Michael S. Rohrbach

Serum Angiotensin-Converting Enzyme Activity in Evaluating the Clinical Course of Sarcoidosis

Thirty-five patients with sarcoidosis were serially monitored for serum angiotensin-converting enzyme activity by a simple radiochemical assay, and we analyzed the relation of such activity to the clinical status as judged from changes in chest roentgenograms, pulmonary function, or symptoms. Converting enzyme levels closely paralleled and occasionally antedated changes in clinical status in patients either undergoing spontaneous remission or being treated with prednisone. Converting enzyme activity seems to be a sensitive index for evaluating the clinical course of sarcoidosis. Converting enzyme determinations may be helpful in corroborating the diagnosis, assessing the likelihood of spontaneous remission, confirming the clinical status of the patient, and determining the adequacy of glucocorticoid therapy.

[Glycine-1-14C]hippuryl-l-histidyl-l-leucine: A substrate for the radlochemical assay of angiotensin converting enzyme

Abstract [ Glycine -1- 14 C]hippuryl- l -histidyl- l -leucine was synthesized and evaluated as a substrate for the radiochemical assay of angiotensin converting enzyme. Hydrolysis is measured by quantitation of the liberated [ glycine -1- 14 C]hippuric acid by liquid seintillation counting and is linear up to 30% hydrolysis. The advantages of the radiochemical assay over the spectrophotometric quantitation of the liberated hippuric acid are its increased sensitivity and lack of interference by nonionic detergents or lipids.

Zymosan induces selective release of arachidonic acid from rabbit alveolar macrophages via stimulation of a β-glucan receptor

Zymosan, which is composed primarily of α‐mannan and β‐glucan polymers, is a well recognized activator of macrophages. The type receptor by which unopsonized zymosan induces arachidonic acid release was investigated. It was found that particulate β‐glucan and zymosan stimulated an identical dose‐dependent release of arachidonic acid. This release of arachidonic acid by zymosan was blocked by soluble β‐glucans whereas soluble mannan had no effect. This inhibition was not due to a general toxic effect of the soluble β‐glucans as they had no effect on calcium ionophore‐induced release of arachidonic acid. β‐glucan‐induced fatty acid release from these cells was shown to be fairly specific for arachidonic acid. These data reveal that zymosan stimulates the specific release of arachidonic acid from rabbit alveolar macrophages, at least in part, via a β‐glucan receptor.

CANDIDA ALBICANS INDUCES THE RELEASE OF INFLAMMATORY MEDIATORS FROM HUMAN PERIPHERAL BLOOD MONOCYTES

Candida albicans (C. albicans) is a major nosocomial pathogen. We examined arachidonic acid (AA) and cytokine production by monocytes stimulated with C. albicans. [14C]-AA labeled monocytes released 8.9 ±2.3% of the incorporated AA following stimulation with live C. albicans (C. albicans: monocyte of 16∶1) (P=0.0002). Prior studies indicate that solubleα-mannans andβ-glucans antagonize mannose andβ-glucan receptors, respectively. Preincubation of monocytes withα-mannan (100μg/ml) caused 45.8 ±5.7% inhibition of [14C]-AA release, whereasβ-glucan (100μg/ml) yielded 43.7 ±6.0% inhibition (P<0.05 for each compared to control). Additionally, monocytes stimulated with C. albicans also released interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), interleukin-6 (IL-6) and interleukin-8 (IL-8). However, a-mannan orβ-glucan failed to inhibit IL-1β release. These data indicate that C. albicans induces monocytes to release AA and inflammatory cytokines. Furthermore, AA, but not cytokine liberation, is partially mediated by a-mannan andβ-glucan components of the fungus.

Tannin is the major agent present in cotton mill dust responsible for human platelet 5-hydroxytryptamine secretion and thromboxane formation

Aqueous extracts of cotton mill dust were examined for their ability to activate washed human platelets as indicated both by the secretion of 5-hydroxytryptamine and the generation of thromboxane A2. The cotton dust extract promoted 5-hydroxytryptamine secretion in a dose-dependent manner. When expressed in terms of its tannin concentration, the dose curve for cotton dust extract was identical to that obtained with purified tannin. Cotton dust extract also promoted the generation of low levels of thromboxane A2. Fractionation of the extract by Amicon ultrafiltration into a > 10,000 MW fraction and a < 10,000 MW fraction revealed that the < 10,000 MW fraction contained an inhibitor of the thromboxane assay. The dose-response curve for thromboxane A2 generation mediated by the > 10,000 MW cotton dust extract fraction was indistinguishable from that of purified tannin. Thus, 2 criteria show that platelet activation by cotton mill dust appears due to the tannin present in the dust.

Characteristics of Monocyte Angiotensin‐Converting Enzyme (ACE) Induction by Dexamethasone

Monocyte maturation to macrophages and transformation into epithelioid granuloma cells in some granulomatous diseases are accompanied by the induction of membrane‐bound angiotensin‐converting enzyme (ACE). The physiologic and pathophysiologic roles of ACE generated in these processes are not known. The pattern and the mechanism of ACE induction in human monocytes are also not well understood. Dexamethasone is one of the agents reported to induce elevated ACE activity in human monocytes, and therefore a suitable tool for studying the phenomenon. This study shows that dexamethasone augments monocyte ACE in a Diphasic dose‐dependent manner with maximum effect at 10‐5 M concentration. Although it enhances the level of ACE activity, dexamethasone doss not alter the time course for ACE induction from that found in unstimulated monocytes. The ACE activity of monocytes cultivated in 10 nM dexamethasone and then exposed to 10‐3 M diazosulfanilic acid (DASA) is reduced approximately by 80% in comparison with cells not treated with DASA, demonstrating that dexamethasone‐induced ACE is an ectoenzyme. Dexamethasone does not increase the activity of other monocyte ectoenzymes: γ‐glutamyltransferase, alkaline phosphodiesterase‐I, and leucine aminopeptktase, showing that dexamethasone induction of ACE is a specific, rather than generalized, effect on plasma membrane enzymes. It is suggested that the increase in ACE activity is due to the increased rate of enzyme synthesis.

Metabolism and subcellular localization of angiotensin converting enzyme in cultured human monocytes

The enhancement of monocyte ACE activity during culture by autologous T-lymphocytes was shown to be due to a stimulation of the rate of ACE synthesis. The rate of synthesis increased from 0.020 mU/10(6) monocytes/hr in monocytes cultured alone to 0.063 mU/10(6) monocytes/hr in monocytes co-cultured with T-lymphocytes. The presence of T-lymphocytes during culture did not alter the rate of ACE degradation observed in monocytes cultured alone. The ACE induced in monocytes by T-lymphocytes appears to be an ecto-enzyme. Brief exposure to diazosulfanilic acid (10(-3) M) and papain (250 micrograms/ml) reduced ACE activity 89% and 66%, respectively, without appreciably altering the activity of the cytosolic enzyme, lactate dehydrogenase.

Structural determinants of the platelet agonist activity of cotton bract condensed tannin

Previous studies on the platelet agonist activity of cotton bract condensed tannin have used tannins with apparent molecular weights greater than 10,000 Da as measured by dialysis and/or ultrafiltration. However, because tannins in cotton dust are heterogeneous in terms of molecular weight, it was important to determine if platelet agonist activity was related to polymer length. To accomplish this, aqueous extracts of cotton dust were fractionated by a series of graded Amicon ultrafiltrations and the resulting fractions were examined for tannin content and platelet agonist activity. All tannins with apparent molecular weight greater than 1000 Da were equally potent platelet agonists while those less than 1000 Da had no measurable platelet agonist activity. This polymer length dependence for activity was supported by the observation that two procyanidin dimers had only minimal platelet agonist activity. In addition, methylation of the tannin hydroxyl groups resulted in the loss of platelet agonist activity. Thus, the platelet agonist activity of tannin requires a minimum polymer length and the presence of free hydroxyl groups.

Cotton Bract Tannin: A Novel Human T-Lymphocyte Mitogen and a Possible Causative Agent of Byssinosis

Cotton bract tannin, a major organic component of cotton dust, was tested for mitogenic activity upon human T lymphocytes. Tannin caused polyclonal activation of human T lymphocytes in vitro. The pattern of T-cell response to tannin was similar to the pattern seen in lymphocytes stimulated with the well-known plant lectin T-cell mitogens, PHA and ConA. The response to tannin was shown to be dependent upon tannin dose and presence of monocytes. The maximum response occurred after 3-4 days in culture with the magnitude comparable to the one achieved by ConA. Lipopolysaccharide from Enterobacter agglomerans, a major contaminant of cotton dust, was shown not to be a mediator of tannin mitogenicity in vitro. A role for tannin as a polyclonal cell activator and, therefore, a possible etiologic agent of byssinosis is suggested by these observations.

Cotton Condensed Tannin: A Potent Modulator of Alveolar Macrophage Host-Defense Function

Alveolar macrophages are the resident airway cells primarily responsible for the protection of the lungs against inhaled toxins and other biologically active material. A number of functional capabilities constitute their host-defense function. They can phagocytize and inactivate foreign material by production of reactive oxygen intermediates or the action of hydrolytic enzymes. In the absence of phagocytosis, macrophages can secrete reactive oxygen intermediates or enzymes that inactivate extracellular biologically active material. They also can secrete metabolites of arachidonic acid and other cytokines that contribute to the inflammatory response of the lungs. Macrophages also secrete a variety of peptide and lipid chemotactic factors that lead to the recruitment of other inflammatory cells into the airways. The condensed tannins, which constitute a significant percentage of the water soluble compounds present in respirable cotton mill dust, dramatically alter the host-defense function of alveolar macrophages in vitro. Tannin inhibits both phagocytosis and production of reactive oxidants in a dose-dependent manner with EC50s of 16 micrograms/mL and 3 micrograms/mL, respectively. This inhibition dramatically decreases the ability of resident alveolar macrophages to clear and detoxify potentially harmful inhaled particles. However, at similar concentrations, tannin stimulates the dose-dependent secretion (EC50 = 15 micrograms/mL) of a low molecular weight lipid neutrophil chemotactic factor that could result in an inflammatory reaction with the recruitment of neutrophils into the lungs. At slightly higher concentrations, tannin promotes the dose-dependent release of arachidonic acid from the macrophage membranes (EC50 = 65 micrograms/mL), which could also contribute to the local inflammatory reaction. Finally, tannin also causes secretion of the cytokine, interleukin-1, from the monocyte precursors of macrophages with an EC50 of 32 micrograms/mL. Interleukin-1 has been implicated as one of the causative agents in the development of fever.