Reduced release of leukotrienes B4 and C4 from alveolar macrophages of rats with silicosis.

Silicosis leads to altered release of fibrogenic and immunomodulating mediators from alveolar macrophages (AM). Since 5-lipoxygenase metabolites have been shown to possess proinflammatory effects and to promote the release of cytokines such as tumor necrosis factor-alpha (TNF-alpha) from mononuclear phagocytes, we determined leukotriene secretion from silica-exposed AM. Rats were exposed to an aerosol of silica particles for 8 days and AM were harvested by bronchoalveolar lavage 5 to 7 mo after exposure. AM from both air-sham control and silica-exposed rats displayed minimal spontaneous leukotriene release upon in vitro culture. Stimulation with opsonized zymosan particles induced leukotriene B4 (LTB4) and leukotriene C4 (LTC4) secretion, which was much greater in control AM than in AM from silica-dusted rats. The reverse was found for zymosan-induced TNF-alpha production, which was higher in AM from silica-exposed than from control rats. To study the interrelation between leukotriene and TNF-alpha release, we incubated zymosan-stimulated AM with the 5-lipoxygenase inhibitor VZ 65. VZ 65 suppressed zymosan-induced TNF-alpha release from AM in a dose-dependent manner, and TNF-alpha production could be restored almost completely by addition of LTB4. These experiments demonstrate that silica exposure resulted in a decreased LTB4 and LTC4 production from AM, which may represent a regulatory mechanism to counterbalance enhanced TNF-alpha production during silicosis.

Enhanced release of prostaglandin E2 from macrophages of rats with silicosis.

The pathogenesis of silicosis results, in part, from interactions between silica particles and alveolar macrophages (AM) with release of cytokines and other mediators. Different arachidonic acid metabolites have been shown to promote or to suppress inflammation and fibrosis. We designed experiments to study the production of cyclooxygenase metabolites and tumor necrosis factor-alpha (TNF-alpha) from macrophages during active silicosis. Macrophages were harvested from rats 5 to 7 mo after an 8-day silica aerosol exposure. Upon in vitro culture of AM, the spontaneous release of prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and prostaglandin D2 (PGD2) of silica-exposed animals was higher than that of sham-exposed animals. Moreover, AM from silicotic rats displayed an increased sensitivity to low concentrations of lipopolysaccharide (LPS, 10 ng/ml) and released copious amounts of PGE2 and TXB2. When compared with similarly enhanced release of TNF-alpha from AM of silica-exposed rats, PGE2 production occurred later and started to increase when TNF-alpha production declined. Addition of the cyclooxygenase blocker indomethacin augmented TNF-alpha production, whereas the addition of PGE2 counteracted TNF-alpha release. Also peritoneal macrophages, which did not have direct contact with silica particles, released enhanced levels of PGE2 in response to low LPS doses. We conclude that AM and other macrophages from silica-exposed rats are preactivated and display an enhanced prostanoid production that could serve anti-inflammatory or immunomodulating roles in silicosis.

Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes.

Infections by coxsackievirus B3 (CVB3) have previously been shown to cause acute and chronic myocarditis characterized by a heavy mononuclear leukocyte infiltration and myocyte necrosis. Because clinical and experimental evidence suggested that cardiac damage may result from immunologic rather than viral mechanisms, we examined in this study the in vitro interaction of CVB3 with human monocytes. CVB3 was capable of infecting freshly harvested monocytes as revealed by immunofluorescence and release of infectious virus particles. Virus infection did not reduce monocyte viability but, on the contrary, enhanced spreading and adherence. In a dose-dependent manner, CVB3 stimulated the release of cytokines from monocytes. Whereas a potent production of TNF-alpha, IL-1 beta, and IL-6 was dependent on exposure to infectious CVB3, IFN release was also induced by UV-inactivated virus. On a molecular level, CVB3 stimulated cytokine gene expression as shown by a marked TNF-alpha, IL-1 beta, and IL-6 mRNA accumulation. Supernatants of CVB3-infected monocytes displayed cytotoxic activity against Girardi heart cells which could be abrogated by an anti-TNF-alpha antiserum. These data suggest that CVB3-induced cytokine release from monocytes may participate in virus-induced organ damage such as myocarditis, which may either occur by a direct cytotoxicity of cytokines or by activation of cytotoxic lymphocytes.

Systemic macrophage stimulation in rats with silicosis: enhanced release of tumor necrosis factor-alpha from alveolar and peritoneal macrophages.

In silicosis, alveolar macrophages (AM) are thought to induce chronic inflammation and fibrosis by release of cytokines. Rats were exposed to aerosols of alpha-quartz and examined 4 to 9 mo later for persistence of silica particles and release of tumor necrosis factor-alpha (TNF-alpha) from macrophages. Silica particles were detected in AM, lung parenchyma, and thoracic lymphoid organs, whereas extrathoracic lymphoid tissues and organs were free of the mineral. When AM were tested functionally, no spontaneous release of TNF-alpha was observed. However, upon in vitro stimulation of AM from silicotic rats with a low concentration of lipopolysaccharide (10 ng/ml), abundant TNF-alpha production was found that was higher and occurred more rapidly than with AM from sham-exposed animals. Peritoneal macrophages, which did not have contact with silica particles, displayed a similarly enhanced TNF-alpha release in response to low doses of lipopolysaccharide. These data demonstrate a state of systemic preactivation ("priming") of macrophages that supports the notion that silicosis is associated with a general immunostimulation.

Fabrication, evaluation, and use of extracellular K+ and H+ ion-selective electrodes.

Ion-selective mini-electrodes have been widely employed to measure extracellular K+ and H+ during myocardial ischemia. However, the recent availability of this technology has not been accompanied by uniform fabrication, amplification, and calibration standards. In their fabrication, the chloride tips of Teflon-coated silver wires should be covered with a cellulose acetate-titanium dioxide sponge followed by a polyvinyl chloride (PVC)-valinomycin (K+) or PVC-tridodecylamine (H+) ion-selective membrane. Critical analysis of the nonworking electrodes using scanning electron micrographs has revealed membrane holes, membrane and sponge contamination, Teflon plaque, poor membrane-sponge-Teflon adhesion, and improperly applied or torn membrane. We have also found that signal amplification must have variable-gain filtration (0-1 Hz) with 0.5-pA input offset current and 10(12)-omega input resistance. Furthermore, in vitro calibration in 3 and 10 mM KCl (K+) or pH 8 and 6 buffer (H+) should produce a Nernstian slope +/- 5 or 10%, respectively, at 26 degrees C with a response time less than or equal to 50 ms, resistance greater than or equal to 10(12) omega, and drifts less than or equal to 1 mV/h. In vivo performance and calibration criteria (delineated for K+ only) include 1) transient response to bolus injections of KCl (0.12 mM/kg body wt) yielding peak amplitude changes of 2.5-3.0 mM, response times less than or equal to 10 s, and washout time constants less than or equal to 3 min, and 2) in vivo calibration to artificial independently confirmed systemic [K+] producing a Nernstian slope +/- 15% at 38 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of verapamil and propranolol on changes in extracellular K+, pH, and local activation during graded coronary flow in the pig.

The beta-adrenergic and calcium channel blocking agents are known to reduce heart rate and alter myocardial contractility. More recent evidence suggests that both agents affect the metabolic consequences of ischemia, independent of their effects on heart rate and contractility. We used a low-flow model of ischemia in swine with heart rate held constant by atrial pacing. Blood was shunted from the carotid artery to the left anterior descending coronary artery through a controlled-flow roller pump to assess the threshold flow for the rise in extracellular potassium ([K+]e) and fall in extracellular pH (pHe) associated with ischemia during control situations and after the administration of either propranolol or verapamil. We also measured the changes in activation delay and contractility associated with graded flow reductions in the presence and absence of these drugs. We found that when heart rate is held constant, 1) verapamil shifts the threshold flow for [K+]e and pHe to lower levels, but propranolol does not; 2) verapamil lessens activation delay, while propranolol aggravates the delay; and 3) verapamil reduces afterload and selectively depresses contractility in the reperfused ischemic zone. We conclude that the calcium channel blockers and the beta-adrenergic-blocking agents have different effects and possibly different modes of action and should not be considered interchangeable when evaluating therapeutic options for patients with ischemic heart disease.

Relationship between extracellular potassium accumulation and local TQ-segment potential during acute myocardial ischemia in the porcine.

Depolarization of resting membrane potential during acute myocardial ischemia is strongly correlated with the accumulation of extracellular potassium ([K+]e). Also, diastolic currents of injury flowing across the ischemic border that occur as a result of local differences in resting membrane potentials cause changes in the TQ-segments of unipolar, DC-coupled, extracellular electrograms. Further, the changes in [K+]e and TQ-segment potentials during acute ischemia and reperfusion follow a similar time course. For these reasons, a predictable relationship between [K+]e and TQ potentials might be expected to exist. If found, easily obtainable local TQ potential measurements could serve as an index of the resting membrane depolarization induced by [K+]e accumulation and, by extension, as an index of ischemic injury. We measured local [K+]e and TQ potentials from 30 mid-myocardial sites in central and marginal ischemic zones in 2 isolated, Langendorff-perfused porcine hearts during a single, 10-min ligation of the left anterior descending coronary artery. In general, we found a linear relationship between [K+]e and TQ potential for both ischemic zones when data was taken as a whole, but the slopes (S) and correlation coefficients (R) were markedly different between the two locations (-2.24 vs -1.28 and -0.73 vs -0.51 for central and marginal zones, respectively). Further, we found a time dependent change in both S and R that was biphasic. Both were low during the first minutes, attained their maximum values at 4 mins, and then fell during the remainder of the occlusion. We conclude, therefore, that local TQ potentials cannot be used as an index of the severity of ischemic changes.(ABSTRACT TRUNCATED AT 250 WORDS)

The intracellular and extracellular changes associated with ischaemia--effects of catecholamines in arrhythmogenesis.

Beta sympathetic stimulation and the beta-adrenergic agonists exert a variety of electrophysiologic effects which contribute to the genesis of ischaemia-related arrhythmias. These are both primary, most likely related to enhancement of the slow calcium current, and secondary, resulting from changes in the metabolism of the ischaemic myocardium as reflected in the accumulation of ions and other end products of metabolism in the extracellular space. In addition, the regional influence of these changes may contribute to the ionic and electrical inhomogeneities. Further studies are needed to define more precisely the factors responsible for the enhanced arrhythmogenicity attributed to beta-adrenergic stimulation.

Effects of verapamil on ischemia-induced changes in extracellular K+, pH, and local activation in the pig.

In experimental animals, the calcium channel-blocking agents lessen the arrhythmogenic, ionic, metabolic, and electrical changes that occur during acute myocardial ischemia. To date, these effects have been studied separately, and the effects of these agents on local activation have not been correlated with ionic or metabolic effects. In open-chest, anesthetized swine, we used bipolar and ion-selective plunge electrodes to simultaneously measure ischemia-induced changes in left ventricular local activation, extracellular K+ ([K+]e), and extracellular pH (pHe). The effects of verapamil (0.2 mg/kg) on these variables were studied during a series of 10 min occlusions of the left anterior descending coronary artery. Compared with control occlusions, verapamil (1) slowed the rise in [K+]e at the center of the ischemic zone and at its lateral margin and decreased the peak [K+]e by 0.9 mM at the center (p less than .05) and by 0.1 mM at the margin (p = .10); (2) slowed the development of acidosis and decreased the peak level of acidosis beyond that expected solely as a result of serial occlusions by 0.19 pH units at the center (p less than .05) and by 0.07 pH units at the margin (p = .10); and (3) slowed the development of local activation delay and often prevented the local activation block that was observed during control occlusions. Effects on local activation became less marked at [K+]e levels greater than 9.0 mM, and the effects of verapamil on local activation were not explained solely by its effects on the local rise in [K+]e or fall in pHe. A possible mechanism for this additional effect on local activation is suggested by preliminary results showing a diminution by verapamil of ionic inhomogeneity.

Effect of serial brief ischemic episodes on extracellular K+, pH, and activation in the pig.

This study was performed to determine the reproducibility of the ionic and electrical changes associated with serial ischemic episodes. We used ion-selective and bipolar plunge electrodes to determine the changes in left ventricular extracellular potassium ([K+]e), extracellular pH (pHe), and local activation during sequential 10 min occlusions of the left anterior descending coronary artery separated by 50 min of reperfusion in open-chest anesthetized pigs. We found that uniformly during the initial occlusion, and in approximately 50% of animals during the second occlusion, [K+]e rose more rapidly but to a lower level than in subsequent occlusions. By the third occlusion the changes in [K+]e were reproducible. Extracellular acidosis was greatest in the first occlusion and decreased progressively with each subsequent occlusion. Local activation was characterized by a decrease in spontaneous improvement and increase in block with each successive occlusion. The occurrence of ventricular fibrillation could not be directly attributed to the magnitude of the change in [K+]e or pHe. Moreover, the occurrence of ventricular fibrillation in one occlusion did not necessarily predict its occurrence thereafter. Our results indicate that serial episodes of ischemia are associated with different but predictable changes in ionic and electrical events that may be clinically relevant and that must be appreciated before the results from similar protocols with serial ischemic episodes can be interpreted meaningfully.