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H A Chapman

Publications and source records attributed to H A Chapman.

18 recordsLinked to original sources
PubMed1992

Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease.

Human alveolar macrophages (HAM) express an elastase activity of acidic pH optimum inhibitable by cysteine protease inhibitors. Recent studies indicate that the only known eukaryotic elastinolytic cysteine protease, cathepsin L, cannot completely account for this activity. In order to search for additional cysteine proteases with elastinolytic activity, low degeneracy oligonucleotide primers based on regions of strong homology among the known cysteine proteases were used to screen reverse-transcribed HAM RNA for cysteine proteases by the polymerase chain reaction. Among the cDNA sequences generated was a 493-base pair product highly homologous to bovine cathepsin S. Screening of a HAM cDNA eukaryotic expression library with this cDNA yielded a 1.7-kilobase full-length cDNA highly homologous to bovine cathepsin S (approximately 85% identical). This cDNA predicts a 331-amino acid preprocathepsin. Expression of this cDNA in COS cells revealed the active enzyme to be a single chain 28-kDa protease, as judged by active site labeling with a novel iodinated analogue of N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucylamido-(4-gua nido)butane (E-64). The recombinant enzyme was found to be elastinolytic toward 3H-labeled elastin (bovine ligamentum nuchae) at pH 5.5 but with 25% of this activity retained at pH 7.0. Labeling of HAM with the active site probe revealed these cells express a 28-kDa cysteine protease, and Northern blot analysis revealed the presence of a approximately 1.7-kilobase cathepsin S mRNA. These data establish that human macrophages express at least two cysteine proteases with elastinolytic activity. The relatively broad pH range of human cathepsin S activity suggests this enzyme may contribute to the contact-dependent elastase activity of live human alveolar macrophages.

Amino Acid Sequence↗
PubMed1992

Visual information decoding deficits in schizophrenia.

Schizophrenic and control subjects were tested on two-flash fusion (TFF) and visual backward masking (VBM) tasks in a repeated measures design. Each subject was tested in a single session. Both tasks used the same equipment and stimuli. There was no difference between the groups in their ability to detect the presence of two separate stimuli in the TFF task. Schizophrenic subjects did require longer interstimulus intervals (ISI) than control subjects to accurately report one of the two targets in the VBM task. Analysis of individual targets reveals that the VBM deficit is a function of the similarity of the target and mask. The more feature detail discrimination necessary, the longer an ISI is required in VBM. The data are interpreted as supporting the conclusion that since the groups did not differ in their performance of the TFF task, which would also have been affected by a sensory abnormality, the deficit in VBM must be explained by reference to a higher level of information processing. The VBM deficit is a failure to decode the target stimulus, and is not simply a function of abnormalities due to an overactive transient channel system.

Adult↗
PubMed1991

A serine esterase released by human alveolar macrophages is closely related to liver microsomal carboxylesterases.

We identified a 60-kDa diisopropylfluorophosphate-(DFP) reactive protein in human bronchoalveolar lavage fluid, at a yield of 50-100 pmol/lavage. The protein is associated with the cell-free lavage fluid sediment, which consists mainly of surfactant. [3H]DFP labeling is inhibited by heating to 56 degrees C, 2 mM phenylmethylsulfonylfluoride and 1 mM bis(4-nitrophenyl)-phosphate. An identical 60-kDa [3H]DFP-reactive protein is present in the insoluble fraction of alveolar macrophage-conditioned culture medium and in total membrane preparations of alveolar macrophages. The [3H]DFP-labeled protein was purified approximately 30-fold from lavage fluid sediment by size-exclusion (Sephacryl S-200) and ion-exchange (Mono-Q) chromatography. Cyanogen bromide treatment of the partially purified protein produced a major labeled peptide of 14 kDa with an NH2-terminal sequence 90% identical to a region of form 1 rabbit liver microsomal carboxylesterase. Esterase activity in unlabeled starting material, detected using p-nitrophenyl valerate as substrate, copurified with the [3H]DFP-labeled enzyme. Degenerate oligonucleotide primers were designed based on the partial amino acid sequence and on a highly conserved region of known liver carboxylesterase sequences. Polymerase chain reaction using these primers and reverse-transcribed human alveolar macrophage mRNA yielded a 354-base pair product which was then used to screen a human alveolar macrophage cDNA library. A complete esterase sequence was obtained from two incomplete, overlapping clones, and is virtually identical to human liver carboxylesterase partial sequences. Northern blot analysis demonstrated a single approximately 1.7-kilobase transcript in human monocytes and alveolar macrophages, with much higher levels in the latter. These data indicate that human alveolar macrophages both contain and release a serine esterase that is apparently identical to liver microsomal carboxylesterase. Its enzymatic profile suggests it is a major component of alveolar macrophage-nonspecific esterase activity. We hypothesize that it acts as a detoxication enzyme in the lung.

Adult↗
PubMed1991

Role of enzyme receptors and inhibitors in regulating proteolytic activities of macrophages.

Expression of proteases by neutrophils and other cells with a prominent regulated secretory pathway is determined largely by stimulus-response secretion of proteins prepackaged in high concentration. The regulated secretory pathway is apparently minor in macrophages, and instead proteases are either channeled into lysosomes or secreted constitutively. Posttranslational regulation of macrophage proteases then depends on compartmentalizing enzymes to their sites of primary function. Available data suggest that cells use both specific receptors and inhibitors to accomplish this. Viewed in this context protease inhibitors primarily function to inhibit enzyme not bound to their receptor. Consonant with this model of regulation, connective tissue turnover by macrophages is a contact-dependent process relatively resistant to exogenous macromolecular inhibitors. Although limited information is available regarding determinants that modulate matrix metabolism by human macrophages, this model suggests that determinants of adhesion and colocalization of enzyme and substrate would be as or more important than alterations of inhibitors in the microenvironment of the cell.

Animals↗
PubMed1991

Cigarette smoking induces an elastolytic cysteine proteinase in macrophages distinct from cathepsin L.

Degradation of the interstitium of the lung by elastolytic enzymes is thought to be a critical component of the pathogenesis of emphysema. Alveolar macrophages are increased in numbers in cigarette smokers and contain the elastolytic cysteine proteinase cathepsin L. We sought to determine if cigarette smoking induces a change in cathepsin L levels in alveolar macrophages which would, in turn, alter the expression of elastolytic activity. Lysates of smokers' macrophages, assayed at pH 5.50, degraded more than seven times as much [3H]elastin as did lysates from nonsmokers' macrophages (44 +/- 20.8 vs. 6 +/- 1.6 micrograms.10(6) cells-1.24 h-1). Little or no activity was demonstrable at neutral pH. Immunoblots of macrophage lysates demonstrated that smokers' cells contain 3.7 +/- 1.1 times as much 25-kDa cathepsin L antigen as nonsmokers' cells. However, as judged by active site labeling, levels of active cathepsin L in smokers and nonsmokers are indistinguishable, suggesting that most of the 25-kDa antigen found in smokers' macrophages is inactive. Inhibitors of cathepsin L had little effect on lysate elastolytic activity, confirming that an enzyme other than cathepsin L is responsible for the increased elastolytic activity seen in smokers' macrophages. Further experiments demonstrated that this second enzyme(s) has a profile of inhibition indicating that it is a cysteine proteinase with optimal activity at pH 5.50. It is this second elastolytic cysteine proteinase(s) that is induced by exposure to cigarette smoke and is responsible for the sevenfold increase in elastolytic activity found in smokers' macrophage lysates.

Actins↗
PubMed1991

An autocrine role for urokinase in phorbol ester-mediated differentiation of myeloid cell lines.

The human myeloid cell line HL60 secretes urokinase-type plasminogen activator (uPA) and expresses its receptor. When stimulated with phorbol myristate acetate (PMA), both secretion of uPA and the expression of its receptor are up-regulated, and these cells differentiate to an adherent phenotype. This adhesive response is markedly reduced in the presence of uPA antibodies. The PMA response is restored by the addition of native uPA, an amino-terminal fragment of uPA (residues 1-143) devoid of proteolytic activity, or a synthetic peptide (residues 12-32) from the uPA growth factor domain known to mediate receptor binding. In contrast, the addition of catalytically active low molecular weight uPA, which is missing the growth factor domain, or a peptide from the catalytic domain (residues 247-266) is ineffective. The influence of uPA antibodies on a second marker of macrophage differentiation, cysteine proteinase activity, was also examined. Cysteine proteinase activity of HL60 cells is increased in PMA-treated cells after 24 h but it fails to increase in the presence of anti-uPA. This increase in cathepsin B-like activity is also restored by exogenous uPA. These experiments indicate that an autocrine interaction of the growth factor domain of uPA with its receptor mediates an essential step in PMA-mediated myeloid cell differentiation.

Cell Adhesion↗
PubMed1990

Developmental expression of plasminogen activator inhibitor type 1 by human alveolar macrophages. Possible role in lung injury.

Urokinase activity is regulated by the specific endogenous plasminogen activator inhibitors type 1 (PAI-1) and type 2 (PAI-2). One of these inhibitors, PAI-1, has been directly implicated in connective tissue metabolism by virtue of its ability to bind extracellular matrix proteins. Because the normal lung is relatively rich in urokinase and abnormalities in urokinase activity have been associated with fibrotic lung diseases, we have explored the possibility of local production of PAI-1 and PAI-2 in human lung. Reverse transcription and subsequent amplification by the polymerase chain reaction of total lung RNA revealed PAI-1 mRNA in each of three normal samples and in two specimens from patients with the adult respiratory distress syndrome (ARDS). In situ hybridizations of lung biopsy specimens from a patient with ARDS with cRNA probes to PAI-1 and PAI-2 indicated that alveolar macrophages express PAI-1 mRNA during the acute injury phase. Subsequent reverse transcription and PCR amplification of normal human monocyte and alveolar macrophage mRNA revealed that neither cell type expressed mRNA for urokinase inhibitors. However, after 24 h stimulation with endotoxin in vitro, monocytes were strongly positive for PAI-2 but negative for PAI-1 mRNA whereas, under the same conditions, alveolar macrophages exhibited mRNA for both PAI-1 and PAI-2. Metabolic labeling of endotoxin-stimulated alveolar macrophages with 35S-methionine followed by immunoprecipitation with PAI-1 and PAI-2 antibodies revealed that macrophages synthesized both PAI-1 and PAI-2. As judged by immunoprecipitation and functional studies, PAI-2 was found to be the major intracellular PA inhibitor whereas PAI-1 was found to predominate outside the cell. Thus, mononuclear phagocytes exhibit a developmental potential for PAI-1 expression. The release of PAI-1 by stimulated macrophages, as observed in the setting of ARDS, may be one mechanism by which these cells promote connective tissue accumulation.

Endotoxins↗
PubMed1990

Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome.

Abundant deposition of bronchoalveolar fibrin and fibronectin occurs during the exudative phase of the adult respiratory distress syndrome (ARDS), promoting hyaline-membrane formation and subsequent alveolar fibrosis. To explore the mechanisms that account for the persistence of bronchoalveolar fibrin and fibronectin, we compared the activity of urokinase, which is necessary for plasminogen activation and fibrin degradation, in cell-free bronchoalveolar-lavage fluid from 8 patients with ARDS, 9 patients with acute pulmonary diseases other than ARDS, and 10 normal subjects. The mean level of urokinase activity in the lavage fluid from the patients with ARDS was 0.003 IU per milliliter of fluid (range, 0 to 0.008), which was significantly lower (P = 0.001) than the level in the fluid from either the patients with pulmonary diseases other than ARDS (0.118 IU per milliliter [range, 0.032 to 0.295]) or the normal subjects (0.129 IU per milliliter [range, 0.045 to 0.198]). The lavage fluid from all the patients with ARDS also had antiplasmin activity, which would promote the persistence of fibrin. A true decrease in urokinase activity was confirmed by the failure of the lavage fluid from the patients with ARDS to convert [125I]plasminogen to plasmin. Despite the low urokinase activity, immunochemical assays revealed normal levels of urokinase antigen in the fluid from the patients with ARDS, suggesting the presence of urokinase inhibitors. Inhibitors were demonstrated directly by a fibrin gel-underlay assay that detects complexes of urokinase with inhibitors. Plasminogen-activator inhibitor type 1 was the principal inhibitor identified. We conclude that increased antifibrinolytic activity due to both urokinase inhibitors and antiplasmins in the bronchoalveolar compartment of patients with ARDS contributes to the formation and persistence of hyaline membranes, a key component of alveolar histopathology in ARDS.

Adult↗
PubMed1990

Alveolar macrophage urokinase receptors localize enzyme activity to the cell surface.

Human alveolar macrophages are known to synthesize urokinase (uPA) and a specific plasminogen activator inhibitor, PAI-2. In this study we have identified a uPA receptor expressed by these cells and defined the influence of PAI-2 on the interaction of uPA with its receptor. Alveolar macrophages from four normal volunteers were incubated with 55 kDa 125I-labeled uPA (0.24-8 nM) in the presence or absence of excess unlabeled uPA. Specific and saturable binding was demonstrable in all cases. Scatchard plots were linear; regression analysis revealed a mean Kd of 5.25 nM (range 3.2-6.7) and mean Bmax of 30.7 femtomoles/10(5) cells (range 21.5-34.5). The structure of the uPA receptor was defined by electroblotting membrane fractions of macrophages and sequentially exposing filters to uPA and uPA antibodies. Membranes from macrophages demonstrated binding of either uPA or a 15-kDa amino-terminal fragment of uPA to a 55- to 60-kDa glycosylated membrane protein. Binding of uPA to filters was blocked by a synthetic oligopeptide containing the known receptor binding region of native uPA. Preincubation of 125I-uPA with PAI-2 dramatically reduced the rate of association of uPA with macrophage uPA receptor. Conversely, receptor-bound uPA activity was less susceptible to inhibition by PAI-2 than soluble uPA activity. These data indicate that normal alveolar macrophages express uPA receptors. The receptor preferentially binds and protects free uPA over complexed enzyme, indicating that one function of the receptor is to allow the cells to express active uPA in an inhibitor-rich environment.

Cell Line↗
PubMed1990

Identification of cystatin C, a cysteine proteinase inhibitor, as a major secretory product of human alveolar macrophages in vitro.

The major inhibitor of the cysteine class of proteinases found in human body fluids, such as spinal fluid, milk, and seminal plasma, is cystatin C. In this study we show that human bronchoalveolar fluid also contains cystatin C and examine cystatin C expression by alveolar macrophages in vitro. Immunoprecipitation of extracts of metabolically labeled cells and immunoblotting of cellular extracts and culture media show that cystatin C is synthesized as a 14 (+/- 0.5) kilodalton (kD) protein and that greater than 90% of the protein is released as the 14 kD product into the culture supernatant (26.5 +/- 6.8 ng per 10(6) cells per 24 h). Cystatin C is not one of the most abundant proteins secreted during the first 24 h in vitro, representing approximately 10 to 12% of the total protein released by normal nonsmoker macrophages. Alveolar macrophages obtained from cigarette smokers or nonsmoker macrophages exposed to zymosan in vitro released 10 to 55% less cystatin C than nonsmoker macrophages. We also assayed culture supernatants from macrophages of smokers and nonsmokers for functional cystatin C. Supernatants of nonsmoker macrophages inhibited cathepsin B-like amidolytic activity in a fluorometric assay at pH 5.5. The inhibition was blocked by adsorption with Sepharose-coupled cystatin C antibodies and the inhibitor subsequently recovered from the Sepharose beads. In contrast, supernatants from smoker macrophages had obvious cathepsin B-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchoalveolar Lavage Fluid↗
PubMed1990

Uptake of extracellular enzyme by a novel pathway is a major determinant of cathepsin L levels in human macrophages.

The phorbol myristate acetate (PMA)-differentiated myelomonocytic cell line, THP-1, and human alveolar macrophages contain the cysteine proteinase cathepsin L. This enzyme is synthesized as a 43-kD proenzyme and processed to the active 25-kD form. Differentiation of THP-1 cells in the presence of human serum resulted in an increase in the size of the vacuolar compartment and the accumulation of more 25-kD cathepsin L antigen, as compared with THP-1 cells differentiated in the presence of fetal calf serum. Cells cultured in both types of sera have equivalent levels of cathepsin L mRNA. Metabolic labeling experiments demonstrated equivalent rates of synthesis, processing to the active form, and persistence in both culture conditions. An extracellular source of enzyme was documented by immunoblotting human serum which demonstrated 25-kD cathepsin L antigen; furthermore, we demonstrated that both THP-1 cells, differentiated in human serum, and human alveolar macrophages take up the 43-kD proenzyme and process it to the 25-kD form. Thus, human serum contains a factor(s) that induces both a marked increase in the size of the vacuolar compartment in differentiated THP-1 cells and a novel pathway that is responsible for the uptake and processing of extracellular cathepsin L. The activity of this inducible pathway is a major determinant of levels of intracellular cathepsin L. Cathepsin L is a potent elastase and the regulation of its uptake and processing may play a role in the pathogenesis of disease processes characterized by the destruction of elastin, such as pulmonary emphysema.

Blotting, Western↗
PubMed1979

Modulation of plasminogen activator secretion by activated macrophages: influence of serum factors and correlation with tumoricidal potential.

A high molecular weight fraction of human serum (Fr-1) was found to both inhibit macrophage tumoricidal activity and enhance plasminogen activator activity in supernates over activated macrophages in vitro. Conversely, a 40- to 90-kilodalton serine esterase (Fr-3) also found in normal human serum and endotoxin enhanced tumoricidal potential and suppressed the supernatant plasminogen activator activity. Inactivation of either Fr-1 or Fr-3 by 2-mercaptoethanol or diisopropyl fluorophosphate, respectively, abolished both biologic effects. Examination of cell-associated and culture medium plasminogen activator activity before and after acidification to inactivate proteinase inhibitors indicated that suppression of plasminogen activator activity by Fr-3 or endotoxin most likely represents modulation of macrophage plasminogen activator secretion. The findings demonstrate that activated macrophages are capable of highly coordinated biologic responses to alterations in their microenvironment and suggest that it is in fact the high potential for such responsiveness that reliably characterizes the activated macrophage. The results also suggest that an endogenous regulatory system dependent on the interaction of serine esterases may operate to regulate the functional capabilities of activated macrophages.

Animals↗
PubMed1978

Modulation of macrophage tumoricidal capability by polyene antibiotics: support for membrane lipid as a regulatory determinant of macrophage function.

We have examined the effects of the sterol-binding polyene antibiotics on macrophage tumoricidal capability. Incubation for 2 hr of activated macrophages from bacillus Calmette-Guérin-infected mice with amphotericin B at 0.5--2 microgram/ml or amphotericin B methyl ester at 0.5--10 microgram/ml enhanced the capability of activated macrophages to kill 3T12 cells. These polyenes did not make normal or stimulated macrophages tumoricidal. Experiments with the ionophores gramicidin, alamethecin, nigericin, and valinomycin indicate that the ionophoretic properties of amphotericin B may not account for its enhancing effect on macrophage tumoricidal potential. Two polyenes with a smaller ring structure, filipin and pimaricin, were also ineffective suggesting that stereospecific modifications in membrane lipid organization underlie the enhancing effect of amphotericin B. The results suggest that the clinical efficacy of amphotericin B in promoting resistance to fungal disease and possibly to neoplasia may operate in part through potentiation of macrophage effector functions.

Amphotericin B↗
PubMed1977

Modulation of macrophage tumoricidal capability by components of normal serum: a central role for lipid.

The tumoricidal capabilities of macrophages can be reversibly inhibited by a lipoprotein of high molecular weight, and the inhibition appears to be reproduced by enrichment of macrophage plasma membranes with cholesterol. A second serum component of lower molecular weight enhances macrophage cytotoxicity. The presence of these components in normal serums suggests a physiological role for such factors in the regulation of macrophage function.

Animals↗
PubMed1977

Macrophage tumor killing: influence of the local environment.

Tumor killing by activated macrophages is not a highly determined biologic event, but a relative capability influenced by the local environment. An intrinsic macrophage cytotoxic effector system is modulated by serum and other environmental factors that can either enhance or suppress tumor killing. Activated macrophages kill tumor cells only when a regulating threshold drops to a critically low level.

BCG Vaccine↗