Selective inhibition by antiflamrnin-2 of thromboxane B2 release from isolated and perfused guinea-pig lung

Antiflammin-2 (AF2) is a nonapeptide corresponding to the amino acid residues 246–254 of lipocortin-1 showing anti-inflammatory activity both in vitro and in vivo. The effect of AF2 on the thromboxane B2 (TXB2) and histamine release from isolated and perfused guinea-pig lungs has been studied. AF-2 (10–100 nM) inhibited leukotriene C4- (LTC4) (3 ng) and antigen-induced (ovalbumin, 1 mg) TXB2 release in normal and sensitized lungs, respectively. In contrast AF-2 (100 nM) did not modify TXB2 release induced by histamine (5 μg) or bradykinin (5 μg) in normal lungs. Antigen-induced histamine release was not affected by 100 nM AF-2 infusion. When tested in chopped lung fragments AF-2 (0.1–25 μM) did not modify the release of histamine and TXB2 induced by antigen (ovalbumin, 10 μg ml−1) or calcium ionophore A 23187 (1 μM). Our results show that the inhibitory effect of AF-2 on TXB2 release is selective and depends on the stimulus applied. In this respect AF-2 mimics, at least in part, the actions of both glucocorticoids and lipocortin-1.


Introduction
Uteroglobin and lipocortin-12 belong to a family of structurally related proteins referred to as annexins or lipocortins which are calcium-binding proteins. 3-s Lipocortins are glucocorticoid-inducible proteins which, by inhibiting phospholipase A2 (PLA2), are believed to be involved in the suppression of prostaglandin and leukotriene synthesis associated with some aspects of the anti-inflammatory activity of corticosteroids. 6 Recently nonapeptide fragments of uteroglobin and lipocortin-1, named antiflammins,7 have been shown to inhibit PLA2 in vitro and to possess anti-inflammatory activity in vivo. [7][8][9] One of these nonapeptides, antiflammin-2 (AF-2, HDMNKVLDL) corresponds to the amino acid residues 246-254 of lipocortin-l.7 However, conflicting results have been reported concerning the biological activity of AF-2. The peptide has been shown to inhibit carrageenan-induced rat paw oedema, 9 porcine pancreatic PLA27 and human polymorphonuclear leucocyte PLA 2 activity as well as PEA2 activity in extracts of human psoriatic epidermis. 1 In contrast, other authors have reported that AF-2 is unable to inhibit porcine PLA2, eicosanoid-release by different cell types, and carrageenan-induced rat paw oedema. >3 In the light of these conflicting results we decided to evaluate the biological activity of AF-2 in the isolated perfused guinea-pig lung model where human recombinant lipocortin-1 has been shown to be very active in inhibiting LTC4-induced TXB 2 (C) 1992 Rapid Communications of Oxford Ltd release. 4 We have also tested the effect of the peptide on histamine release in the same experimental model.

Materials and Methods
Animals: Male guinea-pigs (Dunkin Hartley, 300-400g) were used. In some experiments animals were sensitized by subcutaneous and intraperitoneal injections of equal doses (100 mg) of ovalbumin 15 and the lungs were then removed .3 weeks later.
Mediator release from perfused lungs: Lungs from normal or sensitized guinea-pigs were cannulated through the pulmonary artery, excised and suspended in a chamber where they were immediately perfused with oxygenated Krebs bicarbonate solution at 37C using a Watson and Marlow 503 S peristaltic pump. The rate of perfusion was constant at 5 ml min -1. AF-2 was infused at 0.1 ml min -1 30min before stimulating TXB2 or histamine release and throughout the experiment. Fractions of 5 ml, corresponding to a 1 min collection time, were collected and stored at -80C for assay. Stimuli such as LTC4 (3 ng), ovalbumin (1 mg), histamine (5/g) or bradykinin (5/g) were applied as a bolus injection (0.1 ml).
Mediator release from chopped lungs: Lungs from normal or sensitized male guinea-pig were cannulated and perfused with Krebs bicarbonate as described above. Lungs were then removed, cut into small pieces with a sharp blade and washed extensively. Triplicate samples (0.6 g wet weight) were incubated in Krebs bicarbonate solution at 37C for 10 min in the presence of AF-2 (0.1-25 and subsequently challenged with ovalbumin (10/g/m1-1 sensitized lungs) or calcium ionophore A23187 (1/4M, normal lungs). The incubation was stopped 30 min later by transferring the samples to an ice bath. Incubation media were then centrifuged (50 g) and aliquots for TXB 2 and histamine determination immediately frozen and kept at -80C until analysis. Lung fragments were resuspended in Krebs bicarbonate solution, boiled for 10 min and filtered. The filtrate was centrifuged (50 g') and aliquots for histamine assay were kept at -80C until analysis.
In some experiments lungs were cannulated in situ through the pulmonary artery and perfused with AF-2 for 30 min. The lungs were then rapidly removed, cut into small pieces and incubated and processed as described above.
Radioimmunoassay of thromboxane Be: Thromboxane .B 2 was measured by radioimmunoassay without prior extraction or purification as previously described 16 and expressed as ng min-in perfusion experiments.
Fluorimetric anasis of histamine: Histamine release was measured fluorimetrically 17 and corrected for spontaneous release occurring in the absence of the inducer and expressed as/g min-1 in the perfusion experiments.
Since it has been reported that a reduction of the biological activity of AF-2 is caused by the oxidation of the methionine residue some experiments were performed in the presence of dithiothreithol (10:1). However, we could not detect any diflerences in AF-2 activity.
When the lung was infused with AF-2 the LTC4-induced TXB2 release was greatly reduced throughout the time course of the response (Fig. 1). The peak response was reduced by the peptide in a concentration-related fashion as inhibition of 36%, 54% and 61% were induced by 10,30 and 100 nM infusion of AF-2.
Thromboxane B e release from sensitized unchallenged lungs (14.5 -k 1.6 ng min-1., n 4) was not affected by 100nM infusion AF-2 (13.4-k 1.1ngmin-l" n=4) Antiflammin-2 infusion (100 nM) did not modify either the peak response or the time course of the antigen-induced histamine release from sensitized lungs (Fig. 2B). significantly modify TXBp. and histamine released induced by these agents. Similar results were obtained when lungs were perfused in situ with AF-2 at the above concentrations and fragments subsequently challenged with the same stimuli (data not shown).

Discussion
These data show the ability of AF-2 to inhibit the TXB 2 release induced by LTC 4 or antigen in normal and sensitized guinea-pig lungs respectively. However, when other stimuli, such as histamine and bradykinin were used, no inhibition of TXB2 release was observed.
Human recombinant lipocortin-1 (annexin-1) when infused through normal guinea-pig lung preparation inhibited LTC4-induced TXB 2 release. 14 Glucocorticoids have been shown to inhibit TXBp. release from guinea-pig isolated perfused lungs challenged by different stimuli such as leukotrienes, histamine or antigen while they did not prevent TXB2 generation induced by bradykinin. 18 For these reasons we studied the effect of the nonapeptide, referred to as antiflammin-2, corresponding to lipocortin-1 sequence 246-254, on TXB2 generation from guinea-pig lungs challenged with different stimuli in order to compare its biological activity to the effect of glucocorticoids and lipocortin-1.
Our results suggest that the inhibitory activity of AF-2 on TXB 2 release is dependent on the challenge used and mimics, at least in part, the action of both glucocorticoids and lipocortin-1. In fact AF-2, like glucocorticoids, significantly inhibits TXB2 release induced by EWe 4 and it is unable to block the releasing action of bradykinin in normal lungs. In this respect it is interesting to observe that dexamethasone has an inhibitory activity on the bradykinin-induced eicosanoid release from the inflamed lungs while it is ineffective in normal lungs. 19 The reason for the lack of activity of both glucocorticoids and AF-2 in normal lungs is not clear.
It has been suggested that separated PLA2 pools may exist or that a phospholipase C linked pathway could be involved. 18'2 The main difference between AF-2 and dexamethasone was observed when the lung vas stimulated with histamine. In fact AF-2 was unable to block the histamine-induced TXB2 release in normal lungs, which has been reported to be inhibited by glucocorticoids. 8 We found that AF-2 was unable to inhibit the antigen-induced histamine release from sensitized lungs. This observation is consistent with previous results reported by us suggesting that another glucocorticold-induced protein (vasocortin) different from lipocortin 2 is responsible for the glucocorticoid inhibition of histamine release. 22 The release for the conflicting results reported by different authors on the biological activity of AF-2 is not clear. In fact, the mode of action of lipocortins and lipocortin-derived peptides as PLA2 inhibitors it is not yet understood. Thus until now it has not been clearly demonstrated if the PLA2 inhibition depends on a direct interaction between the enzyme and the inhibitor or it is due to the binding of the inhibitor to the substrate. 23 Another aspect which could affect the biological activity of the peptide might be its oxidation possibly occurring during the experimental procedures. In fact it has been suggested that AF-2 might be inactivated by spontaneous oxidation of its methionine residue in the position 3 and consequently it might be effective only in the presence of a reducing agent. In this respect we did not observe any significant difference in the inhibition of TXB2 release by AF-2 in the presence or absence of dithiothreitol.
Furthermore the inhibitory action of AF-2 on TXB 2 generation seems to be dependent not only on the stimulus but also on the experimental model used. Indeed, the peptide was completely ineffective when assayed on TXB2 release from chopped lungs.
Corticosteroids are very powerful drugs in asthma therapy but their mechanism is not yet clearly understood and their clinical use mostly empirical. Antiflammin-2 could be used as a useful tool in exploring the mechanism through which glucocorticosteroids could affect the airway regulatory mechanisms.