysis suggest that normal cerebral metabolic and functional interrelationships are altered by shunting. Ammonia stimulation of the hypothalamic satiety centers may suppress appetite and lead to cachexia. Reductions in the ammonia detoxification capacity of skeletal muscle may increase the probability of developing future episodes of hyperammonemia, perpetuating the process. Direct effects of ammonia on specific brain centers such as the dorsomedial hypothalamus and reticular activating system may combine with global disruptions of cerebral metabolic-functional relationships to produce the protean manifestations of portal-systemic encephalopathy.
A H Lockwood, M D Ginsberg, H M Rhoades, M T Gutierrez
Clinical and biochemical characteristics of familial hypercholesterolemia (FH) heterozygotes possessing an abnormally high molecular weight low density lipoprotein receptor (HMWR) are reported. The disorder is transmitted as an autosomal dominant trait and is not distinguishable from classic heterozygous FH on clinical grounds. The average plasma low density lipoprotein (LDL) level is 360 mg/dl and tendon xanthomata and early coronary disease are present. LDL receptor activity is higher than expected. In skin fibroblast cultures two types of functional LDL receptors are present, one with a normal apparent native molecular weight of 140,000, and the other of 176,000. When immobilized on nitrocellulose paper both receptors bind LDL. Maximum 125I-LDL binding capacity of fibroblast monolayers is reduced only 20%, compared with 50% in typical heterozygous FH. Affinity for 125I-LDL is increased and a 38% reduction in the Michaelis constant for LDL is observed. When autologous 125I-LDL was injected intravenously, the fractional catabolic rate of LDL was 205% and the LDL apoprotein B production rate was 328% of that found in a typical heterozygous FH subject. Thus, both in vitro and in vivo testing indicated only a modest deficiency of LDL receptor activity. Kindred members possessing the HMWR had an associated abnormality of cholesterol biosynthesis. Cholesterol balance studies in three individuals with the HMWR trait demonstrated elevated cholesterol biosynthesis of two to three times the mean of normal subjects. These findings suggest that increased LDL production and increased cholesterol production may assume a significant role in the pathologic manifestations of heterozygous FH. Functional abnormalities in LDL receptor activity as measured in fibroblast culture may be relatively small.
R A Levy, R E Ostlund Jr, C F Semenkovich, J L Witztum
J F Gusella
Epidemiologic studies suggest that women who smoke have lower endogenous estrogen than nonsmokers. To explore the possible link between cigarette smoking and decreased endogenous estrogens, we have examined the effects of constituents of tobacco on estrogen production in human choriocarcinoma cells and term placental microsomes. In choriocarcinoma cell cultures, nicotine, cotinine (a major metabolite of nicotine), and anabasine (a minor component of cigarette tobacco) all inhibited androstenedione conversion to estrogen in a dose-dependent fashion. Removal of nicotine, cotinine, and anabasine from the culture medium resulted in the complete reversal of the inhibition of aromatase. In the choriocarcinoma cell cultures, a supraphysiologic concentration of androstenedione (73 microM) in the culture medium blocked the inhibition of aromatase caused by nicotine, cotinine, and anabasine. In preparations of term placental microsomes, nicotine, cotinine, and anabasine inhibited the conversion of testosterone to estrogen. Kinetic analysis demonstrated the inhibition to be competitive with respect to the substrate. These findings suggest that some nicotinic alkaloids directly inhibit aromatase. This mechanism may explain, in part, the decreased estrogen observed in women who smoke.
R L Barbieri, J Gochberg, K J Ryan
Complementary DNA coding for human monocyte interleukin 1 (IL-1), pI 7 form, was expressed in Escherichia coli. During purification, IL-1 activity on murine T cells was associated with the recombinant protein. Homogeneous human recombinant IL-1 (hrIL-1) was tested in several assays to demonstrate the immunological and inflammatory properties attributed to this molecule. hrIL-1 induced proliferative responses in a cloned murine T cell in the presence of suboptimal concentrations of mitogen, whereas no effect was observed with hrIL-1 alone. At concentrations of 0.05 ng/ml, hrIL-1 doubled the response to mitogen (5 X 10(6) half maximal units/mg). Human peripheral blood T cells depleted of adherent cells underwent a blastogenic response and released interleukin 2 in the presence of hrIL-1 and mitogen. hrIL-1 was a potent inflammatory agent by its ability to induce human dermal fibroblast prostaglandin E2 production in vitro and to produce monophasic (endogenous pyrogen) fever when injected into rabbits or endotoxin-resistant mice. These studies establish that the dominant pI 7 form of recombinant human IL-1 possesses immunological and inflammatory properties and acts on the central nervous system to produce fever.
C A Dinarello, J G Cannon, J W Mier, H A Bernheim, G LoPreste, D L Lynn, R N Love, A C Webb, P E Auron, R C Reuben
The c-fms gene product is related, and possibly identical, to the receptor for the mononuclear phagocyte colony stimulating factor, CSF-1. Using antisera to a recombinant v-fms--coded polypeptide, glycoproteins encoded by the human c-fms locus were detected in mononuclear cells from normal peripheral blood and in promyelocytic HL-60 cells 24 h after induction of monocytic differentiation with phorbol ester. The 150-kD human c-fms--coded glycoprotein was expressed at the cell surface, was active as a tyrosine-specific protein kinase in vitro, and shared primary structural features with the product of the feline retroviral v-fms oncogene. A biochemically indistinguishable glycoprotein was detected in human choriocarcinoma cell lines. Like peripheral blood mononuclear cells and phorbol ester-treated HL-60 cells, the choriocarcinoma cells expressed high affinity binding sites for human CSF-1. In addition to serving as a lineage specific growth factor in hematopoiesis, CSF-1 may play a role in normal trophoblast development.
C W Rettenmier, R Sacca, W L Furman, M F Roussel, J T Holt, A W Nienhuis, E R Stanley, C J Sherr