The turnover of apolipoprotein B (apo B) in very low density, intermediate density, and low density lipoproteins (VLDL, IDL, and LDL) and in the light and heavy fractions of LDL was determined in seven patients with hyperapobetalipoproteinemia (hyperapo B), six normolipidemic subjects, and five patients with heterozygous familial hypercholesterolemia (FH). After receiving an injection of 125I-VLDL, hyperapo B patients were found to have a higher rate of synthesis of VLDL-apo B than controls (40.1 vs. 21.5 mg/kg per d, P less than 0.05) but a reduced fractional catabolic rate (FCR) (0.230 vs. 0.366/h, P less than 0.01). After receiving an injection of 131I-LDL, hyperapo B patients had higher rates of LDL-apo B synthesis than controls (23.1 vs. 13.0 mg/kg per d, P less than 0.001), as did FH patients (22.7 mg/kg per d). The FCR of LDL was similar in hyperapo B patients and controls (0.386 vs. 0.366/d) but was markedly decreased in FH patients (0.192/d). Most subjects exhibited precursor-product relationships between VLDL and IDL, and all did between IDL and light LDL; an analogous relationship between light and heavy LDL was evident in most hyperapo B patients and controls but not in FH patients. Simultaneous injection of differentially labeled LDL fractions and deconvolution analysis showed increased light LDL synthesis with normal conversion into heavy LDL in hyperapo B, whereas in FH conversion of light LDL was reduced and there was independent synthesis of heavy LDL. These data show that the increased concentration of LDL-apo B in hyperapo B is solely due to increased LDL synthesis, which is secondary to increased VLDL synthesis; in contrast, in FH there is both an increase in synthesis of LDL (which is partly VLDL-independent) and reduced catabolism.
B Teng, A D Sniderman, A K Soutar, G R Thompson
Fetal liver and thymus transplantation can be successfully employed for the treatment of severe combined immunodeficiency disease. In virtually all cases, donor and recipient cells are HLA mismatched. In a patient suffering from a severe combined immunodeficiency disease, full immunological reconstitution was obtained after fetal liver and thymus transplantation. HLA typing revealed that the patient's T cells were of donor origin, while the B cells and monocytes were of host origin. Despite this complete HLA mismatch, the patient was found to mount a subnormal to normal antibody response in vivo. This finding is in contrast with the concept that antigen recognition by T cells is major histocompatibility complex (MHC) restricted. To define the mechanism responsible for this in vivo antibody response, antibody production by peripheral blood mononuclear cells from the patient was tested in vitro after in vivo booster. The in vitro anti-tetanus toxoid antibody production was similar to that of the control group. In addition, specific proliferative responses to tetanus toxoid were obtained. Immunoglobulin allotype determination showed that antibodies were synthetized by host B cells. The results of the present study indicate that transplanted T lymphocytes and recipient cells cooperate despite complete HLA mismatch.
M G Roncarolo, J L Touraine, J Banchereau
A comparison of the receptor-mediated interaction of malondialdehyde-low density lipoprotein and maleyl-albumin has been examined in human monocytes during differentiation in vitro. The recognition of both ligands by the scavenger receptor of these cells has been confirmed. We now report that human monocytes express a second cellular surface receptor for maleyl-albumin that is distinct from the scavenger receptor. The activity of the maleyl-albumin receptor, determined by both binding and lysosomal hydrolytic assays, substantially exceeds that of the scavenger receptor in freshly isolated monocytes. A dramatic and rapid decline in the activity of the maleyl-albumin receptor occurs within 72 to 96 h during differentiation in vitro. At day 7, while only 5-10% of the original activity of the maleyl-albumin receptor remains, it is similar to that of the maximally expressed scavenger receptor. Both the binding and hydrolysis of ligand mediated by the maleyl-albumin receptor are specifically inhibited by alpha-casein and alkaline-treated albumin; neither of these proteins is recognized by the scavenger receptor. The occurrence of the exceptionally active maleyl-albumin receptor on freshly isolated human monocytes suggests that it participates in processes necessary to the function of the cells that diminish in importance after differentiation of the monocytes into macrophages in vitro. Furthermore, while maleyl-albumin is a useful adjunct to studies of cellular events mediated by the scavenger receptor, the presence of a second receptor for maleyl-albumin must be taken into account as a potential contributing and complicating event.
M E Haberland, R R Rasmussen, C L Olch, A M Fogelman
Splanchnic and peripheral exchange of glucose and gluconeogenic substrates was examined in 12 healthy subjects during 2 h of arm or leg exercise on a bicycle ergometer and during a 40-min postexercise recovery period. The work intensity corresponded to 30% of the maximal pulmonary oxygen uptake. The regional exchange of substrates was evaluated using catheter technique and indicator dilution methods for blood flow measurements. Our findings indicate that prolonged arm exercise as compared with exercise with the legs results in a greater increase in heart rate (25-40%) and a more marked reduction in splanchnic blood flow (10-30%) as well as higher arterial concentrations of lactate, free fatty acids, and catecholamines. The respiratory exchange ratio was consistently higher with arm exercise. In addition, arm exercise results in a greater fractional extraction and utilization of glucose by exercising muscle as well as a greater hepatic gluconeogenesis from lactate and glycerol. During recovery from prolonged arm exercise, leg muscle becomes an important site of lactate release to the splanchnic bed, despite a lack of net glucose uptake by the leg. Simultaneously, arm muscle shows an increase in glucose uptake in the absence of a net release of lactate. These coincident but discordant processes in the leg and arm during recovery suggest the occurrence of a redistribution of muscle glycogen from previously resting (leg) muscle to previously exercising (arm) muscle.
G Ahlborg, J Wahren, P Felig
Tissue fibrosis results, in part, from an interaction between growth regulatory molecules released by mononuclear phagocytes and fibroblasts. In the chronic interstitial lung disorders, alveolar macrophages, the mononuclear phagocytes of the lung, are known to spontaneously release two growth factors for fibroblasts, fibronectin and alveolar macrophage-derived growth factor (AMDGF) that together stimulate nonreplicating lung fibroblasts to divide. In addition to these two primary growth promoting signals, alveolar macrophages are able to release other mediators that may have a potential role in modulating lung fibroblast replication in response to these primary signals, including interferon gamma (IFN gamma), prostaglandin E2 (PGE2), and interleukin 1 (IL-1). To evaluate this possibility, we examined the effect of each of these other mediators on lung fibroblast replication in response to fibronectin and AMDGF in serum-free, defined medium. IFN gamma had no effect on fibroblast replication. In contrast, PGE2 resulted in a dose-dependent inhibition of fibroblast replication in response to fibronectin and AMDGF with 50% of the maximum inhibition observed at a PGE2 concentration of less than 10 ng/ml. IL-1, while not active as a primary growth promoting signal, at concentrations of 4-10 U/ml, augmented fibroblast replication in response to fibronectin and AMDGF by 10 to 15%. Temporally, the growth augmenting effect of IL-1 occurred early in the G1 phase of the cell cycle. These data indicate that lung fibroblast replication in response to two of the primary growth promoting signals spontaneously released by alveolar macrophages in the interstitial lung disorders, while uninfluenced by IFN gamma, can be inhibited by PGE2 and modestly augmented by IL-1. Understanding the relevant fibroblast growth modulatory signals within the alveolar microenvironment in the chronic interstitial disorders may lead to rational therapeutic strategies designed to interrupt the fibrotic process.
P B Bitterman, M D Wewers, S I Rennard, S Adelberg, R G Crystal
Studies were undertaken in Munich-Wistar rats to determine whether maintenance of chronic metabolic alkalosis (CMA) is associated with an increase in proximal HCO3- reabsorption, or whether a reduction in glomerular filtration rate (GFR) is required to sustain the elevated plasma HCO3- concentration. Superficial single nephron glomerular filtration rate (SNGFR), and absolute proximal HCO-3 (APRHCO3) and water (APRH2O) reabsorption were measured 20 +/- 3 d after the induction of CMA in eight rats and the results compared with seven age-matched control animals. Plasma [HCO3-] was 39.1 +/- 1.8 mM in CMA rats compared with 26.0 +/- 0.4 mM in controls (P less than 0.001). In the CMA rats, SNGFR was 44.8 +/- 1.1 vs. 38.2 +/- 2.1 nl/min in controls (P less than 0.025). As a result, the single nephron filtered load of HCO3- (FLHCO3) increased from 1,147 +/- 61 pmol/min in control to 2,040 +/- 108 pmol/min in CMA (P less than 0.001). APRHCO3 increased by greater than 65%, from 970 +/- 65 pmol/min in control to 1,624 +/- 86 pmol/min in CMA (P less than 0.001). APRH2O increased from 18.4 +/- 1.6 nl/min in control to 24.0 +/- 0.8 nl/min in CMA (P less than 0.005). Tubular hypertrophy resulted in an increase in the length of the proximal convoluted tubule from 5.6 +/- 0.2 to 6.5 +/- 0.2 mm (P less than 0.005). The pattern of HCO3- reabsorption along the length of the proximal convoluted tubule in CMA was indistinguishable from that found in normal rats in which FLHCO3 was varied acutely by altering SNGFR. The increase in tubular length accounted for only 30% of the increase in APRH2O and 15% of the increase in APRHCO3. We conclude that a sustained reduction in GFR is not required for maintenance of CMA in the rat. If GFR is chronically restored to normal levels, the alkalosis is maintained by an increase in APRHCO3. The increase in reabsorption is accounted for by tubular hypertrophy, a chronic adaptive response, and a load-dependent response that is indistinguishable from that seen in normal rats when FLHCO3 is increased acutely by increasing SNGFR.
D A Maddox, F J Gennari
Circulating insulin immunoreactivity (IRI) in type I diabetic patients (insulin-dependent diabetes mellitus [IDDM]) includes a covalent aggregate about twice the size of insulin. These studies were designed to determine the source and conditions promoting the accumulation of this material. Among 31 IDDMs, the aggregate made up 28 +/- 3.6% of the mean fasting plasma IRI. Five of these patients were restudied after 5 d of treatment with equidose intravenous insulin. The relative amount of the aggregate during subcutaneous treatment (40 +/- 8.0%) was indistinguishable (P greater than 0.7) from that at the termination of intravenous treatment (41 +/- 6.8%). To determine whether previous exposure to therapeutic insulin influenced the appearance and accumulation of the aggregate, we intravenously or subcutaneously infused insulin for 5 h in nine healthy volunteers (euglycemic clamp). At the termination of the high-dose intravenous infusion (10 mU X kg-1 X min-1), the concentration of the aggregate was 81 +/- 18 microU/ml, and it accounted for 2.9% of total IRI. At the conclusion of the other infusion protocols, the absolute amounts of aggregate were somewhat less, but they accounted for similar percentages. On polyacrylamide gel electrophoresis, the circulating aggregate was indistinguishable from a material of similar molecular weight contaminating commercial insulin. We conclude that the insulin aggregate found in the blood of IDDMs originates in commercial insulin. Its appearance is independent of the route of insulin administration. Prolonged and continuous use of insulin may increase its concentration but is not necessary for its appearance. The potential biologic and immunologic consequences of the aggregate are important matters that need to be addressed.
M Maislos, P M Mead, D H Gaynor, D C Robbins
Diabetes mellitus is associated with important changes in renal hemodynamics and transport function. Disturbances in solute transport have also been characterized in nonrenal tissues during hyperglycemia and insulinopenia. The purpose of this study was to determine if diabetes is associated with adaptive changes in function of the brush-border membrane of the proximal tubule. We studied Na+ and glucose transport in rat microvillus membrane vesicles isolated from the renal cortex of streptozotocin-induced and BB/W autoimmune diabetic rats. Untreated diabetes was associated with an increase in pH-stimulated total and amiloride-sensitive 22Na+ uptake into vesicles. Insulin treatment returned vesicle 22Na+ uptake to control levels. The increased Na+/H+ exchange was shown to be a result of increased net renal acid production rather than a specific response to insulinopenia because treatment with NaHCO3 also returned 22Na+ uptake to control levels. On the other hand, Na+-glucose cotransport, which was depressed in vesicles from untreated diabetics, returned to control levels with insulin but not NaHCO3 administration. This decreased Na+-glucose cotransport was not secondary to reduction in transport sites in untreated diabetics. These results show that in diabetes mellitus, increased Na+/H+ exchange activity is not the direct result of insulinopenia. However, the diabetic state appears to alter the functioning of the luminal Na+-glucose cotransporter.
R C Harris, B M Brenner, J L Seifter
Since mammalian atria were recently found to contain vasoactive and natriuretic peptides, we investigated the following in normal humans: plasma human atrial natriuretic peptide concentrations, effective renal plasma flow (ERPF), glomerular filtration rate (GFR), urinary water and electrolyte excretion, blood pressure (BP), and catecholamine, antidiuretic hormone (ADH), angiotensin II, and aldosterone levels before, during, and after intravenous administration of the newly synthetized alpha-human atrial natriuretic peptide (alpha hANP). In 10 subjects alpha hANP given as an initial bolus of 50 micrograms followed by a 45-min maintenance infusion at 6.25 micrograms/min increased plasma alpha hANP from 58 +/- 12 to 625 +/- 87 (mean +/- SEM) pg/ml; caused an acute fall in diastolic BP (-12%, P less than 0.001) and a hemoconcentration (hematocrit +7%, P less than 0.01) not fully explained by a negative body fluid balance; increased GFR (+15%, P less than 0.05) despite unchanged or decreased ERPF (filtration fraction +37%, P less than 0.001); augmented (P less than 0.05- less than 0.001) urinary chloride (+317%), sodium (+224%), calcium (+158%), magnesium (+110%), phosphate excretion (+88%), and free water clearance (from -0.76 to +2.23 ml/min, P less than 0.001) with only little change in potassium excretion; and increased plasma norepinephrine (P less than 0.001) while plasma and urinary epinephrine and dopamine, and plasma ADH, angiotensin II, and aldosterone levels were unchanged. The magnitude and pattern of electrolyte and water excretion during alpha hANP infusion could not be accounted for by increased GFR alone. Therefore, in normal man, endogenous alpha hANP seems to circulate in blood. alpha hANP can cause a BP reduction and hemoconcentration which occur, at least in part, independently of diuresis and are accompanied by sympathetic activation. An increase in GFR that occurs in the presence of unchanged or even decreased total renal blood flow is an important but not sole mechanism of natriuresis and diuresis induced by alpha hANP in man.
P Weidmann, L Hasler, M P Gnädinger, R E Lang, D E Uehlinger, S Shaw, W Rascher, F C Reubi
von Willebrand factor (vWF) is a large, multimeric glycoprotein that helps platelets adhere to vascular subendothelium. Although vWF binding to platelet receptors and connective tissue constituents is of fundamental importance in adhesion, there is little information regarding the nature of these vWF binding sites. In this paper, we have compared the structural requirements for vWF binding with platelet glycoprotein Ib (GpIb), heparin, and collagen and have shown that fragments derived from large vWF multimers retain biologic activity. We have shown that a 440,000-D subunit produced by disulfide reduction and alkylation of vWF polymer binds to platelet GpIb. When analyzed by polyacrylamide gel electrophoresis and Sepharose CL6B chromatography, the 440,000-D vWF oligomer is a dimer of the 220,000 subunit of fully reduced native vWF. This vWF dimer competes with 125I-vWF for binding to GpIb with an IC50 of 100 micrograms/ml (227 nM). The GpIb binding domain on vWF was further localized by digestion of native vWF polymers with Staphylococcal V8 protease. A 285,000-D fragment of vWF multimer was separated from heterogeneous 210,000-225,000-D fragments by its ability to bind to heparin. The 285,000-D fragment that bound to heparin Sepharose was composed of two disulfide-linked 175,000- and 115,000-D polypeptides. The heterogeneous fragments contained disulfide-linked 96,000, 66,000, and 53,000-D polypeptides when analyzed on polyacrylamide gel electrophoresis. The 285,000-D fragment competed with 125I-vWF for binding to GpIb with an IC50 of 22 micrograms/ml (77 nM), while the other fragments did not compete for binding. Neither the vWF dimer nor the proteolytic fragments competed with native 125I-vWF polymer for binding to collagen.
P Bockenstedt, J M Greenberg, R I Handin
Human platelets that were preincubated with porcine elastase aggregated spontaneously upon the addition of fibrinogen. Maximal aggregation to fibrinogen was observed with platelets pretreated with an elastase concentration of 111 micrograms/ml, and half-maximal aggregation occurred after treatment with 11 micrograms/ml elastase. Binding of radiolabeled fibrinogen to elastase-treated platelets was specific, saturable, and showed a single class of 48,400 +/- 9,697 fibrinogen-binding sites per platelet with a dissociation constant of 6.30 +/- 1.48 X 10(-7) M. ATP, apyrase, and the stimulators of platelet adenylate cyclase forskolin, prostaglandin E1, prostacyclin, and N6, 2'-O-dibutyryl cyclic AMP did not inhibit the fibrinogen-induced aggregation of elastase-treated platelets. EDTA completely blocked the initiation of aggregation and reversed the fibrinogen-induced aggregation of elastase-treated platelets. Monoclonal and polyclonal antibodies directed against glycoproteins (GP) IIb and IIIa completely blocked the fibrinogen-induced aggregation of elastase-treated platelets. Immunoprecipitates with these antibodies obtained from detergent extracts of surface-radiolabeled, intact, and elastase-treated platelets contained the glycoproteins IIb and IIIa. We conclude that surface proteolysis by low concentrations of elastase can expose fibrinogen-binding sites associated with GPIIb and GPIIIa on the platelet surface, resulting in spontaneous aggregation upon the addition of fibrinogen. These findings may be relevant to hemostatic changes observed in patients with increased levels of circulating elastase.
E Kornecki, Y H Ehrlich, D D De Mars, R H Lenox
Extracellular superoxide was detected in cultures of monkey and human arterial smooth muscle cells as indicated by superoxide dismutase inhibitable reduction of cytochrome c. Superoxide production by these cells in the presence of Fe or Cu resulted in modification of low density lipoprotein (LDL). The degree of LDL modification was directly proportional to the rate of superoxide production by cells. Superoxide dismutase (100 micrograms/ml), and the general free radical scavengers butylated hydroxytoluene and butylated hydroxyanisole (50 microM), inhibited Fe- and Cu-mediated modification of LDL by monkey smooth muscle cells, while catalase (100 micrograms/ml) and mannitol (25 mM) had no effect. The chelators desferrioxamine and diethylenetriamine pentaacetic acid completely inhibited Fe- and Cu-promoted modification of LDL, while EGTA had no inhibitory effect. EDTA stimulated Fe-promoted modification in the 1-100 microM range while inhibiting Cu-mediated modification of LDL. LDL modified by smooth muscle cells in the presence of 10 microM Fe or Cu stimulated [14C]oleate incorporation into cholesteryl ester by human macrophages and murine J774 cells to a degree comparable to that produced by acetylated LDL. LDL incubated with smooth muscle cells and metal ions in the presence of superoxide dismutase failed to enhance macrophage cholesteryl ester accumulation. Thus, arterial smooth muscle cells in culture generate superoxide and modify LDL by a superoxide-dependent, Fe or Cu catalyzed free radical process, resulting in enhanced uptake of the modified LDL by macrophages. Neither hydroxyl radicals nor H2O2 are likely to be involved. Superoxide-dependent lipid peroxidation may contribute to biological modification of LDL, resulting in foam cell formation and atherogenesis.
J W Heinecke, L Baker, H Rosen, A Chait
To explore possible mechanisms by which complement membrane attack complexes (MAC) that are deposited in the glomerular mesangium might be pathogenic, we stimulated rat glomerular mesangial cells grown in vitro with nascent MACs formed from the purified human complement components C5b6 and normal human serum and measured production of superoxide ion (O2-) and hydrogen peroxide (H2O2). Mesangial cells incubated with C5b6 + serum, which results in cell membrane interaction with the MAC, produce 0.9 +/- 0.15 nmol O2-/10(5) cells per 30 min, which was significantly greater than the amount produced by cells incubated with C5b6 alone, serum alone, or decayed MACs that can no longer interact with the cell membrane (0.3 +/- 0.2, 0.4 +/- 0.1, 0.3 +/- 0.2 nmol O2-/10(5) cells per 30 min, respectively; P less than 0.02). Production of O2- after stimulation with MACs increased during the first 20 min of incubation but then plateaued. Cells exposed to decayed MACs produced small amounts of O2-, which did not increase from 20 to 60 min. Production of H2O2 was also observed after stimulation with MACs, and continued to increase during 60 min of incubation (1.22 +/- 0.16 nmol H2O2/10(5) cells per 60 min), whereas H2O2 production could not be detected after exposure to decayed MACs. Cell viability was not adversely affected by exposure to nascent MACs as determined by trypan blue exclusion or chromium-51 release. These results demonstrate that glomerular mesangial cell membrane interaction with the MAC stimulates the production of the toxic oxygen metabolites O- and H2O2. Activation of the terminal complement pathway by mesangial immune deposits in vivo might lead to tissue injury by stimulation of local production of toxic oxygen-free radicals.
S Adler, P J Baker, R J Johnson, R F Ochi, P Pritzl, W G Couser
The relative roles of phospholipid fatty acyl chain length and phospholipid fatty acyl chain unsaturation in the determination of rat renal brush border membrane order were examined using multilamellar liposomes. Exposure of brush border membranes to sphingomyelinase resulted in a time- and concentration-dependent decrement in sphingomyelin content. Liposomes prepared from lipid extracts of these membranes were reconstituted to defined phosphatidylcholine (PC)/sphingomyelin (SPH) ratios with pure synthetic PCs of defined chain length and degrees of unsaturation. Mixed-acid PCs from bovine liver, egg, and the rat renal brush border membrane were also examined. The steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) at 37 degrees C was used to reflect acyl chain packing. The steady state anisotropy of DPH in liposomes isolated from the rat renal brush border membrane averaged 0.205 +/- 0.001, n = 8. When liposomes were reconstituted to PC/SPH ratios of 1.1, 1.6, and 2.4 with saturated PCs of acyl chain length 16 to 22, differences in anisotropy between groups were not observed. However, when PCs containing unsaturated or mixed-acid fatty acyl chains were introduced, anisotropy decreased in a concentration dependent fashion. These data suggest that phospholipid fatty acyl chain unsaturation, but not acyl chain length, has a powerful influence on renal brush border membrane order and the PC/SPH ratio is an important determinant of renal membrane order by virtue of the unsaturated fatty acids normally present with these phospholipids.
M K Hise, W W Mantulin, E J Weinman
Using a glucose-responsive beta cell line, we tested the hypothesis that the free cytosolic Ca2+ concentration ([Ca2+]i) is the primary signal that couples a stimulus to insulin secretion, and examined the involvement of the extracellular Ca2+ pool in this process. Glucose or depolarization of the beta cell with 40 mM K+ stimulated a monophasic release of insulin directly proportional to the extracellular Ca2+ concentration. 40 mM K+ increased 45Ca2+ uptake and increased [Ca2+]i, which was measured with quin 2, 4.7-fold, from 56 +/- 3 to 238 +/- 17 nM. With high glucose, 45Ca2+ uptake did not increase, and [Ca2+]i was unchanged or fell slightly. There was a striking correlation between inhibitory effects of verapamil, the Ca2+ channel blocker, on insulin secretion and the rise in [Ca2+]i evoked by K+. Higher concentrations of verapamil were required to inhibit glucose- than K+-stimulated insulin secretion (dose giving half-maximal effect of 1.4 X 10(-4) M vs. 6.0 X 10(-7) M). Incubation in Ca2+-free, 1 mM EGTA buffer for 30 min lowered [Ca2+]i to 14 +/- 2 nM, and inhibited acute insulin release to both secretagogues. If high glucose was present in the Ca2+-free period, reintroduction of 2.5 mM Ca2+ in high glucose restored insulin secretion only to the basal rate. However, if low glucose was present during the Ca2+-free period, high glucose and 2.5 mM Ca2+ triggered a full first-phase insulin response. These data suggest that high glucose generates a non-Ca2+ signal that turns over rapidly and provide direct evidence that K+ triggers insulin release by drawing extracellular Ca2+ into the beta cell through verapamil-sensitive Ca2+ channels. However, an increase [Ca2+]i is not the primary signal that evokes glucose-stimulated insulin release in this beta cell line.
A E Boyd 3rd, R S Hill, J M Oberwetter, M Berg
The reducing equivalents used by the human neutrophil respiratory burst oxidase are derived from NADPH generated by the hexose monophosphate shunt. The CO2 generated by the HMP shunt is spontaneously hydrated and the protons (H+) are secreted upon the dissociation of carbonic acid. The mechanism and significance of H+ secretion by the resting and stimulated neutrophil was investigated. A basal rate of H+ secretion by resting neutrophils observed in a choline buffer was augmented with the addition of sodium (Na+) (Km for Na+ was 3.22 +/- 0.32 mM). Amiloride, a Na+/H+ antiporter inhibitor, reduced H+ secretion in Na+-containing buffers with a Ki = 1.02 microM. This Na+/H+ exchange mechanism was also operative in cells stimulated with a variety of agonists, and an increased H+ flux, relative to resting cells, was observed at higher Na+ concentrations. Cytoplasts incorporating acridine orange were also used to assess Na+-H+ flux. Cytoplasts were used to avoid alteration of the fluorescent pH probe by HOCl formed in intact neutrophils. Alkalinization of the cytoplasm was dependent on extracellular Na+ in concentrations similar to that found to augment H+ secretion in intact cells. Also, amiloride competitively inhibited H+ secretion by the cytoplasts. Both superoxide (O2-) production and lysozyme release in cells stimulated with opsonized zymosan or concanavalin A was significantly inhibited in the absence of Na+, restored to normal with the addition of Na+ in low concentrations, and inhibited again in the presence of amiloride. A Na+/H+ antiporter similar to that found in other cell types is present in the human neutrophil and appears linked to activation of the respiratory burst and degranulation.
J Wright, J H Schwartz, R Olson, J M Kosowsky, A I Tauber
Cigarette smoking produces oxidant-mediated changes in the lung important to the pathogenesis of emphysema. Since vitamin E can neutralize reactive oxygen species and prevent peroxidation of unsaturated lipids, it may constitute an important component of the lung's defense against oxidant injury. To better characterize the antioxidant protective role of vitamin E, young asymptomatic smokers and nonsmokers were evaluated by bronchoalveolar lavage before and immediately after a 3-wk course of oral vitamin E (2,400 IU/d). Smoker alveolar fluid at baseline was relatively deficient in vitamin E compared with nonsmoker fluid (3.1 +/- 0.7 ng/ml vs. 20.7 +/- 2.4 ng/ml, P less than 0.005). Although smoker alveolar fluid vitamin E levels increased to 9.3 +/- 2.3 ng/ml after supplementation, the levels remained significantly lower than nonsmoker baseline levels (P less than 0.01). This deficiency was explained, in part, by the increased oxidative metabolism of vitamin E to the quinone form in the lungs of smokers compared with nonsmokers. Although the significance of a lower concentration of alveolar fluid vitamin E is unclear, it may compromise the antioxidant protection afforded by the alveolar fluid as it coats the lung's epithelial surface. The protective role of vitamin E was assessed by cytotoxicity experiments, which demonstrated that the killing of normal rat lung parenchymal cells by smoker alveolar macrophages was inversely related to the vitamin E content of the parenchymal cells. These findings suggest that vitamin E may be an important lower respiratory tract antioxidant, and that the deficiency seen in young smokers may predispose them to an enhanced oxidant attack on their lung parenchymal cells.
E R Pacht, H Kaseki, J R Mohammed, D G Cornwell, W B Davis
Micropuncture and/or morphologic studies were performed in intact Wistar-Kyoto rats (WKY) (group 0), intact spontaneously hypertensive rats (SHR) (groups 1 and 5), uninephrectomized (UNX) WKY (groups 2 and 6), and UNX SHR (groups 3 and 4, 7 and 8). UNX was performed when rats were 5 wk of age. Groups 0-4 were observed for 34 wk after which whole kidney clearance and morphologic studies were performed. Groups 5-8 underwent micropuncture study at 10 wk of age. Groups 4 and 8 were fed a diet containing 6% protein. All other rats ingested standard laboratory diet. 5 wk after UNX, normotensive group 6 had higher single nephron glomerular filtration rate (SNGFR) and initial glomerular plasma flow rate (QA) than intact, hypertensive group 5. Glomerular transcapillary hydraulic pressure difference (delta P) was similar in these two groups. Hypertensive group 7 exhibited less elevation in SNGFR and QA than group 6, but delta P was significantly increased. The presence of glomerular capillary hypertension in UNX SHR at 10 wk was associated with the development of significant proteinuria and an increased incidence of mesangial expansion and glomerular sclerosis at 7 mo (group 3) as compared with groups 0, 1, and 2. Protein restriction prevented the development of increased delta P in UNX SHR (group 8) and also conferred long-term protection from increased urinary protein excretion and glomerular injury (group 4). These studies suggest that glomerular capillary hypertension predisposes to glomerular injury in this model of hypertension with reduced renal mass.
L D Dworkin, H D Feiner
To test the hypothesis that a defibrillation shock is unsuccessful because it fails to annihilate activation fronts within a critical mass of myocardium, we recorded epicardial and transmural activation in 11 open-chest dogs during electrically induced ventricular fibrillation (VF). Shocks of 1-30 J were delivered through defibrillation electrodes on the left ventricular apex and right atrium. Simultaneous recordings were made from septal, intramural, and epicardial electrodes in various combinations. Immediately after all 104 unsuccessful and 116 successful defibrillation shocks, an isoelectric interval much longer than that observed during preshock VF occurred. During this time no epicardial, septal, or intramural activations were observed. This isoelectric window averaged 64 +/- 22 ms after unsuccessful defibrillation and 339 +/- 292 ms after successful defibrillation (P less than 0.02). After the isoelectric window of unsuccessful shocks, earliest activation was recorded from the base of the ventricles, which was the area farthest from the apical defibrillation electrode. Activation was synchronized for one or two cycles following unsuccessful shocks, after which VF regenerated. Thus, after both successful and unsuccessful defibrillation with epicardial shocks of greater than or equal to 1 J, an isoelectric window occurs during which no activation fronts are present; the postshock isoelectric window is shorter for unsuccessful than for successful defibrillation; unsuccessful shocks transiently synchronize activation before fibrillation regenerates; activation leading to the regeneration of VF after the isoelectric window for unsuccessful shocks originates in areas away from the defibrillation electrodes. The isoelectric window does not support the hypothesis that defibrillation fails solely because activation fronts are not halted within a critical mass of myocardium. Rather, unsuccessful epicardial shocks of greater than or equal to 1 J halt all activation fronts after which VF regenerates.
P S Chen, N Shibata, E G Dixon, P D Wolf, N D Danieley, M B Sweeney, W M Smith, R E Ideker
The exact pathway whereby the initial catabolism of the adenine nucleotides proceeds from AMP and the possibility of a recycling of adenosine were investigated in human erythrocytes. Adenine nucleotide catabolism, reflected by the production of hypoxanthine, is very slow under physiologic conditions and can be greatly increased by suppression of glucose or alkalinization of the medium. Experiments with inhibitors of adenosine deaminase and adenosine kinase demonstrated that under physiologic conditions the initial catabolism of AMP proceeds by way of a deamination of AMP, followed by dephosphorylation of inosine monophosphate, and that no recycling occurs between AMP and adenosine. Under glucose deprivation, approximately 75% of the 20-fold increase of the catabolism of the adenine nucleotides proceeded by way of a dephosphorylation of AMP followed by deamination of adenosine, and a small recycling of this nucleoside could be evidenced. Inhibition of adenosine transport showed that the dephosphorylation of AMP occurred intracellularly. When the incubation medium was alkalinized in the presence of glucose, the 15-fold increase in the conversion of AMP to hypoxanthine proceeded exclusively by way of AMP deaminase but a small recycling of adenosine could also be evidenced. The threefold elevation of intraerythrocytic inorganic phosphate (Pi) during glucose deprivation and its 50% decrease during alkalinization as well as experiments in which extracellular Pi was modified, indicate that the dephosphorylation of red blood cell AMP is mainly responsive to variations of AMP, whereas its deamination is more sensitive to Pi.
F Bontemps, G Van den Berghe, H G Hers
Dimethylhydrazine (DMH) is a potent procarcinogen with selectivity for the colon. To determine whether alterations in the lipid composition and fluidity of rat colonic brush border membranes existed before the development of DMH-induced colon cancer, rats were injected s.c. with this agent (20 mg/kg body weight per wk) or diluent for 5, 10, and 15 wk. Animals were killed at these time periods and brush border membranes were prepared from proximal and distal colonocytes of each group. The "static" and "dynamic" components of fluidity of each membrane were then assessed, by steady-state fluorescence polarization techniques using limiting hindered fluorescence anisotropy and order parameter values of the fluorophore 1,6 diphenyl-1,3,5-hexatriene (DPH) and fluorescence anisotropy values of DL-2-(9-anthroyl) stearic acid and DL-12-(9-anthroyl) stearic acid, respectively. Membrane lipids were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. Phospholipid methylation activity in these membranes was also measured using S-adenosyl-L-methionine as the methyl donor. The results of these studies demonstrate that: the lipid composition and both components of fluidity of proximal DMH-treated and control membranes and their liposomes were similar at all time periods examined; at 5, 10, and 15 wk the "dynamic component of fluidity" of distal DMH-treated membranes and their liposomes was found to be higher, similar, and lower, respectively, than their control counterparts; the "static component of fluidity" of distal DMH-treated membranes and their liposomes, however, was similar to control preparations at all three time periods; and alterations in the lipid composition and phospholipid methylation activities appeared to be responsible for these differences in the "dynamic component of fluidity" at these various time periods.
T A Brasitus, P K Dudeja, R Dahiya
Transferrin (Tf) is a growth factor that transports iron in plasma. It is essential for proliferation of activated T lymphocytes. Previous studies have suggested that peripheral blood cells are capable of synthesizing Tf. Using in situ hybridization techniques and human Tf complementary DNAs as probes, peripheral blood cells have been examined for sites of Tf messenger RNA (mRNA) transcription. The studies described here demonstrate that Tf is synthesized by a specific subset of T lymphocytes, the T4+ inducer subset. T lymphocyte proliferation is dependent upon the presence of both interleukin 2 (IL-2) and Tf, even though resting cells do not possess receptors for either. The present studies indicate that during T cell activation, induction of IL-2 mRNA transcription and IL-2 receptor expression precede the transcription of Tf mRNA and expression of Tf receptors, respectively. These events in turn precede the initiation of DNA synthesis. Transferrin and its receptor appear to be involved in an autocrine pathway which is functionally linked to the IL-2/IL-2 receptor autocrine loop.
J B Lum, A J Infante, D M Makker, F Yang, B H Bowman
Our previous studies had suggested a link between bile salt stimulation of colonic epithelial proliferation and the release and oxygenation of arachidonate via the lipoxygenase pathway. In the present study, we examined the role of reactive oxygen versus end products of arachidonate metabolism via the cyclooxygenase and lipoxygenase pathways in bile salt stimulation of rat colonic epithelial proliferation. Intracolonic instillation of 5 mM deoxycholate increased mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA. Responses to deoxycholate were abolished by the superoxide dismutase mimetic CuII (3,5 diisopropylsalicylic acid)2 (CuDIPS), and by phenidone or esculetin, which inhibit both lipoxygenase and cyclooxygenase activities. By contrast, indomethacin potentiated the response. Phenidone and esculetin suppressed deoxycholate-induced increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5, 12, and 15-hydroxyeicosatetraenoic acid (HETE), whereas CuDIPS had no effect. Indomethacin suppressed only PGE2. Deoxycholate (0.5-5 mM) increased superoxide dismutase sensitive chemiluminescence 2-10-fold and stimulated superoxide production as measured by cytochrome c reduction in colonic mucosal scrapings or crypt epithelium. Bile salt-induced increases in chemiluminescence were abolished by CuDIPS, phenidone, and esculetin, but not by indomethacin. Intracolonic generation of reactive oxygen by xanthine-xanthine oxidase increased colonic mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA approximately twofold. These effects were abolished by superoxide dismutase. The findings support a key role for reactive oxygen, rather than more distal products of either the lipoxygenase or cyclooxygenase pathways, in the stimulation of colonic mucosal proliferation by bile salts.
P A Craven, J Pfanstiel, F R DeRubertis
Human neonatal lymphocytes produced little macrophage activation factor in response to mitogens. This correlated with decreased production of interferon-gamma (IFN gamma): adult lymphokines contained 894.2 +/- 177.1 U/ml, whereas neonatal cord and peripheral lymphokines contained 66.9 +/- 17.0 and 116.7 +/- 29.6 U/ml by bioassay. Results by radioimmunoassay (RIA) for IFN gamma were similar. In contrast, the interleukin 2 content of cord lymphokines was greater (P less than 0.01) and that of neonatal peripheral blood lymphokines similar to that of adults. Interleukin 1 production and interleukin 2 receptor expression and affinity were similar for adult and neonatal cells. Interleukins 1 and 2 in amounts comparable to those in adult lymphokines did not increase production of macrophage activation factor or IFN gamma by neonatal cells. Neonatal cells did not contain intracellular IFN or degrade exogenous IFN. Excess suppressor activity was not found in neonatal cultures. Addition of IFN alpha, 10,000-50,000 U/ml of interleukin 2 or phorbol myristate acetate (PMA) to cord mononuclear cells or of adult monocytes or PMA to cord T cells increased IFN gamma production compared to cells stimulated with concanavalin A (ConA) alone. Nevertheless, under optimal conditions (T cells + PMA + Con A), adult cells produced much more IFN gamma (1,360 +/- 261 U/ml by RIA) than cord cells (122 +/- 37 U/ml). Staphylococcal enterotoxin A (SEA) stimulated cord cell IFN gamma production at low cell densities; nevertheless, adult cells produced more IFN in response to SEA 1,341 +/- 350 U/ml) than cord cells (350 +/- 33 U/ml). Decreased production of IFN gamma by neonatal cells appears to be due both to differences in their intrinsic capacity to produce IFN gamma and to differences in regulatory mechanisms.
C B Wilson, J Westall, L Johnston, D B Lewis, S K Dower, A R Alpert
A differential solute clearance technique was used to evaluate glomerular capillary wall function in 20 patients with membranous glomerulopathy and massive proteinuria. The clearance of inulin, the filtration fraction, and the fractional clearance of uncharged dextrans of a radius of 28-48 A were depressed significantly below control values in 20 healthy volunteers (P less than 0.01). In contrast, the fractional clearance of dextrans of radius greater than 50 A was elevated markedly. A theoretical model of solute transport that depicts the major portion of the glomerular capillary wall as an isoporous membrane and the minor portion as a nondiscriminatory shunt pathway revealed the calculated glomerular ultrafiltration coefficient to be five times lower and mean pore radius of the major membrane component to be 4 A smaller than control values. However, the fraction of filtrate volume permeating the shunt pathway was three- to fourfold above control values and correlated strongly in individual patients with the fractional clearance of albumin (r = 0.76) and of IgG (r = 0.80). Lowering renal plasma flow by 24% during indomethacin therapy in seven patients resulted in a 74% reduction in proteinuria accompanied by a corresponding diminution of filtrate formed through the shunt pathway. Morphometric analysis of glomerular ultrastructure revealed the magnitude of depression of the glomerular filtration rate and of urinary protein leakage to be related strongly to changes in the epithelial layer of the glomerular capillary wall, but not to the density of subepithelial immune deposits. We conclude that glomerular capillaries in membranous glomerulopathy are characterized by a loss of ultrafiltration capacity and of barrier size-selectivity, and that subepithelial immune deposits do not provide a structural basis for these functional alterations.
O Shemesh, J C Ross, W M Deen, G W Grant, B D Myers
Because of controversy regarding the relationship of cytoplasmic ionized calcium concentration ([Cai2+]) to platelet activation, we studied the correlation of platelet aggregation and ATP secretion with [Cai2+] as determined by 2-methyl-6-methoxy 8-nitroquinoline (quin2) and aequorin in response to ADP, epinephrine, collagen, the Ca2+ ionophore A23187, and thrombin. Both indicators showed a concentration-dependent increase in [Cai2+] in response to all agonists except epinephrine when gel-filtered platelets were suspended in media containing 1 mM Ca2+. With epinephrine, a rise in [Cai2+] was indicated by aequorin, but not by quin2; [Cai2+] signals, aggregation, and secretion were suppressed by EGTA. ADP [0.5 microM] produced a rise in [Cai2+] that was registered by both aequorin and quin2 in platelets in Ca2+-containing media; addition of EGTA to the medium raised the threshold concentration of ADP to 5.0 microM for both indicators. Collagen produced progressive concentration-related increases in [Cai2+] and aggregation in aspirin-treated aequorin-loaded platelets. Quin2 failed to indicate a rise in [Cai2+]at lower collagen concentrations with EGTA or aspirin. [Cai2+] response to A23187 and thrombin was reduced by addition of EGTA to platelets loaded with either aequorin or quin2. With all five agonists in all conditions tested, aequorin [Cai2+] signals occurred at the same agonist concentration as that or lower than that which produced platelet shape change, aggregation, or secretion. Platelet activation was better correlated with changes in [Cai2+] indicated by aequorin than with the response of quin2, possibly because aequorin is more sensitive to local zones of [Cai2+] elevation.
J A Ware, P C Johnson, M Smith, E W Salzman
A hereditary (three family members) deficiency of antithrombin III (AT-III) in which AT-III antigen (AT-III ag) is normal in spite of low heparin cofactor and antithrombin activity is described. Plasma levels were: AT-III ag, 0.92-0.96 U/ml; AT-III heparin cofactor activity, 0.54-0.62 U/ml; progressive antithrombin activity index, 0.13-0.18; anti-Xa activity, 0.50-0.56 U/ml. Plasma crossed immunoelectrophoresis (CIE) patterns performed with and without added heparin were normal, but serum CIE revealed a decreased complex peak. Purification of the patient's plasma AT-III by heparin-sepharose affinity chromatography showed a normal protein recovery and elution profile, but the purified AT-III fraction showed only 50% of the normal progressive thrombin neutralization and anti-Xa activity. When thrombin-antithrombin (TAT) complexes were formed by incubating with excess thrombin, SDS-polyacrylamide gel electrophoresis (PAGE) analysis revealed that half the patient AT-III formed TAT complexes while the remainder migrated as free AT-III. All the control AT-III formed TAT complexes. The patient's nonreacting AT-III (AT-III "Denver"), isolated by affinity chromatography, showed CIE and SDS-PAGE migration patterns characteristic of normal AT-III but failed to bind thrombin or Xa. Calculations from turnover studies in one patient and normal subjects with autologous 131I-AT-III suggested that AT-III "Denver" is removed from the plasma slightly more rapidly than normal. These studies indicate that the patients' variant AT-III molecule was characterized by normal heparin interaction but defective binding and inhibition of thrombin and Xa. These characteristics allow isolation of the nonreactive variant molecule by heparin-sepharose affinity chromatography.
J E Sambrano, L J Jacobson, E B Reeve, M J Manco-Johnson, W E Hathaway
When spleen cells of 5-fluorouracil (5-FU)-treated mice were cultured in the presence of interleukin 3 (IL-3), most colonies revealed IL-3 concentration-dependent colony formation except for mast cell colonies and blast cell colonies. While most colonies were smaller in lower concentrations of IL-3, the size of the blast cell colonies were similar between high and low IL-3 groups. These data suggested that blast cell colony development requires less IL-3 than the development of multilineage colonies from blast cell colonies. This notion was supported by experiments in which IL-3 was added twice to cultures of spleen cells of 5-FU-treated mice. When low concentrations of IL-3 were added on day 7, there was a reduction in the number of multilineage colonies formed without an effect on the number of blast cell colonies. Using this information, we developed a culture system that favors blast cell colony formation by cells of normal mice. When low (20 U/ml) concentrations of IL-3 were added to cultures of spleen cells of normal mice on day 7 of incubation in media containing 2-5% fetal calf serum, blast cell colonies were the predominant colony type. The blast cell colonies revealed high but variable secondary replating efficiencies. These data suggest that multipotential progenitors may become less sensitive to IL-3 as they differentiate in culture.
K Koike, J N Ihle, M Ogawa
This experiment was performed to determine if plasma glucose homeostasis is maintained in normal human volunteers during light exercise (40% maximal oxygen consumption [VO2 max]) when changes in insulin and glucagon are prevented. Hormonal control was achieved by the infusion of somatostatin, insulin, and glucagon. Glucose kinetics and oxidation rates were determined with stable isotopic tracers of glucose, and by indirect calorimetry. Two different rates of replacement of insulin and glucagon were used; in one group, insulin was clamped at 19.8 +/- 2.6 microU/ml (high-insulin group), and in the other group insulin was clamped at 9.2 +/- 1.3 microU/ml (low-insulin group). Glucagon was maintained at 261 +/- 16.2 and 124 +/- 6.4 pg/ml, respectively, in the high-insulin and low-insulin groups. Without hormonal control, plasma glucose homeostasis was maintained during exercise because the increase in glucose uptake was balanced by a corresponding increase in glucose production. When changes in insulin and glucagon were prevented, plasma glucose concentration fell, particularly in the high-insulin group. Glucose uptake increased to a greater extent than when hormones were not controlled, and glucose production did not increase sufficiently to compensate. The increase in glucose uptake in the hormonal control groups was associated with an increased rate of glucose oxidation. When euglycemia was maintained by glucose infusion in the hormonal control subjects, the modest increase in glucose production that otherwise occurred was prevented. It is concluded that during light exercise there must be a reduction in insulin concentration and/or an increase in glucagon concentration if plasma glucose homeostasis is to be maintained. If such changes do not occur, hypoglycemia, and hence exhaustion, may occur.
R R Wolfe, E R Nadel, J H Shaw, L A Stephenson, M H Wolfe
We have proposed that chronic hyperglycemia alters the ability of glucose to modulate insulin secretion, and have now examined the effects of different levels of hyperglycemia on B cell function in normal rats using chronic glucose infusions. Rats weighing 220-300 g were infused with 0.45% NaCl or 20, 30, 35, or 50% glucose at 2 ml/h for 48 h, which raised the plasma glucose by 18 mg/dl in the 30% rats, 37 mg/dl in the 35% rats, and 224 mg/dl in the 50% group. Insulin secretion was then examined using the in vitro isolated perfused pancreas. Glucose-induced insulin secretion remained intact in the normoglycemic 20% glucose rats and it was potentiated in the mildly hyperglycemic 30% glucose rats. However, with even greater hyperglycemia in the 35% glucose group the insulin response to a high glucose perfusate was severely blunted, and it was totally lost in the most hyperglycemic 50% glucose rats. In a second protocol that examined glucose potentiation of arginine-stimulated insulin release, a similar impairment in the ability of glucose to modulate the insulin response to arginine was found with increasing levels of chronic hyperglycemia. On the other hand, the ability of a high glucose concentration to inhibit arginine-stimulated glucagon release was preserved in all glucose-infused rats, but the glucagon levels attained in response to the arginine at 2.8 mM glucose were much less in the 50% glucose rats than in all the other groups. These data clearly show that after 48 h of marked hyperglycemia, glucose influence upon insulin secretion in the rat is severely impaired. This model provides a relatively easy and reproducible method to study the effects of long-term hyperglycemia on B cell function.
J L Leahy, H E Cooper, D A Deal, G C Weir
We sought direct evidence for anti-islet cellular cytotoxicity in diabetic Bio-Breeding/Worcester (BB/W) rats by comparing the effects of splenic lymphoid cells from BB/W diabetic (D), diabetes-prone (DP), and diabetes-resistant (DR) rats on the release of 51Cr from damaged islet cells in vitro. D and DP splenic lymphoid cells were cytotoxic to major histocompatibility complex (MHC)-compatible Wistar-Furth (WF) rat islet cells and also to MHC-incompatible Lewis rat islet cells and a rat islet cell line (RIN 5F), whereas WF and Lewis rat spleen cells and a rat pituitary cell line (GH3) were not lysed by lymphoid cells from D or DP rats. The cytotoxic cells were identified as natural killer (NK) cells since NK-sensitive cells (G1-TC and YAC-1 cell lines) were lysed by D and DP spleen cells, YAC-1 cells competed for the lysis of RIN islet cells by D spleen cells, lysis of RIN cells was increased by using D spleen cells from the low density fraction (large lymphocytes/monocytes) of a Percoll density gradient, and incubation of D spleen cells with an antiserum to NK cells (anti-asialo GM1 serum) and complement decreased monoclonal antibody-defined subsets containing NK cells (W3/13+ OX19- and OX8+), and this was accompanied by similar decreases in cytotoxicity to YAC-1, RIN, and WF islet cells. These studies demonstrate that NK cell activity is increased in BB/W diabetic and DP rats, and that islet cells can serve as targets for these NK cells. The findings suggest that NK cells may participate in the islet-directed cellular cytotoxic response leading to beta cell destruction and diabetes.
P MacKay, J Jacobson, A Rabinovitch
After circulating in the vascular system a short time, polymorphonuclear leukocytes (PMN) migrate to extravascular sites in response to chemotactic stimuli. Prestimulation of PMN in vitro by secretagogues has been shown to increase their number of N-formylmethionylleucylphenylalanine (fmet-leu-phe) and complement component C3bi (CR3) receptors. We investigated whether the same phenomenon occurred in vivo, comparing characteristics of human skin chamber and guinea pig peritoneal exudate and blood PMN. Exudate PMN of both species contained approximately 28% less of the specific granule marker vitamin B12-binding protein (P less than 0.01) but a similar amount of the azurophil granule marker beta-glucuronidase. The total number of fmet-leu-phe receptors was 5.9 times higher in guinea pig exudate than in blood PMN (P less than 0.01) and 2.9 times higher in human exudate than in blood PMN (P less than 0.02). All exudate PMN and most blood PMN preparations showed a high affinity receptor (Kd approximately 2.3 X 10(-8) M) and a low affinity receptor (approximately 1.5 X 10(-7) M). The upregulation of fmet-leu-phe receptors in exudate PMN correlated with an improved responsiveness to fmet-leu-phe induced membrane depolarization, oxidative metabolism, and chemotaxis. In addition, the concentration of fmet-leu-phe that produced a half-maximal response of chemotaxis, superoxide production, and membrane potential depolarization was 10-fold lower in exudate PMN than in blood PMN. Human exudate PMN had a twofold increased C3bi receptor expression compared with blood PMN. Thus, a preferential loss of specific granules is associated with increased number of high and low affinity fmet-leu-phe receptors and increased C3bi receptor expression not only in vitro, but also in vivo. The data indicate that exudation primes PMN for their subsequent responsiveness to fmet-leu-phe, a modification that may be crucial for efficient antimicrobial host defense.
W Zimmerli, B Seligmann, J I Gallin
Zomepirac is a nonsteroidal anti-inflammatory drug recently withdrawn from use because of an unexplained high incidence of immunological reactions. It is metabolized in humans to a reactive, unstable acyl glucuronide which accumulates in plasma. Because of the similarity of zomepirac glucuronide to bilirubin glucuronide in structure and stability and the documented irreversible binding of bilirubin to albumin through its acyl glucuronide, we studied the reaction of zomepirac acyl glucuronide with albumin in vitro from pH 5 to 9 and in vivo in six healthy human volunteers who had received a single 100-mg oral dose of zomepirac. Irreversible binding of zomepirac to protein was determined by exhaustive washing of protein, followed by hydrolysis of bound zomepirac-protein adduct with base, extraction of the liberated drug, and chromatographic measurement. Irreversible binding was observed both in vitro and in vivo. The extent of binding in vitro was time- and pH-dependent. In vitro drug binding was also observed for the isomers of zomepirac glucuronide which were formed by intramolecular acyl migration. Irreversible binding in vivo correlated with overall exposure to zomepirac glucuronide when exposure was expressed as the area under the plasma concentration vs. time curve. When probenecid (500 mg, twice daily), which decreases the plasma clearance of zomepirac glucuronide, was administered concurrently with zomepirac, irreversible binding of zomepirac was increased. The nature of the zomepirac protein binding is probably covalent. Formation of irreversibly protein-bound zomepirac occurs via the acyl glucuronide as previously shown for bilirubin glucuronide, and the reaction may be general for other drugs that are metabolized to acyl glucuronides.
P C Smith, A F McDonagh, L Z Benet
A T cell surface membrane-associated glycoprotein, Tp40 (40,000 mol wt), also designated as CD-7, was not expressed by the T cells of a patient with severe combined immunodeficiency. In addition to this abnormality, T cell proliferative responses to mitogens were defective and the IL-2 receptor expression was deficient on the patient's T lymphocytes. However, his T cells were found to provide help for the differentiation of normal B cells to Ig-secreting cells. Abundant circulating B cells were detected. These B cells proliferated normally in the presence of anti-mu antibodies and B cell growth factors, but did not differentiate into antibody-secreting cells when provided with the help of normal T cells. In addition, his activated B cells did not proliferate to IL-2 even though IL-2 receptors were expressed. A successful allogeneic histocompatible bone marrow transplantation resulting in T cell engraftment corrected both the T and B cell immunodeficiencies. These findings support the hypothesis that the Tp40 deficiency present in this patient is related to a defect of the T cell precursors, and that Tp40 plays important roles not only essential to T cell interactions but also to certain aspects of T-B cell interaction during the early lymphoid development.
L K Jung, S M Fu, T Hara, N Kapoor, R A Good
We have evaluated the subunit composition of plasma von Willebrand factor (vWF) and found evidence that cleavage is present in normal individuals, increased in IIA and IIB von Willebrand disease (vWD), but decreased or absent in variants with aberrant structure of individual oligomers. vWF was rapidly purified from plasma on an analytical scale by monoclonal antibody immunoaffinity chromatography in the presence of protease inhibitors. After reduction and electrophoresis in 5% polyacrylamide gels containing sodium dodecyl sulfate, fragments of 189, 176, and 140 kD, as well as the predominant 225-kD subunit, were identified in plasma vWF from 25 normal individuals. The vWF polypeptides were detected by immunoblotting with a mixture of 55 anti-vWF monoclonal antibodies followed by 125I-rabbit anti-mouse antibody and autoradiography. In five individuals with type IIA and five individuals with type IIB vWD, the proportions of 176 and 140-kD fragments were increased relative to the intact 225-kD subunit, as determined by excising each band and quantitating incorporated radioactivity. In contrast, these fragments were either not detectable or were present in only trace amounts in variants with abnormal structure of individual oligomers (types IIC and IID, and a new variant, type IIE vWD). The results reported here provide evidence that absence of large vWF multimers in these two groups of variants results from different mechanisms. In addition, they demonstrate that partial cleavage of the plasma vWF subunit is a normal event.
T S Zimmerman, J A Dent, Z M Ruggeri, L H Nannini
Affinity-purified IgE-binding factors from the plasma of patients with the hyper IgE syndrome (HIE) were assessed for their capacity to enhance IgE synthesis by B cells derived from patients with allergic rhinitis or normal nonatopic donors. IgE-binding factors from three of four HIE patients enhanced IgE synthesis by B cells from patients with perennial allergic rhinitis, or with seasonal allergic rhinitis (SAR) and recent pollen exposure, but did not enhance IgE synthesis by B cells from nonatopic donors or from SAR patients with no recent pollen exposure. IgG synthesis was not affected by HIE IgE binding factors. In contrast, IgE binding factors from three of three nonatopic donors failed to enhance IgE or IgG synthesis. Plasma IgE-binding factors from the fourth patient with HIE contained a mixture of IgE-potentiating activity and IgE-suppressive activity. These two activities could be separated on concanavalin A Sepharose or peanut agglutinin agarose columns. Human IgE potentiating factor, but not IgE suppressive factor, had affinity for concanavalin A but not peanut agglutinin and fractionated into two peaks on gel filtration over Sephadex G-75: one peak with a molecular size of approximately 15,000 D and the other with a molecular size of approximately 60,000 D. The isolation of functional IgE binding factors which potentiate IgE synthesis from the plasma of patients with HIE suggests that IgE-binding factors play an important role in the in vivo regulation of IgE synthesis in man.
D Y Leung, R Frankel, N Wood, R S Geha
Inoculation of golden Syrian hamsters with Venezuelan encephalitis (VE) virus results in a sustained diminution in glucose-stimulated insulin release that is correctable by cyclic (c) AMP analogs and phosphodiesterase inhibitors. This suggested the importance of directly measuring cAMP content in VE-infected and control islets in response to insulin secretagogues. The basal cAMP content of VE-infected islets (0.14 +/- 0.02 pmol/micrograms islet DNA) was approximately half that of control islets (0.27 +/- 0.02 pmol/micrograms islet DNA) (P less than 0.05). In the presence of 10 microM glucagon (and 3 mM glucose), the rate of cAMP generation in VE-infected islets was only half that of control islets. With 10 mM alpha-ketoisocaproic acid, the rates of cAMP generation were indistinguishable between control and experimental groups. In response to 20 mM glucose and 3-isobutyl-1-methylxanthine (IBMX) (a phosphodiesterase inhibitor), cAMP generation in VE-infected islets was 81% (NS) of the control rate. When a more specific phosphodiesterase inhibitor, RO 20-1724, was used with 20 mM glucose, cAMP generation in the infected islets was only 44% (P less than 0.001) of the control value. Insulin secretion over the perifusion period paralleled the cAMP levels. In the presence of 10 mM alpha-ketoisocaproic acid, there was no difference in insulin secretion between VE-infected and control islets, while there was a statistically significant (P less than 0.05) difference with 10 microM glucagon or 20 mM glucose (in 1 mM RO 20-1724). These data point to a defect in the cAMP generation system of VE-infected islets, although additional factors involved in insulin secretion may also be impaired by the virus.
E J Rayfield, K J Kelly
The uptake of 14C-palmitate by rat liver cell monolayers is depressed by binding of the fatty acid to albumin. When the uptake flux is divided by the concentration of free palmitate in the bathing medium, however, the resulting clearance is approximately 14 times greater in the presence of albumin than in its absence. These findings are not accounted for by the different diffusion rates of free and bound palmitate across an unstirred fluid layer, nor attributable to nonequilibrium binding. Instead we argue that the most plausible explanation is accelerated dissociation of albumin-palmitate complexes mediated by the cell surface--an interpretation that also explains the uptake kinetics of other albumin-bound organic anions by perfused rat liver.
A B Fleischer, W O Shurmantine, B A Luxon, E L Forker
Sn(tin)-protoporphyrin, a potent competitive inhibitor of heme oxygenase, can suppress hyperbilirubinemia in animal neonates and significantly reduce plasma bilirubin levels in animals and man. To further explore the biological actions and metabolic disposition of Sn-protoporphyrin, we have examined its effect in the suckling neonate when administered to the mother either 24-48 h before or immediately after birth. Sn-protoporphyrin, when administered before birth, crossed the placental membranes, inhibited fetal heme oxygenase, and suppressed the transient hyperbilirubinemia that occurs in the neonate after birth in a dose-dependent manner. Tissue heme oxygenase activity in the neonate was also lowered in a dose-dependent manner. The blood-brain barrier of the neonate was permeable to Sn-protoporphyrin for a period of between 20-28 d of postnatal life. Sn-protoporphyrin, however, was not retained in brain, but left the brain space with a t1/2 of 1.7 d. In addition, Sn-protoporphyrin administered once at birth to neonates inhibited brain heme oxygenase in a dose-dependent manner. The results of this study demonstrate that Sn-protoporphyrin can cross the placental membranes, inhibit tissue heme oxygenase activity in the fetus, and can also, following such prenatal treatment, suppress the hyperbilirubinemia of the newborn animal.
G S Drummond, A Kappas
The stability in vivo and circulatory clearance of immunotoxins were assessed in rhesus monkeys. The immunotoxins studied were T cell-specific monoclonal anti-T11 antibodies conjugated by disulfide linkage to ribosome-inactivating toxins. Intact immunotoxin was detectable in the circulation of the monkeys following a single intravenous infusion. This was demonstrated by quantitative flow-cytometric analysis, gel-filtration, and sodium dodecyl sulfate-gel electrophoresis. This intact conjugate was shown to be functional in the plasma of the infused animals in an in vitro cytotoxicity assay. However, a number of factors contributed to bring the level of circulating immunotoxin to a less than optimal level. When conjugated to a ribosome-inactivating toxin, the antibody was cleared more rapidly than was the native antibody. Furthermore, following infusion, some breakdown of the conjugate occurred, resulting in the generation of detectable levels of circulating free antibody. The present data indicate the feasibility of using immunotoxins as therapeutic tools in man.
N L Letvin, V S Goldmacher, J Ritz, J M Yetz, S F Schlossman, J M Lambert
Serum bone gamma-carboxyglutamic acid-containing (Gla) protein (sBGP), a sensitive and specific marker of bone turnover, was measured in 25 patients with primary hyperparathyroidism and in 24 patients with bone metastases with or without hypercalcemia. Despite similar levels of hypercalcemia, sBGP was increased in primary hyperparathyroidism (14.2 +/- 9.6 ng/ml, P less than 0.001), was decreased in malignant hypercalcemia (3.1 +/- 2.8 ng/ml, P less than 0.001), and was normal in patients with bone metastases without hypercalcemia (6.6 +/- 2.7 ng/ml). In primary hyperparathyroidism, sBGP was correlated with serum immuno-reactive parathyroid hormone (r = 0.90), calcium (r = 0.73), and with the adenoma weight (r = 0.79). After parathyroidectomy, sBGP slowly returned to normal values within 2-6 mo, suggesting that sBGP reflects increased bone turnover rather than a direct effect of parathyroid hormone on BGP synthesis at the cell level. An iliac crest biopsy was performed in 11 patients with primary hyperparathyroidism and in 9 cancer patients in a noninvaded area. sBGP was significantly correlated with all parameters reflecting bone formation but not with bone resorption. Patients with bone metastases were analyzed according to the presence or the absence of hypercalcemia. In contrast to normocalcemic patients who had normal sBGP, hypercalcemic patients had decreased sBGP (P less than 0.001) and a lower bone formation at the cellular level (P less than 0.05). Thus, biochemical and histological data suggest that an unknown humoral factor might be responsible for this uncoupling between increased resorption and decreased formation. This uncoupling, rather than local release of calcium by the metastatic process, might be responsible for hypercalcemia in patients with bone metastases.
P D Delmas, B Demiaux, L Malaval, M C Chapuy, C Edouard, P J Meunier
In the present study we used a bioassay system for measuring plasma cholecystokinin (CCK) to evaluate whether CCK has a physiologic role in regulating gastric emptying in humans. Plasma CCK levels and gastric emptying after ingestion of a mixed liquid meal were determined in five normal male volunteers. Fasting CCK levels averaged 0.8 +/- 0.1 pM and increased to 6.5 +/- 1.0 pM within 10 min of drinking the mixed meal. CCK levels remained elevated for up to 90 min. Gastric emptying after a meal was slow; at the end of the 90 min 68% of the original volume remained in the stomach. The rate of gastric emptying of water was then measured in the same individuals with a simultaneous infusion of either saline, or one of two doses of CCK (12 pmol/kg per h and 24 pmol/kg per h). With the saline infusion, plasma CCK levels did not increase above basal and gastric contents emptied rapidly. At the end of 90 min only 7% of the original volume remained in the stomach. The lower dose of CCK resulted in a plasma level of 3.4 pM which both reproduced the average postprandial plasma level and caused a significant delay in gastric emptying. The higher dose of CCK achieved plasma levels of 8 pM and resulted in a delay in gastric emptying that was similar to that seen with the mixed meal. Since exogenous CCK at concentrations which occur postprandially delays gastric emptying, we conclude that CCK is a physiologic regulator of gastric emptying.
R A Liddle, E T Morita, C K Conrad, J A Williams
The insulin receptor from human brain tumors of glial origin was examined for the first time using intact cells (from an established cultured human glioblastoma cell line) and partially purified solubilized membranes (from cultured cells and freshly isolated human brain tumors). The structure of the glial insulin receptor subunits was assessed by affinity cross-linking of 125I-insulin with the alpha-subunit of the receptor, neuraminidase treatment of the cross-linked receptor, behavior of the receptor on lectin columns, and electrophoretic mobility of the phosphorylated beta-subunit. The functions of the insulin receptor were examined by measuring specific 125I-insulin binding (receptor concentration, affinity, specificity, pH-, time-, and temperature dependence), insulin-induced down-regulation of the receptor, insulin-stimulated autophosphorylation of the beta-subunit, and phosphorylation of exogenous substrates as well as insulin-stimulated glucose uptake in glioblastoma cells. All of these properties were typical for the insulin receptor from target tissues for insulin action. The insulin receptor of the normal human brain showed the altered electrophoretic mobility and lack of neuraminidase sensitivity of its alpha-subunit previously reported for the rat brain receptor. There was no difference, however, in the functions of the receptor subunits (binding, phosphorylation) from the normal brain tissue and the eight human gliomal tumors. Since the glial elements compose a majority of the brain cells, the "normal" structure and function of their insulin receptor might provide a key to understanding the role of insulin in the carbohydrate metabolism of the human central nervous system.
G Grunberger, W L Lowe Jr, A McElduff, R P Glick
When blood coagulation takes place in the presence of calcium ions, alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked to fibrin by activated coagulation Factor XIII (XIIIa) and thereby contributes to the resistance of fibrin to fibrinolysis. It was previously shown that the cross-linking reaction is a reversible one, since the alpha 2PI-fibrinogen cross-linked complex could be dissociated. In the present study we have shown that the alpha 2PI-fibrin cross-linking reaction is also a reversible reaction and alpha 2PI which had been cross-linked to fibrin can be released from fibrin by disrupting the equilibrium, resulting in a decrease of its resistance to fibrinolysis. When the fibrin clot formed from normal plasma in the presence of calcium ions was suspended in alpha 2PI-deficient plasma of buffered saline, alpha 2PI was gradually released from fibrin on incubation. When alpha 2PI was present in the suspending milieu, the release was decreased inversely to the concentrations of alpha 2PI in the suspending milieu. The release was accelerated by supplementing XIIIa or the presence of a high concentration of the NH2-terminal 12-residue peptide of alpha 2PI (N-peptide) which is cross-linked to fibrin in exchange for the release of alpha 2PI. When the release of alpha 2PI from fibrin was accelerated by XIIIa or N-peptide, the fibrin became less resistant to the fibrinolytic process, resulting in an acceleration of fibrinolysis which was proportional to the degree of the release of alpha 2PI. These results suggest the possibility that alpha 2PI could be released from fibrin in vivo by disrupting the equilibrium of the alpha 2PI-fibrin cross-linking reaction, and that the release would result in accelerated thrombolysis.
J Mimuro, S Kimura, N Aoki
Fibrin formation and turnover are intimately associated with inflammation and wound healing. To explore whether fibrin(ogen)-derived peptides exert direct effects upon cells involved in inflammation and tissue repair we examined the capacity of human fibrinopeptide B (hFpB), a thrombin-derived proteolytic cleavage product of the fibrinogen B beta-chain, to stimulate neutrophils (PMN), monocytes, and fibroblasts. hFpB caused directed cell migration of PMN and fibroblasts that was optimal at approximately 10(-8) M. This chemotactic activity was blocked by preincubating hFpB with antiserum to hFpB. hFpB was not chemotactic for monocytes. The chemotactic potency of hFpB for PMN was equivalent to that of anaphylatoxin from the fifth component of human complement (C5a), leukotriene B4 (LTB4), and formyl-methionyl-leucyl-phenylalanine (fMLP), and for fibroblasts its chemotactic activity was comparable to that of platelet-derived growth factor. hFpB did not interact with PMN receptors for C5a, LTB4, or fMLP as (a) desensitization with 10(-7) M hFpB abolished chemotaxis to hFpB but had no effect upon chemotaxis to C5a, LTB4, or fMLP and (b) induction of chemotactic responses to fMLP and LTB4 in neutrophilic leukemic cells (HL-60 cells) by incubation with dimethylsulfoxide did not extend to hFpB. Like fMLP, hFpB caused a rapid, dose-dependent increase in PMN cytoskeletal associated actin, but unlike fMLP, hFpB did not cause PMN aggregation, release of lysosomal enzymes (lysozyme and beta-glucuronidase), or the production of superoxide anion. These results suggest that hFpB may have a role in recruiting PMN and fibroblasts at sites of fibrin deposition and turnover. The capacity of hFpB to cause PMN chemotaxis without causing concurrent release of lysosomal enzymes or the production of superoxide anion is further evidence for the complexity of PMN responses to chemotactic agents.
R M Senior, W F Skogen, G L Griffin, G D Wilner
Epidermal cell-derived thymocyte activating factor (ETAF), a cytokine produced by keratinocytes, has previously been shown to be biochemically and functionally very similar, if not identical, to interleukin 1 (IL-1). Both ETAF and IL-1 have been demonstrated to be chemotactic for neutrophils and mononuclear cells in vitro. In order to demonstrate that this activity has physiological relevance we have used a simple in vivo model. The present study demonstrates that injection of high-titer ETAF or purified recombinant murine IL-1 into the mouse footpad results in an influx of neutrophils into the site with peak accumulation at 4 h. Footpad swelling also occurs with a time course roughly paralleling that of the neutrophil accumulation. Injection of control proteins failed to reproduce this phenomenon. Margination of neutrophils within blood vessels was seen within 1 h of injection of ETAF or IL-1, followed by entry into the stroma by 4 h. This suggests that chemotactic activity and not merely increased adherence or inhibition of migration is occurring. 5-10 d of daily, subcutaneous injection of ETAF on the mouse flank resulted in an infiltrate of neutrophils, and to a lesser degree, mononuclear cells in association with epidermal hyperplasia, subcutaneous fibrosis, and focal muscle necrosis in the panniculus carnosus. These findings were not seen in control sites injected with media. These findings provide direct in vivo experimental evidence suggesting a physiologic role for ETAF/IL-1 in local inflammation.
R D Granstein, R Margolis, S B Mizel, D N Sauder
The molecular mechanism of volatile anesthetic action remains unknown. Attempts to elucidate this mechanism have been complicated by the absence of models in which changes in neuronal cellular properties can be correlated with changes in whole animal anesthetic effect. In this study we describe a model where diet-induced alterations in rat brain fatty acid composition are correlated with alterations in volatile anesthetic potency. Rats maintained on a fat-free diet showed significant depletion of arachidonic acid (20:4 omega 6; 5,8,11,14-eicosatetraenoic acid) and docosahexaenoic acid (22:6 omega 3; 4,7,10,13,16,19,-docosahexaenoic acid) in brain, and a corresponding increase in Mead acid (20: 3 omega 9; 5,8,11-eicosatrienoic acid). These fat-deprived rats were significantly more sensitive to all volatile anesthetics tested than were age-controlled rats on a normal diet. Parenteral supplementation of the fat-deprived animals with linolenic acid (18: 3 omega 3, 9,12,15-octadecatrienoic acid) completely reconstituted the docosahexaenoic acid content of brain without affecting anesthetic sensitivity. In contrast, supplementation of the fat-deprived rats with linoleic acid (18: omega 6; 9,12-octadecadienoic acid) caused a dramatic decrease in anesthetic sensitivity, but only a small change in whole brain arachidonate content. Further analysis revealed that linoleate supplementation of fat-deprived animals resulted in a preferential normalization of the arachidonate content of brain phosphatidylinositol as compared with other brain phosphoglycerides. These results demonstrate for the first time a correlation between changes in membrane composition and anesthetic effect, and indicate that the precise fatty acid composition (perhaps in specific phospholipids) of brain is important in the mechanism of volatile anesthetic action.
A S Evers, W J Elliott, J B Lefkowith, P Needleman
In order to determine whether the A cell may be directly suppressed by glucose in the absence of insulin, canine pancreata were perfused in vitro, both antegrade, through the arterial system and retrograde, through the venous system. Studies of the islet microvasculature have suggested that blood flows from the B cell core to the mantle; thus, the A cell may be tonically inhibited by intra-islet insulin. Retrograde perfusion may then be expected to prevent insulin from reaching the A cell, releasing it from inhibition. Retrograde perfusion with 88 mg/dl glucose markedly increased both insulin and glucagon secretion relative to antegrade levels. In a series of experiments, glucose concentrations were changed from 88 to 200 mg/dl. An antegrade glucose change resulted in increased insulin (134+/-21%; P less than 0.0025) and decreased glucagon (-26+/-9%, P less than 0.025) secretion. A retrograde glucose increase resulted in increased secretion of both insulin (91+/-15%; P less than 0.0005) and glucagon (23+/-9%; P less than 0.0125). To confirm that retrograde perfusion deprived the A cell of endogenous core derived, vascularly delivered insulin, possibly resulting in increased insulin sensitivity, 0.3 mU/ml exogenous porcine insulin was infused. Antegrade, 0.3 mU/ml insulin, had no effect on glucagon secretion (P less than 0.250), while retrograde infusion of 0.3 mU/ml insulin significantly inhibited glucagon secretion (-31 + 8%; P less than 0.0005). The results of our study support the concept that the direction of blood flow and of flow-dependent intra-islet hormone interactions are from the islet B cell core to the mantle. It was further concluded that the normal A cell may not be suppressed by glucose in the absence of insulin.
J I Stagner, E Samols
Pancreatic polypeptide and neuropeptide Y share 50% amino acid homology (18 out of 36 residues), suggesting that they may have common ancestral origins. cDNA clones complementary to human mRNAs encoding pancreatic polypeptide and neuropeptide Y were used to detect specific human genomic DNA sequences in human-mouse somatic cell hybrid lines. The pancreatic polypeptide gene (PPY) segregated with human chromosome 17, while the neuropeptide Y gene (NPY) segregated with human chromosome 7. Examination of cell hybrids with chromosomal rearrangements assigned PPY to the p11.1-qter region and NPY to the pter-q22 region of their respective chromosomes.
T Takeuchi, D L Gumucio, T Yamada, M H Meisler, C D Minth, J E Dixon, R E Eddy, T B Shows
Seropositive rheumatoid arthritis (RA) in adult and juvenile patients is associated with the serologic marker HLA-DR4. This association is incomplete; about one-third of the patients lack the disease-associated HLA-DR4 haplotype. The main biological function of class II molecules is to restrict the recognition of antigen by T lymphocytes. We therefore tested the hypothesis that patients with seropositive RA share T cell recognition sites for an unknown antigen and that such T cell "epitopes" are not identified by conventional serologic typing. We generated alloreactive human T cell clones by stimulating peripheral blood lymphocytes of normal donors against a lymphoblastoid cell line from a juvenile patient with seropositive RA. A panel of clones that recognized only HLA-Dw14 cells on a panel of homozygous typing cells was used to analyze class II molecules of adult patients with seropositive RA. By inhibition studies using monoclonal antibodies, the epitopes recognized by the different clones could be further characterized and assigned either to DR- or to DQ-encoded cell surface products. By using four different clones, it was possible to identify Dw14-associated T cell epitopes on all seropositive rheumatoid patients tested who typed HLA-DR4-positive and also on all eight DR4-negative patients tested. Approximately one-half of nonrheumatoid DR4-positive donors carried one or more determinants recognized by these clones; the expression of these allodeterminants in DR4-negative nonrheumatoid patients was rare (less than 10%). Thus, alloreactive human T cell clones are powerful tools to define T cell recognition sites on class II molecules that are not identified by conventional typing. Using T cell clones with specificities for determinants expressed on Dw14 homozygous typing lines, we were able to demonstrate shared epitopes on cells of all patients tested with seropositive RA irrespective of their HLA-D or HLA-DR type. These data suggest that major histocompatibility complex class II antigens of RA patients might be much more homogeneous than demonstrated by the incomplete HLA-DR4 association.
J Goronzy, C M Weyand, C G Fathman