Rohrbach, Michael S.

Candida albicans is an increasingly important fungal pathogen. Alveolar macrophages respond to fungal components such as zymosan by releasing arachidonic acid (AA) and AA metabolites. However, few studies hypothesized that macrophages respond to C. albicans by releasing AA and generating AA metabolites as a consequence of interaction of mannose and beta-glucan receptors with fungal cell wall components. [14C]AA-labeled rabbit alveolar macrophages released AA following stimulation with either live or heat-killed C. albicans. High-pressure liquid chromatography analysis revealed that 55% of the AA released was metabolized via cyclooxygenase and lipoxygenase pathways. The metabolites consisted of prostaglandin E2, prostaglandin F2 alpha, 6-ketoprostaglandin F1 alpha, thromboxane B2, and leukotrienes B4 and D4. We further examined the roles of alpha-mannan and beta-glucan components of C. albicans in mediating these alterations of eicosanoid metabolism. Prior work in our laboratory has shown that soluble alpha-mannan and beta-glucan inhibit macrophage mannose and beta-glucan receptors, respectively. Incubation of alveolar macrophages with soluble alpha-mannan derived from C. albicans (1 mg/ml) resulted in 49.8% +/- 2.6% inhibition of macrophage AA release during stimulation with intact C. albicans (P = 0.0001 versus control). Macrophage AA release in response to C. albicans was also inhibited to a significant but lesser degree by soluble beta-glucan (36.2% +/- 1.3%; P = 0.008 versus control). These results indicate that C. albicans stimulates macrophage AA metabolism and that these effects are partly mediated by alpha-mannan and beta-glucan constituents of the fungus.

Abstract 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.

The role of Ca2+ was investigated in the response of alveolar macrophages to cotton tannin, an agent implicated in the lung disease byssinosis in textile mill workers. A physiological concentration of extracellular Ca2+ was found to be required for tannin-mediated release of radiolabeled arachidonic acid (AA). Flow cytometry using indo 1 indicated that tannin caused a rapid and dose-dependent Ca2+ increase in macrophages that also required extracellular Ca2+. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid virtually abolished the Ca2+ influx mediated by tannin but had little effect on intracellular Ca2+ release induced by thapsigargin, N-formylmethionyl-leucylphenylalanine, or thimerosal. A mechanism for extracellular Ca2+ influx was demonstrated by rapid Mn2+ quenching of indo 1 by tannin. Verapamil inhibited tannin-mediated Ca2+ influx and AA release, but the effective concentration was 100 microM. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid chelated all Ca2+ in the cells and effectively abolished the tannin response. Exposure to tannin was not associated with cytotoxicity, as judged by 51Cr release. The data suggest that tannin induces Ca2+ influx in alveolar macrophages, which represents an important prerequisite for a cell-signaling pathway resulting in the accumulation of free AA.

Abstract Inhalation of cotton mill dust leads to the development of the occupational lung disease byssinosis in a portion of the exposed workers. Condensed tannins present in the dust have biologic activities consistent with the hypothesis that they are one of the etiologic agents for the disease. Inhalation of either cotton dust or tannin provokes an acute inflammatory response characterized by the influx of neutrophils into the airways. The secretion of a low-molecular-weight, lipid neutrophil chemotactic factor from the alveolar macrophages in response to tannin stimulation appears to be important in this inflammatory process. In these studies, the effect of tannin on the ability of alveolar macrophages and neutrophils to produce hydrogen peroxide was examined. Low concentrations of tannin itself induced a modest production of hydrogen peroxide from conditioned rabbit alveolar macrophages, while higher concentrations failed to induce peroxide production. In the presence of an independent stimulator of peroxide production (concanavalin A), tannin inhibited peroxide production at all concentrations examined. Aqueous extracts of cotton mill dust (CDE) had an identical effect on peroxide production in a manner that indicated that the tannin present in the dust was responsible for the effect. Like its direct effect on macrophage peroxide production, tannin induced modest peroxide production in human neutrophils. However, unlike its effect on macrophages, tannin enhanced the peroxide production induced by the presence of an independent stimulator (phorbol myristate acetate). CDE had a similar effect on peroxide production, but the dose-response curves suggested that only the high-molecular-weight polymers of tannin present in the CDE were able to enhance peroxide production. These results indicate that inhaled tannin would decrease the ability of resident alveolar macrophages to inactivate inhaled, potentially harmful cotton dust particles but would enhance the host-defense function of the recruited neutrophils. This difference in the action of tannin on neutrophils and macrophages may be of importance in the pathogenesis of byssinosis.

Abstract The condensed tannin present in cotton mill dust profoundly alters the functional capabilities of resident alveolar macrophages. Previous studies from this laboratory have shown that in vitro exposure of rabbit resident alveolar macrophages to condensed tannin significantly inhibits the ability of these cells to produce reactive oxygen intermediates or to ingest particles. In the present study, we demonstrate that condensed tannin also alters arachidonic acid (C20:4) metabolism in these cells. Exposure of rabbit resident alveolar macrophages to condensed tannin results in the time- and dose-dependent release of C20:4 from the membrane phospholipids. The release of C20:4 occurred only at tannin concentrations > 25 µg/ml and was maximal 90 min after the onset of exposure. The EC50 for release was 75 µg/ml. Exposure to 100 µg/ml tannin resulted in the release of 20 ± 3% of the [14C]C20:4 incorporated in the cell membrane. In comparison, exposure to 160 µg/ml zymosan resulted in the release of 14 ± 4% of the [14C]C20:4. For both tannin and zymosan, phosphatidylcholine and phosphatidylinositol were the principal sources of the released C20:4. Approximately 63% of the C20:4 released after zymosan stimulation was further metabolized, mainly via the cyclooxygenase pathway. The major metabolites were 6-ketoprostaglandin F1α, prostaglandin F2α, and prostaglandin E2. In contrast, only 24% of the C20:4 released by tannin was subsequently further metabolized. The metabolites formed were essentially evenly distributed between products of the cyclooxygenase pathway and the lipoxygenase pathway. Exposure of alveolar macrophages to 50 µg/ml tannin for 30 min reduced the ability of the cells to subsequently incorporate C20:4 by 50 to 70%. In contrast, exposure of the cells to 160 mg/ml zymosan for 30 min had only a minimal effect on the subsequent ability of these cells to incorporate C20:4. These results indicate that tannin promotes C20:4 release, at least in part, by inhibiting its reacylation back into phospholipids, a mechanism that differs from that of zymosan.

Abstract Pneumocystis carinii is an opportunistic organism that causes severe lung injury in immunocompromised hosts. Macrophage responses to P. carinii are poorly defined. Arachidonic acid (AA) and its metabolites are potent mediators of inflammation and have been implicated in host response to microorganisms. We therefore examined the production of eicosanoids from rat and rabbit alveolar macrophages stimulated with purified P. carinii. [14C]AA-labeled rabbit macrophages released 8.50 ± 1.33% of the incorporated [14C]AA after 90 min in response to P. carinii (P = 0.0001 compared with unstimulated controls). In contrast, a similar number of rat alveolar macrophages exhibited a smaller but significant response to P. carinii, releasing 3.84 ± 1.54% of their [14C]AA after 90 min (P = 0.001 compared with control). We further determined that P. carinii stimulated substantial production of prostaglandin E2 and concurrently a small amount of leukotriene B4 release from alveolar macrophages. To further investigate whether serum opsonization of P. carinii enhances these alterations in AA metabolism, we assessed the effect of P. carinii immune serum on P. carinii-induced AA release. P. carinii opsonized with this antiserum caused significantly greater AA release from rat alveolar macrophages than either unopsonized P. carinii or organisms opsonized with nonimmune serum. Previous studies suggest that P. carinii interacts with macrophage β-glucan and mannose receptors. However, incubation of macrophages with P. carinii in the presence of either soluble β-glucan or α-mannan failed to alter the release of AA from macrophages in response to P. carinii. Macrophage release of eicosanoids represents a potentially important host inflammatory response to P. carinii infection.

SUMMARYThe ability of monocyte subpopulations to be induced selectively by T lymphocytes to synthesize enhanced levels of angiotensin-converting enzyme (ACE) was examined using an in vitro model employing normal peripheral blood monocytes and T lymphocytes. Separation of monocytes into subpopulations on the basis of buoyant density indicated no difference in the ability of the resulting monocyte subpopulations to produce basal levels of ACE when cultured in the absence of T lymphocytes. However, the subpopulations differed significantly in their ability to synthesize enhanced levels of ACE in response to the presence of autologous T lymphocytes; low-density monocytes were induced by T lymphocytes to synthesize three-fold more ACE than were highdensity monocytes. Surface antigen labelling using MoAbs demonstrated that the low-density monocyte subpopulations also had a significantly higher percentage of Leu-M2+ monocytes compared with the high-density monocyte subpopulations. When monocytes were separated on the basis of the presence of the Leu-M2 antigen using an immune rosetting technique, T lymphocytes were able to induce significantly elevated levels of ACE in the Leu-M2+ enriched monocyte subpopulation but were unable to induce ACE beyond basal levels in the Leu-M2+-depleted monocyte subpopulation. These results demonstrate that monocytes are heterogeneous with respect to their ability to be induced by T lymphocytes to synthesize ACE. This raises the possibility that selective accumulation of a monocyte subpopulation in the granulomatous inflammation of sarcoidosis may be one of the factors required for elevated ACE synthesis in the resulting granuloma epithelioid cells.

SUMMARY The T lymphocyte-mediated induction of angiotensin-converting enzyme (ACE) in cultured autologous peripheral blood monocytes has been proposed as a model system for the investigation of the in vivo induction of ACE in the monocyte-derived granuloma epithelioid cells of some granulomatous diseases such as sarcoidosis. The studies described here were designed to evaluate the specificity of the model system by comparing the parameters for induction of ACE with those for the induction of another monocyte metallo-ecto-peptidase, leucine aminopeptidase (LAP). The concentration of LAP in freshly isolated monocytes was 009 mU/106 monocytes (s.e.m. 0.04) and increased to a maximal value of 019 mU/106 monocytes (s.e.m. 0.32) after 3 days when monocytes were cultured alone. ACE was not detectable in freshly isolated monocytes. However, after 6 days of culture, monocytes contained 0.22 m U ACE/106 monocytes (s.e.m. 0.04). Comparison of the levels of ACE and LAP induced during culture of monocytes alone indicated that the induction of these two enzymes was correlated. The induction of both enzymes was further enhanced by the presence of T lymphocytes in a dose-dependent manner. At 4 × 106 T lymphocytes per culture, ACE levels increased to 1.81 mU/106 monocytes (s.e.m. 0.24) and LAP levels to 1.03 mU/106 monocytes (s.e.m. 0.35). The enhancement of ACE activity required autologous lymphocytes, while heterologous T lymphocytes were equally effective in inducing LAP. Comparison of the levels of ACE and LAP induced during co-culture of autologous T lymphocytes and monocytes from 21 independent donors, demonstrated no correlation between the induction of ACE and LAP. These data indicate that, although T lymphocytes also enhance the induction of LAP, the underlying mechanism must differ from that of ACE induction.

AbstractPrevious studies with macrophages that had been prelabeled with [14C]arachidonic acid (20∶4) have shown that condensed tannin is a potent agonist for the release of arachidonic acid. However, it has not been demonstrated that the percentage release of [14C]20∶4 accurately reflects the metabolic activity of the endogenous 20∶4 pool. In order to measure the 20∶4 mass release relative to the total cellular 20∶4 pool, the free fatty acids of freshly isolated alveolar macrophages were derivatized with a fluorescent reagent, and then separated and quantified by high‐performance liquid chromatography. The amounts of esterified fatty acids were measured by gas chromatography of the methyl esters. Free fatty acid levels were compared to those of the total esterified plus unesterified fatty acids to determine the actual percentage released of each fatty acid. Tannin‐stimulated release of 20∶4 mass reflected that previously reported for the release of [14C]20∶4 label but at a slower rate and at a much lower percentage indicating that [14C]20∶4 had been incorporated into part of a more reactive pool. The specificity of the fatty acid release induced by tannin and β‐1,3‐glucan, a known agonist for 20∶4 release, was also examined. Both agonists promoted an increase in the levels of free 20∶4 and of other fatty acids. A comparison of the absolute increases of each of the fatty acids indicated that tannin caused a preferential increase in the mass of free 20∶4, whereas β‐1,3‐glucan evoked a selective increase in the mass of 16∶0.