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Biomedical subjects

C Kroegel

Publications and source records attributed to C Kroegel.

At least 109 records · Page 6Linked to original sources
PubMed1990

Characterization of eosinophil cell activation by peptides. Differential effects of substance P, melittin, and FMET-Leu-Phe.

The ability of three different peptides, substance P (SP), FMLP and melittin, to activate eosinophils purified from the peritoneal cavity of human serum-treated guinea pigs was investigated. Degranulation (eosinophil peroxidase, EPO), oxidative burst (O2-), [Ca2+]i mobilization, and arachidonic acid metabolism (thromboxane B2, TXB2) were used as indices of eosinophil activation. SP (100 nM to 100 microM), FMLP (1 to 100 microM) and melittin (10 nM to 100 microM) induced EPO release but only FMLP (1 to 100 microM) led to an elevation of [Ca2+]i. The melittin- and SP-induced EPO secretion occurred at both cytotoxic and noncytotoxic concentrations as assessed by lactate dehydrogenase release. In addition, the effect of SP was not inhibited by the SP analogue (D-Pro4, D-Trp7,9,10)SP(4-11) and SP failed to promote the generation and subsequent release of TXA2. In contrast, FMLP (10 to 100 microM) stimulated the release of TXB2 from guinea pig eosinophils that was selectively inhibited by pretreatment of the cells with BOC-FMLP. On an equimolar basis (1 microM), melittin was approximately fivefold more active at promoting TXB2 release than FMLP. The results indicate that eosinophils respond to the three peptides, SP, melittin, and FMLP in differential fashion. We conclude that activation of guinea pig eosinophils by FMLP is likely to be receptor-mediated whereas the actions of SP and melittin may act through nonspecific peptide-membrane phospholipid interactions.

Animals↗
PubMed1990

Platelet activating factor stimulates cyclo-oxygenase activity in guinea pig eosinophils. Concerted biosynthesis of thromboxane A2 and E-series prostaglandins.

The effect of platelet activating factor (PAF) on the generation of cyclo-oxygenase-derived arachidonic acid metabolites was examined on purified eosinophils harvested from the peritoneal cavity of male guinea pigs. PAF produced a concentration-dependent increase in the amount of immunoreactive thromboxane B2 (TXB2) and PGE1/E2 released from these inflammatory cells at a relative molar ratio of 30:1. The EC50 of PAF was 20 to 40 nM and maximum stimulation (4.5-fold) of both prostanoids occurred at 1 microM PAF. The ability of PAF to generate TXA2 was rapid (t 1/2 = 9 s), transient (40 s), noncytotoxic, and noncompetitively antagonized by the PAF-receptor blocking drug, WEB 2086. On an equimolar (100 nM) basis, PAF was significantly more effective than C5a, fMLP, and PMA at stimulating TXB2 release but markedly less potent than the calcium ionophore, calcimycin. Pretreatment of eosinophils with the cyclo-oxygenase inhibitor flurbiprofen (8 microM for 5 min) abolished the ability of PAF to promote both TXB2 and PGE1/E2 release. Likewise, dazmegrel (50 microM for 5 min), a selective inhibitor of thromboxane synthetase, abolished PAF-stimulated TXB2 release but markedly augmented the elaboration of PGE1/E2. Inhibition of cyclo-oxygenase with flurbiprofen affected neither the ability of PAF to elevate the intracellular calcium ion concentration (measured by fura-2 fluorescence) nor its appetency to generate superoxide anions at any PAF concentration examined. It is concluded that activation of guinea pig eosinophils by PAF is receptor-mediated and independent of the concomitant generation of cyclo-oxygenase-derived excitatory prostanoids. Inasmuch as TXA2 may contribute to the pathogenesis of bronchial hyperreactivity, then these data implicate the eosinophil as a potential source of this lipid mediator.

Animals↗
PubMed1990

The role of eosinophils in asthma.

During recent years it has become apparent that the eosinophil may represent a powerful effector cell in the pathogenesis of asthma, particularly in the late asthmatic response. It can be stimulated by a number of stimuli among which PAF appears to be one of the most effective. The eosinophil is a source for a variety of proinflammatory and toxic products with profound effect in the lungs and airways. These eosinophil products mimic some of the features of asthma and the strong association of the eosinophil with asthma has led to the suggestion that asthma would be better classified as "chronic desquamating eosinophilic bronchitis." Although the evidence known to date is persuasive many questions still remain unanswered and await further investigation. Future therapeutic approaches in asthma may aim at interrupting the mechanisms leading to eosinophil bronchial accumulation and activation.

Airway Resistance↗
PubMed1990

Beta 2-adrenergic receptors on eosinophils. Binding and functional studies.

We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand [125I]pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed.

Adenylyl Cyclases↗
PubMed1990

The effects of activated eosinophils and neutrophils on guinea pig airway epithelium in vitro.

Epithelial shedding is a characteristic feature of asthmatic airways and has been attributed to eosinophil products. We have examined the interaction of purified intraperitoneal guinea pig eosinophils with or without platelet-activating factor (PAF, 10(-7) M) or lyso-PAF (10(-7) M) with guinea pig tracheal epithelium in vitro. At 0, 4, 14, and 24 h, the percentage of ciliation of the tracheal circumference (CTC) was measured by light microscopy and the ciliary beat frequency (CBF) by photometry. PAF-activated eosinophils (50 x 10(6) cells/ml) disrupted the epithelium, mean CBF and CTC being reduced by 77.8 +/- 5.8% (mean +/- SEM; P less than 0.001 versus control) and 94.2 +/- 1.4% (P less than 0.001) over 24 h, respectively. PAF (10(-7) M) alone had no significant effect. Lyso-PAF with eosinophils (50 x 10(6) cells/ml) also reduced mean CBF and CTC but to a lesser extent. Eosinophils alone also led to a reduction of 36.2 +/- 11.4% in mean CBF and 53.0 +/- 15.5% in CTC, but these changes were not significant. The PAF antagonist, WEB 2086 (10(-6) M), significantly inhibited the mean CBF and CTC reduction due to PAF-activated eosinophils by 61.5 +/- 17.2% (P less than 0.01) and 20.8 +/- 6.5% (P less than 0.05), respectively. In addition, catalase (1,125 U/ml) partially inhibited the mean CBF and CTC reduction induced by PAF-activated eosinophils. Intraperitoneal neutrophils (PMN) (50 x 10(6) cells/ml) also disrupted epithelium but to a lesser extent (24-h reduction: 34.2 +/- 12.7% for mean CBF and 60.2 +/- 13.2% for CTC, respectively). Stimulation with PAF (10(-7) M) had no further effect. Marked exfoliation of the epithelial layer was observed after 14 h of incubation with activated eosinophils. We concluded the PAF-activated eosinophils are capable of grossly disrupting ciliated epithelium and may contribute to epithelial damage observed in asthma.

Animals↗
PubMed1990

[Degranulation of human eosinophilic granulocytes by the platelet activating factor].

Incubation of purified human eosinophil granulocytes (greater than 93%) with the platelet activating factor (PAF), led at concentrations of 1 pM to 10 microM, to a non-cytotoxic and dose-dependent liberation of proteins both from the "small" and the "specific" granules. The mean EC50 for this PAF-induced effect was 1.47 nM (n = 9), with a maximum at 100 nM PAF. The degranulating effect of PAF can be inhibited competitively by the specific PAF-receptor antagonist, WEB 2086 (KB between 12 and 13 nM), and is Ca2(+)-dependent.

Cell Degranulation↗
PubMed1989

Evidence for two platelet activating factor receptors on eosinophils: dissociation between PAF-induced intracellular calcium mobilization degranulation and superoxides anion generation in eosinophils.

We have compared platelet activating factor (PAF)-induced eosinophil peroxidase (EPO) release and intracellular calcium mobilization with superoxide anion (.O2-) generation from guinea pig eosinophils. EPO release and Ca2+ mobilization occurred at lower concentrations of PAF (EC50 values of 1.3 nM and 11.5 nM, respectively) while .O2- production was observed at higher concentrations (EC50 of 31.7 microM). Receptor characterization with the competitive PAF antagonist, WEB 2086, gave pA2 values of 8.5 and 8.3 for EPO enzyme release and rise in [Ca2+]i, respectively, and 5.8 for the .O2- production. In addition, PAF-induced degranulation and elevation of [Ca2+]i were dependent on extracellular Ca2+ whereas PAF-stimulated .O2- generation was dependent on the presence of extracellular Mg2+ ions. These results suggest the existence either of two subtypes of the PAF receptor or a single receptor that can exist in one of two affinity states on guinea pig eosinophils.

Animals↗
PubMed1989

Stimulation of degranulation from human eosinophils by platelet-activating factor.

Platelet-activating factor (PAF) is a highly active mediator which has been implicated in allergic inflammation and bronchial asthma, possibly by interacting with eosinophils. We have examined the effect of PAF on activation of purified human eosinophils as measured by degranulation (eosinophil peroxidase, eosinophil cationic protein, arylsulfatase B, beta-glucuronidase, and alkaline phosphatase) and oxidative metabolism (superoxide anion production). PAF induced enzyme release at concentrations ranging from 1 pM to 10 microM in a rapid (t1/2 5 to 8 min), Ca2+-dependent and noncytotoxic manner from both the specific and small granules, whereas its biologic precursor and metabolite, lyso-PAF, had no effect. For all enzymes, maximal enzyme release occurred at 100 nM PAF with a mean ED50 value of 1.47 +/- 0.4 nM. At this concentration the mean percentage of total enzyme release by PAF from specific granules was 20.3 +/- 1.6% (17.9% for eosinophil peroxidase, 20.6% for beta-glucuronidase, 22.4% for alkaline phosphatase) and 28.8 +/- 2.2% from small granules (arylsulfatase B). Calcium ionophore A23187, PMA, and opsonized zymosan also induced eosinophil degranulation but their peak effect after 10-min incubation with maximal release 14.7%, 12.9%, or 14.1%, respectively, was lower when compared with PAF. Incubation of eosinophils with the PAF-antagonist WEB 2086 led to a parallel shift of the dose-response curve to the right, indicating a competitive antagonism. PAF also caused generation of superoxide anions by human eosinophils but this occurred at higher concentrations of PAF (1 microM to 30 microM) with an ED50 of 8.4 +/- 0.9 microM. Again, this effect was competitively inhibited by WEB 2086. These studies demonstrate that PAF activates human eosinophils to release granule constituents and generate superoxide anions. Since both PAF and eosinophil products are associated with pathogenesis of bronchial asthma our findings may be of particular pathophysiologic relevance.

Alkaline Phosphatase↗
PubMed1989

Characterization of platelet-activating factor-induced elevation of cytosolic free calcium concentration in eosinophils.

In order to evaluate the role of calcium in the activation processes in eosinophils induced by platelet-activating factor (PAF), we investigated the changes in free cytoplasmatic Ca2+ concentration using fura-2. PAF causes a rapid and transitory rise of the intracellular free calcium ion concentration [( Ca2+]i) in purified guinea pig eosinophils of approx. 1000 nM above a basal level of 120.7 +/- 36.5 nM (n = 10). The effect was dose-related with a maximum rise at 1000 nM PAF and an EC50 of 17.4 nM and specifically inhibited by the PAF antagonist WEB 2086 with an IC50 of 95.5 nM. WEB 2086 did not affect either the leukotriene B4- or the fMet-Leu-Phe-induced elevation of [Ca2+]i. The response to PAF was dependent on external Ca2+ as it was significantly inhibited by EGTA (85.6 +/- 5.4%) and Ni2+ (95.8 +/- 2.1%) but not by the dihydropyridine antagonist nimodipine. We conclude that Ca2+ entry via receptor-operated Ca2+ channels may be involved in PAF-induced degranulation of eosinophils.

Animals↗
PubMed1989

Effect of theophylline and adenosine on eosinophil function.

Eosinophils may play a critical role in asthma and bronchial hyperresponsiveness, yet the effect of theophylline on their function is not certain. We have examined the effects of theophylline on opsonized zymosan-induced superoxide anion (O2-) release from guinea pig eosinophils harvested from the peritoneal cavity and from human eosinophils obtained by differential centrifugation of blood from patients with peripheral eosinophilia. Theophylline at high concentration (10(-3) M) inhibited O2- release by 27.6 +/- 9.4% (mean +/- SEM, p less than 0.05), whereas at clinically relevant concentrations (10(-6) and 10(-5) M), it significantly potentiated this by 26.8 +/- 9.9% (p less than 0.05) and 36.9 +/- 6.3% (p less than 0.01), respectively. 8-phenyltheophylline (10(-7) to 10(-3) M), which like theophylline inhibits adenosine receptors but does not inhibit phosphodiesterase activity, produced potentiation at all concentrations. Preincubation of eosinophils with adenosine deaminase (0.1 U/ml) enhanced O2- release by 72.4 +/- 15.2% (p less than 0.01), whereas addition of adenosine (3 x 10(-8) to 10(-6) M) reversed the potentiation induced by theophylline (10(-5) M) in a concentration-dependent manner. Inhibition was greater with the A2-selective analog N-ethylcarboxamide adenosine than the A1-selective analog phenylisopropyladenosine, suggesting that A2-receptors are involved. In human eosinophils we have demonstrated a similar effect of theophylline and adenosine on O2- release. Our results indicate that therapeutic concentrations of theophylline may potentiate eosinophil activation in vivo by competing with circulating adenosine for eosinophil A2-receptors. This would be consistent with the lack of effect of theophylline on bronchial hyperresponsiveness, which may be related to eosinophilic inflammation.

Adenosine↗
PubMed1989

Density heterogeneity of eosinophil leucocytes: induction of hypodense eosinophils by platelet-activating factor.

We have examined the induction of hypodense eosinophils by platelet-activating factor (PAF), a mediator which may be involved in eosinophil activation in allergic diseases. Guinea-pig eosinophils were incubated with buffer or PAF and applied to continuous Percoll density gradients. Cellular density ranged from 1.0142 to 1.1369 g/ml. Peak eosinophil density in control was 1.0887 +/- 0.0008 g/ml (mean +/- SEM), and 91.1 +/- 1.4% of eosinophils were distributed between 1.0810 and 1.1000 g/ml. Preincubation of eosinophils with PAF(10(-7) M) resulted in a time-dependent and non-cytolytic increase of the number of hypodense eosinophils, with peak densities after incubation for 1 hr and 2 hr of 1.0834 +/- 0.0014 (n = 4, P less than 0.05) and 1.0755 +/- 0.0007 g/ml (n = 6, P less than 0.01), respectively. After incubation for 2 hr, 82.0 +/- 4.9% (n = 6) eosinophils showed a density lower than 1.080 g/ml. Lyso-PAF, the inactive precursor and metabolite of PAF, at a concentration of 10(-7) M had no effect on cell density. The specific PAF receptor antagonist WEB 2086 (10(-6) M) inhibited the PAF-induced density shift by 87.0 +/- 5.3%. Our results demonstrate that a single mediator is able to induce the formation of hypodense eosinophils. We conclude that the appearance of hypodense eosinophils in allergic diseases such as asthma may occur, at least in part, in response to inflammatory mediators which activate these cells.

Animals↗
PubMed1988

The potential pathophysiological role of platelet-activating factor in human diseases.

Platelet-activating factor (PAF) is a new phospholipid mediator released from various cell types and tissues by the catalytic action of phospolipase A2 and acetyl transferase upon immunological and non-immunological stimuli. It activates the cells by binding to specific binding sites. PAF exhibits a broad range of biological and pharmacological activities including platelet and neutrophil aggregation, eosinophil chemotaxis, bronchoconstriction, hypotension, and acute renal failure. In addition, PAF is involved in acute graft rejection, endotoxin shock, and gastrointestinal ulceration. Furthermore, it closely mimics the pathology of bronchial asthma and is cabable of producing most of the phenomena seen in inflammation. So far several PAF antagonists have been described and shown to afford protection. In future, pharmacological studies using such antagonists will help to elucidate the pathophysiological role of PAF in human diseases.

Humans↗