Collagen-stimulated human platelet aggregation is mediated by endogenous calcium-activated neutral protease

To clarify the physiological role of calcium-activated neutral protease (CANP) in human platelets, we loaded the platelets with a Ca2+ -sensitive fluorescent dye, fura-2, and measured the degree of aggregation, cytosolic calcium ion concentration [( Ca2+]i), and proteolysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At physiological concentration of Ca2+ (1 mM) in the incubation medium, [Ca2+]i was below 0.5 microM and platelet aggregation was not shown. Ionomycin (0.15 microM) or collagen (50 micrograms/ml), but not ADP (10 microM), sharply enhanced the [Ca2+]i to near 1 microM and caused the aggregation. A calcium entry blocker, verapamil, completely abolished both the [Ca2+]i rise and the aggregation. NCO-700, a membrane permeable inhibitor against cysteine proteases (including CANP), dose-dependently blocked the aggregation but did not change the [Ca2+]i transient. SDS-PAGE revealed that filamin, talin, and 70 kDa protein were specifically degraded when platelets were aggregated by ionomycin or collagen and that the proteolysis was not observed when the aggregation was blocked by verapamil or NCO-700. These data provided evidence that Ca2+ entry exceeding 0.5 microM is essential, but not sufficient per se, and that activation of cysteine protease, most likely CANP, is involved in the platelet aggregation by collagen or calcium ionophore.     

Identification of a membrane-associated interleukin 1 in macrophages.

We have found a surface membrane-associated interleukin 1 (IL-1) with potent thymocyte and T-cell stimulatory activity on peptone-elicited peritoneal macrophages. The IL-1 activity was demonstrated on both fixed macrophage monolayers and on isolated membranes from unfixed macrophages. Membrane IL-1 was induced by adherence and/or by adding heat-killed Listeria monocytogenes to macrophage cultures. The macrophage membrane IL-1 was similar functionally and antigenically to soluble IL-1, but its expression could be temporally dissociated from IL-1 secretion; membrane IL-1 was induced earlier and persisted longer than IL-1 secretion during in vitro macrophage culture. Moreover, when cultured macrophages that had ceased both secretion and membrane expression of IL-1 were restimulated by adding heat-killed Listeria, substantial membrane IL-1 was induced in the absence of detectable IL-1 secretion. Membrane IL-1 appears to be an integral membrane protein since it was solubilized by detergent but was not eluted by EDTA, high salt, or low pH treatment of the membranes.     

Cleavage of human von Willebrand factor by platelet calcium-activated protease

In human platelet lysates prepared by addition of nonionic detergent (Triton X-100) or by sonication, the multimer composition and electrophoretic mobility of platelet von Willebrand factor (vWF) were consistently modified under conditions that would favor activation of the endogenous calcium-activated, sulfhydryl-dependent neutral protease (CAP). By sodium dodecylsulfate-agarose gel electrophoresis, native platelet vWF contained some multimers that were larger than those characteristic of plasma vWF. Modified platelet vWF contained a multimer population equivalent to or smaller than that of plasma vWF plus an additional fast-migrating band. In crossed immunoelectrophoresis (CIE), modified platelet vWF was characterized by a more anodic distribution and the appearance of a distinct, cross- reactive, anodic component previously designated VIIIR:Ag fragment. In the presence of calcium, radiolabeled purified plasma vWF was also degraded by the protease in question, with a decrease in the apparent molecular weight of the reduced monomer from 230,000 to 205,000. The VIIR:Ag fragment isolated from the same degraded plasma vWF by preparative CIE was shown to be composed of an identical mol wt 205,000 subunit. Because cleavage of plasma or platelet vWF was inhibited by prior addition of leupeptin, EDTA, ethylene glycol bis (beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA), or N-ethylmaleimide (agents known to inhibit platelet CAP) but was unaffected by numerous other protease inhibitors, including soybean trypsin inhibitor, benzamidine, hirudin, phenylmethylsulfonyl fluoride, aprotonin, or epsilon-aminocaproic acid (none of which inhibits platelet CAP), we conclude that proteolysis of vWF observed in this study is a direct effect of CAP and is not mediated by way of secondary proteases.     

Degradation of sarcoplasmic reticulum calcium-pumping ATPase in ischemic-reperfused myocardium: Role of calcium-activated neutral protease

Summary In an attempt to clarify the mechanism of sarcoplasmic reticulum (SR) dysfunction during the genesis of irreversible damage in the ischemic-reperfused myocardium, the changes in SR Ca²⁺-pumping ATPase (Ca²⁺-activated, Mg²⁺-dependent ATPase; Ca²⁺-ATPase) activity were studied during ischemia and subsequent reperfusion in the isolated perfused guinea pig heart preparation and correlated with the accumulation of calcium in the myocardium. Although the SR Ca²⁺-ATPase activity was not affected by ischemia of 40 min, reperfusion of the ischemic myocardium resulted in a definite time-dependent decrease in the enzyme activity. The reduction of SR Ca²⁺-ATPase activity was associated with a concomitant decrease in the enzyme concentration in the isolated SR and was in a good correlation with a substantial accumulation within the mhocardium. As the results indicated the possibility that proteolytic degradation by a calcium-activated protease(s) was responsible for the reduction of enzyme activity, we examined for the possible involvement of calcium-activated neutral protease (CANP). However, SR Ca²⁺-ATPase obtained either from the normal hearts or from the hearts after 40-min ischemia was found to be quite resistant to proteolytic actions of the two forms of CANP, i.e., CANP and mCANP partially purified from the guinea pig heart. These results suggest that a destructive process leading to the degradation of SR Ca²⁺-ATPase is activated by reperfusion, but not by ischemia per se, and that CANP is not implicated in the degradation of SR Ca²⁺-ATPase.This study was supported in part by a Grant-in-Aid to Y. Y. from the Ministry of Education, Science and Culture of Japan.     

Identification of the discontinuous binding site in human interleukin 1 beta for the type I interleukin 1 receptor.

Human interleukin 1 beta (IL-1 beta) exerts its diverse biological effects by binding to specific receptors on target cells. Two types of IL-1 receptor (IL-1R) have been identified: the type I IL-1R (p80) and the type II IL-1R (p68). Using site-specific mutagenesis, we have identified the binding site on IL-1 beta for the murine type I IL-1R. Analogs of the IL-1 beta protein containing defined amino acid substitutions were produced and tested for competitive binding to the two IL-1Rs. Substitutions of the amino acids at seven positions resulted in analogs that had greater than or equal to 100-fold reductions in competitive binding to the type I IL-1R, while maintaining substantial binding to the type II IL-1R. These seven amino acids (Arg-4, Leu-6, Phe-46, Ile-56, Lys-93, Lys-103, and Glu-105) are clustered in the IL-1 beta molecule, forming a discontinuous binding site. The side chains of all seven residues are exposed on the surface of IL-1 beta. The cumulative binding energies contributed by each of the residues predict a binding affinity that is consistent with the observed Kd of the wild-type protein for the type I IL-1R.     

Cleavage of fibrinogen by the human neutrophil neutral peptide-generating protease.

The human neutrophil peptide-generating protease, which generates a low molecular weight vasoactive peptide from a plasma protein substrate, is directly fibrinolytic and cleaves human fibrinogen in a manner distinct from plasmin. Fibrinogen was reduced from 340,000 Mr to derivatives of 270,000-325,000 Mr during interaction with the protease at enzyme-to-substrate ratios of 0.3 or 1.0 microgram/1.0 mg. The 310,000-325,000 Mr cleavage fragments exhibited prolonged thrombin-induced clotting activity but were able to be coagulated, whereas the 270,000-290,000 Mr fragments were not able to be coagulated. Anticoagulants were not generated at either enzyme dose. As analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis in 4-30% gradient gels and 10% gels stained for protein and carbohydrate, the diminution to 310,000-325,000 Mr and the prolongation of thrombin-induced clotting time resulted from cleavage of the fibrinogen A alpha chain. The further decrease in size to 270,000-290,000 Mr was associated with B beta-chain and gamma-chain cleavage and an inability to form gamma-gamma dimers. The neutral peptide-generating protease, a distinct human neutrophil neutral protease with fibrinolytic and fibrinogenolytic activities comparable to those of plasmin on a weight basis, cleaves fibrinogen in a manner that is distinct from the action of plasmin, leukocyte elastase, and leukocyte granule extracts. It may be that the concerted action of this neutrophil protease to generate a vasoactive peptide and to digest fibrinogen and fibrin facilitates neutrophil movement through vascular and extravascular sites.     

Negative regulation of early B lymphopoiesis by interleukin 3 and interleukin 1 alpha.

We recently developed a two-step methyl cellulose culture system for murine lymphohemopoietic progenitors that are capable of differentiation along the myeloid and B-lymphoid lineages. In this system, two-factor combinations, which include steel factor plus interleukin (IL) 6, IL-11, or granulocyte colony-stimulating factor effectively supported the lymphomyeloid potential of primary colonies. Interestingly, IL-3 could neither replace nor act synergistically with steel factor in maintaining the B-lymphoid potential of the primary colonies although the frequency of colony formation was the same with IL-3 and steel factor. We now report that addition of IL-3 or IL-1 alpha to a permissive system suppresses the B-lymphoid potential of primitive progenitor cells in primary culture in dose-dependent fashion. In vivo transfer of the primary colonies to scid mice confirmed the suppressive effects of IL-3 and IL-1 alpha. In addition, IL-1 alpha inhibited pre-B-cell colony formation in the secondary culture. Once pre-B-cell colonies had formed in secondary culture, neither factor affected the proliferation of the pre-B cells. These results suggest negative regulatory roles for IL-3 and IL-1 alpha in early stages of B lymphopoiesis.     

Myocardial hypertrophy in transgenic mice overexpressing human interleukin 1[alpha ]

BACKGROUND: Interleukin (IL)-1 has profound effects on nonimmune cells and organs, including the heart. The effects of IL-1 on transgenic hearts have not yet been described. METHODS AND RESULTS: We generated transgenic mice overexpressing the human IL-1 gene under control of the cytomegalovirus enhancer/chicken beta-actin promoter. Heart weight-body weight ratio increased 1.4- to 2.2-fold in transgenic mice compared with wild-type mice. Lung weight-body weight ratio also increased in transgenic mice, all of which died within 14 days of birth. Light microscopy revealed concentric hypertrophy with cardiomyocyte hypertrophy in all transgenic mice and pulmonary edema in some of them. Electron microscopy showed myofilament loss and an increased number of giant mitochondria, but no sarcomere disarray. Northern blotting showed that gene expression had been reprogrammed in the left ventricle of transgenic mice. Expression of fetal-type genes such as prepro-atrial natriuretic factor and beta-myosin heavy chain were increased, but voltage-dependent calcium channel messenger RNA expression was decreased in the left ventricle of transgenic mice compared with that of wild-type mice. CONCLUSIONS: IL-1 may cause structural and functional alterations in cardiac myocytes.     

Inhibition of interleukin 1 induced chondrocyte protease activity by a corticosteroid and a nonsteroidal antiinflammatory drug.

We report that administration of the corticosteroid, methylprednisolone (PRED) inhibited interleukin 1 (IL-1) induction of chondrocyte caseinolytic activity (25-55%) and collagenolytic activity (15-24%). The nonsteroidal antiinflammatory drug (NSAID), naproxen (NAP) had no effect on either enzyme activity over a therapeutic range (7-30 micrograms/ml) but at 120 micrograms/ml inhibited IL-1 induced caseinolytic and collagenolytic activity by 17 and 19%, respectively. However, PRED (2 micrograms/ml) in combination with NAP (30 micrograms/ml) significantly increased the inhibition of caseinolytic activity (p less than 0.001) compared to that observed with PRED (2 micrograms/ml) alone. The suppression of IL-1 induced collagenolytic activity noted with PRED in combination with NAP did not exceed that observed with PRED alone.     

Reduction of experimentally produced acute myocardial infarction size by a new synthetic inhibitor, NCO-700, against calcium-activated neutral protease

Calcium-activated neutral protease (CANP) might be involved in the irreversible degradation of myocardial proteins in the ischemic region, leading to the loss of contractility. The new compound, NCO-700, and its analogues were synthetized against CANP. Among these analogues, NCO-700 was the most potent to reduce the size of acute myocardial infarction, which was produced by coronary artery ligation in rabbits, in vivo, although it showed less powerful action to inhibit CANP activity in vitro. The new reagent, NCO-700 might be promising to reduce acute myocardial infarction size and beneficial for the clinical studies, because it had no action to reduce cardiac muscle contractility, compared with beta antagonist or calcium-channel blockades.     

Two types of human mast cells that have distinct neutral protease compositions.

Two human mast cell types were identified by immunohistochemical techniques in skin, lung, and small intestine. One type contains the neutral proteases, tryptase and chymotryptic proteinase, and is termed the TC mast cell. The second type contains only tryptase and is termed the T mast cell. Both types are fixed better by Carnoy's fluid than by formalin. The percentage of mast cells accounted for by the T type was 12 in skin; 98 in mucosa and 13 in submucosa of small intestine; and 77 in bronchial/bronchiolar subepithelium, about 97 in bronchial/bronchiolar epithelium, and 93 in alveoli of lung. Dispersed lung cells contained 90% T mast cells. The mean area of TC mast cells (76 micron2) was slightly larger than that of T mast cells (66 micron2); however, there was such extensive overlap that individual mast cells belonging to different types could not be distinguished on the basis of size. The recognition of human mast cell types with distinct protease compositions suggests a higher level of complexity of human mast cell-mediated reactions than heretofore appreciated.     

Localization of the enzyme neutral endopeptidase to the human synovium.

Neuropeptides, contained within sensory nerve fibers in the synovium, are present in inflammatory joint fluids. The potency of these peptides in vitro has led to the hypothesis that enzyme degradation systems are operative in vivo. To address this question we localized neutral endopeptidase (NEP; EC 3.4.24.11) in human synovium. The normal human synovium failed to show any immunoreactivity for NEP. In the disease groups there was intense staining of cells surrounding blood vessels. Our data are consistent with the hypothesis that a proportion of synovial fibroblasts are the major source of this enzyme in the arthritic joint.     

Identification of a 31,500 molecular weight islet cell protease as cathepsin B.

A method for the preparation of a radioisotopically labeled active-site directed reagent for proteases (125I-Tyr-Ala-Lys-ArgCH2Cl) is described, and an example of its use as a sensitive method for identifying trypsin-like proteases is provided. This high specific activity reagent was then used in an attempt to identify proteases in rat islets of Langerhans involved in the conversion of proinsulin to insulin. Previous studies have indicated that the endoprotease involved in proinsulin conversion is a cysteine proteinase and that 125I-Tyr-Ala-Lys-ArgCH2Cl affinity labels an islet crude granule fraction protein having a molecular weight of 31,500. Here we demonstrate, using a probe of higher specific activity, that the major affinity-labeled proteins of the islet crude granule fraction, when displayed by sodium dodecyl sulfate gel electrophoresis, have molecular weights of approximately 39,000 (5%), 31,500 (53%), and 5,000-6,000 (37%), with several other minor proteins (less than 5%) also labeled. The two predominant labeled proteins were mainly soluble rather than membrane bound, and they exhibited patterns of competition with various inhibitors that were similar to the pattern shown by the conversion of proinsulin to insulin in vitro. A rabbit antibody to rat liver cathepsin B immunoprecipitated both affinity-labeled 31,500 and 5,000-6,000 molecular weight proteins, and on the basis of this and structural considerations the 31,500 molecular weight cysteine protease is identified as cathepsin B. The 5,000-6,000 molecular weight peptide is an NH2-terminal, active site cysteine-containing, proteolytic fragment of the 31,500 molecular weight protein. Because cathepsin B is not per se a candidate for the proinsulin convertase because of its excessively broad substrate specificity, these studies suggest that a similar enzyme or a modified form of this enzyme is active within the secretory progranules, whereas the more typical cathepsin B may be largely confined to lysosomal contaminants in our granule preparations.     

Regulation of hippocampal glutamate receptors: evidence for the involvement of a calcium-activated protease.

Specific [3H]glutamate binding to rat hippocampal membranes and the calcium-induced increase in this binding are markedly temperature-sensitive and are inhibited by alkylating or reducing agents as well as by various protease inhibitors. N-Ethylmaleimide, chloromethyl ketone derivatives of lysine and phenylalanine, and tosylarginine methyl ester decrease the maximum number of [3H]glutamate binding sites without changing their affinity for glutamate. Preincubation of the membranes with glutamate does not protect the glutamate "receptors" from the suppressive effects of these agents. The proteases trypsin and alpha-chymotrypsin increase the maximum number of [3H]glutamate binding sites. The effects of calcium on glutamate binding are different across brain regions. Cerebellar membranes are almost insensitive whereas hippocampal and striatal membranes exhibit a strong increase in the number of binding sites after exposure to even low concentrations of calcium. These results suggest that an endogenous membrane-associated thiol protease regulates the number of [3H]glutamate-associated thiol protease regulates the number of [3H]glutamate binding sites in hippocampal membranes and that this is the mechanism by which calcium stimulates glutamate binding. The possibility is discussed that the postulated mechanisms participate in synaptic physiology and in particular may be related to the long-term potentiation of transmission found in hippocampus under certain conditions.     

Potential antiinflammatory effects of interleukin 4: suppression of human monocyte tumor necrosis factor alpha, interleukin 1, and prostaglandin E2.

Stimulated human monocytes/macrophages are a source of mediators such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and prostaglandin E2 (PGE2), which can modulate inflammatory and immune reactions. Therefore, the ability to control the production of such mediators by monocytes/macrophages may have therapeutic benefits, and it has been proposed that glucocorticoids may act in this way. Purified human monocytes, when stimulated in vitro with lipopolysaccharide (LPS) or with LPS and gamma interferon (IFN-gamma), produce TNF-alpha, IL-1, and PGE2. Cotreatment of stimulated cells with the purified human lymphokine, interleukin 4 (IL-4 greater than or equal to 0.1-0.5 unit/ml; 12-60 pM) dramatically blocked the increased levels of these three mediators; for TNF-alpha and IL-1, the inhibition was manifest at the level of mRNA. Thus, IL-4 can suppress some parameters of monocyte activation and, as for B cells, have opposite effects to IFN-gamma. The effects of IL-4 on human monocytes are similar to those obtained with the glucocorticoid dexamethasone (0.1 microM).     

Differential effects of interleukin 1 alpha and 1 beta on cultured human and rat thyroid epithelial cells

The effects of human recombinant interleukin 1 alpha (20 pg/1-2 micrograms/l) and 1 beta (200 pg/1-20 micrograms/l) on two systems of thyroid cells have been compared. The thyroglobulin and cAMP secretion and the DNA content of human thyroid cells cultured in monolayer and of continuously grown rat thyroid cells, Fischer rat thyroid cell line have been studied. The growth of the rat thyroid cell line was inhibited by interleukin 1 beta (20 ng/1-20 micrograms/l), but not by interleukin 1 alpha. None of the cytokines changed the cAMP production of the rat thyroid cells. In contrast, both cAMP production and thyroglobulin secretion were inhibited dose-dependently by the cytokines in human thyroid cells in secondary cultures. These results caution the interpretation and extrapolation of changes induced by interleukin 1 from one cell system to the other.