[67]
EICOSANOID
STUDIES
USING HUMAN/ANIMAL
LUNG
621
Acknowledgment Supported by N I H grants H L 34215 and H L 18828 to R L and H L 31591 to KM.
[67] Preparation of Human and Animal Lung Tissue for Eicosanoid Research By T. V1GAN~), M. T. CRIVELLARI,M. MEZZETTI, and G. C. FOLCO
Eicosanoids are fatty acids of great biological significance because of their ubiquity, high potency, and multitude of other effects. Among all the organs that have been investigated, the lungs are the most active in forming and metabolizing eicosanoids; human and animal lung tissue can oxidize arachidonic acid (AA), generating cyclooxygenase as well as lipoxygenase-derived products that appear to play a role in the development of asthma, allergic reactions, and inflammation. In fact, leukotrienes (LT) have potent spasmogenic actions on human lung tissue in vitro, affect mucus production and clearance, and may be important factors in the pathogenesis of airway hyperresponsiveness. A sufficient number of in vitro methods has been developed utilizing human and animal lung tissue for eicosanoid research; this chapter will describe techniques that are useful in understanding the physiopathological role of eicosanoids in the pulmonary system as well as their functional effects. Preparation of Human Lung Parenchyma to Produce Eicosanoids Normal human lung parenchyma is a preferential tissue to use and probably represents the richest source of sulfidopeptide LT and of PGD2. The tissue is usually available following surgery for pulmonary carcinoma or bronchiectasis; the lung parenchyma should be immediately collected in a buffered solution such as Tyrode's [composition (in g/liter): NaC1, 8.0, KC1, 0.25, CaC1 0.15 MgCI2-6 H20, 0.01, NaH2PO4"H20, 0.066, glucose, 1.0, NaHCO3, 1.0] or Krebs-Henseleit [composition (in g/liter): NaCI, 6.9, KCI, 0.35, KHzPO4, 0.16, MgSO4.7 H20, 0.29, CaC12, 0.28, glucose, 1.0, NaHCO3,2.1], pH 7.4, kept at 4° and gassed with a mixture of 95% 02-5% CO2. It is imperative that the tissue not be frozen as it loses its capacity to respond to different stimuli. METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
622
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[67]
Small biopsies of approximately 100 mg each are sampled at random from the excised lung portion and used for histological examination to exclude the possibility of neoplastic infiltrations. Usually the available peripheral tissue is free of macroscopically evident bronchi or vessels and is chopped manually to fragments of approximately I00 mg each. The fragments are washed again using the same buffer and can now be utilized for release experiments. It is possible to follow at least two different experimental protocols in order to mimic a release of lipid mediators that is likely to occur during immediate hypersensitivity reactions. The fragments can be passively sensitized using diluted reaginic serum from hyperimmune patients (1500-5000 ng/ml of specific IgE). The tissue is incubated overnight (1 g tissue/5 ml diluted serum) at room temperature in a Dubnoff shaking incubator without need for direct oxygenation; alternatively, the passive sensitization can be carried out using a shorter incubation step (3 hr) at 37°. Lung parenchyma is then washed extensively using the same buffer and divided into portions of about 1 g resuspended in 10 ml of fresh Tyrode's or Krebs buffer. The parenchymal fragments are preincubated for 10 min under gentle agitation and challenged with the appropriate antigen (usually 500-1000 ng/ml), agonists or vehicle. At the end of the incubation, an aliquot (max. 0.5 ml) is collected for prostaglandin assay using RIA I or EIA 2 while another is immediately tested for the presence of eicosanoid-like activity using appropriate bioassay systems. 3 The remaining volume (approximately 8 ml) can be added to the required internal standards (PGB2 500 ng or tracer amounts of radiolabeled LT, 20,000 dpm) and frozen or further processed for quantitative assays of prostanoids using physicochemical methods. It is also possible to trigger the immunological release of different autacoids from pulmonary tissue using specific anti-human IgE antibodies which are usually raised in sheep or goat. Our personal experience, accumulated in about 40 experiments with an equal number of human specimens, indicates that the mediator release obtained in this experimental condition does not require the time-consuming step of passive sensitization. Usually after mincing and careful washing of the lung fragments, the tissue is preconditioned in Tyrode's buffer for approximately 30-60 min at 37° (1 g/5 ml), rewashed, and subjected to final incubation in Tyrode's buffer (1 g/10 ml). The challenge is performed using a final anti-human IgE 1 E. Granstrom and H. Kindahl, Adv. Prostaglandin Thromboxane Res. 5, 119 (1978). 2 p. Pradelles, J. Grassi, and J. Maclouf, Anal. Chem. 57, 1170 (1985). 3 S. Moncada, S. H. Ferreira, and J. R. Vane, Adv. Prostaglandin Thromboxane Res. 5, 211 (1978).
[67]
EICOSANOID STUDIES USING HUMAN/ANIMAL LUNG
623
antibody dilution varying between 1:300 to 1:1000. Preparation and storage of aliquots is carried out as described above. As the availability of good hyperimmune sera as well as that of antihuman IgE antibodies can present a problem, a preliminary check of the tissue releasability should be performed with the calcium ionophore A23187 (I-5/~M), stopping the reaction 20-30 min after challenge. By the concomitant use of a bioassay setup which utilizes a strip of longitudinal smooth muscle of guinea pig ileum, it is possible to follow rather carefully the development of the experiment. It is advisable to carry on the immunological challenge only in tissues that have responded successfully to A23187. Experience with approximately 200 human lung parenchymal specimens, indicates that while good releasability after A23187 treatment occurs in about 80-90% of the cases, a successful immunological challenge takes place in about 50% of the tissues. Preparation of Human Bronchial Tissue Both large and small human airways are suitable models to assess the pharmacological activity of eicosanoids as well as their role in pathophysiology. Bronchi of different caliber, from primary bronchi to terminal bronchioles, are utilized for such studies, however the latter, with an internal caliber of 1 to 2 mm, are better suited because they have no cartilage plates. These can be identified and differentiated from pulmonary arteries and veins of similar caliber. The vascular lumen can be pinched with small forceps and with gentle pulling can be detached from the surrounding parenchyma, whereas, the bronchioles cannot be freed. In addition, particularly following surgery for bronchiectasis, the bronchial lumen shows presence of mucous plugs. A fine polyethylene catheter is gently slipped through the full length of the bronchus in order to facilitate its localization. Further confirmation that one is dealing with a bronchiole comes from the fact that a gentle administration of air into the catheter leads to tissue inflation. The cannula is left in place and the bronchioles are carefully dissected free of parenchyma and blood vessels; it is advisable to leave a minimum layer of parenchyma to avoid inadvertent damage of the bronchiole smooth muscle. During this, as well as the following preparation steps, the tissue is kept moist with lukewarm saline. The polyethylene tubing is now fixed in a vertical position and the bronchiole spiralized to obtain a strip 2-3 mm wide and of varying length (usually 20-30 mm). Great care is taken to avoid the stretching of the preparation as this may damage its contractile function; a silk surgical thread is tied to both ends of the strip.
624
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[67]
The smooth muscle strips are suspended in a 10-ml organ bath, under an isometric resting tension of 200 to 500 mg, at 37°, in a Krebs-Henseleit solution oxygenated with a mixture of 95% 02-5% CO2. The time required to attain a steady baseline tone may vary considerably. Whenever possible the tissues should be used the day of surgery or stored overnight at 4 ° under a slow constant flow of oxygenated buffer and then set up the following day, without appreciable loss of sensitivity or contractility. Transducers are coupled to a multichannel pen recorder and during the stabilization period (I-2 hr) it is advisable to change the buffer every I015 min. Pharmacological studies can be performed by adding agonist eicosanoids as well as their potential antagonists in small volumes (0.05-0.1 ml). In this way, cumulative dose-response curves (for sulfidopeptide LT, 0.1-100 riM) can be constructed as tachyphylaxis for LT or bronchoactive prostaglandins (e.g., PGD2) has not been described in human bronchi. By using a similar technique, Dahlen e t al. 4 w e r e able to demonstrate that bronchi from allergic asthmatics respond to antigen exposure with a contraction that correlates with the release of sulfidopeptide LT. Human bronchiolar strips represent a relatively homogeneous structure and their functional changes are not affected by vascular smooth muscles that lie in close proximity, as is the case of the parenchyma. They can be used as detector organs for eicosanoids and, in general, for bronchoactive substances, such as histamine or PAF. The classical nonflow organ bath technique and also the perifusion technique (i.e., the tissue is set up in a tissue chamber where the buffer is constantly changed by means of a pump) require that the active compound be diluted in a volume that rarely is smaller than 5 ml. On the other hand, superfusion techniques involve only a very transient contact of the tissue with the substances added as bolus, but also require a rather high flow, 5-10 ml/min, once again leading to high dilution of the samples being tested. A technique that we have used successfully is laminar flow superfusion, first suggested by Ferreira 5 in 1976. The isolated assay tissue is immersed in mineral oil thermostatted at 37° and superfused with a drip of Tyrode's solution at a very low rate of flow (0.1-0.2 ml/min). Each drop of aqueous buffer floats slowly downward at the interface between the tissue and the oil, allowing a contact time that is approx. 1 sec/1 cm of strip length. It is important to ensure a close contact between the catheter delivering the buffer and the thread which connects the tissue with the 4 S. E. Dahlen, G. Hansson, P. Hedqvist, T. Bjorck, E. Granstrom, and B. Dahlen, Proc. Natl. Acad. Sci. U.S.A. 80, 1712 (1983). 5 S. H. Ferreira and F. De Souza Costa, Eur. J. Pharmacol. 39, 379 (1976).
[67]
EICOSANOID STUDIES USING HUMAN/ANIMAL LUNG
625
transducer, because lateral drops may be formed. This particular assay method should be used only if the partition coefficient between the water and oil phases of the compound under study guarantees full retention in the buffer. Spiral strips of human bronchioles are easily adjusted to the laminar flow technique and amounts as small as 0.3-1 pmol of sulfidopeptide leukotrienes can be detected. This method is preferred when only very minute amounts of different eicosanoids or drugs are available. Preparation of Guinea Pig Isolated Trachea, Parenchyma, and Lung The airway tree of the guinea pig has been extensively studied, particularly for eicosanoid research. The first evidence of the existence of thromboxane (Tx) A2 and sulfidopeptide LT came from experiments using this particular organ. The tracheopulmonary system of the guinea pig can be easily excised and the trachea and peripheral parenchymal strips can be isolated. These isolated preparations represent suitable models of the central and peripheral airways, respectively. Guinea pig lungs may also be used whole, perfused through the pulmonary artery, and occasionally even ventilated with a respiratory pump. It is, therefore, possible to perform functional studies at different airway levels as well as to investigate the capacity of the organ to respond to different stimuli with a secondary formation of eicosanoids. The tracheal smooth muscle of the guinea pig is well known for its outstanding responsiveness to multiple agonists; a contractile doseresponse curve to sulfidopeptide LT ranges from 0.03 to 100 nM and no tachyphylaxis occurs. Moreover, this response is likely to be modulated by the concomitant formation of other dilator prostanoids (perhaps PGE2) as the maximal response is potentiated by pretreatment of the tissue with indomethacin or meclofenamic acid (1/zM). Several preparations have been described using the isolated guinea pig trachea and with careful dissection the muscle fibers can be arranged to run longitudinally. A chain of tracheal sections can be made by cutting individual rings of cartilage and tying them together. At least three rings are required to obtain contractions that are adequately recorded: resting tension 1-2 g and isotonic tone recording. Tyrode's or Krebs physiological solutions are normally used. Alternatively, the trachea can be set up as a spiral or a zigzag section and this technique brings the muscle sections close to each other; the smooth muscle fibers are located dorsally and usually the cartilaginous rings are cut ventrally along the middle, from the larynx to the first bifurcation. The organ is opened, fixed on a cork disc
626
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[67]
with fine pins at its comers and zigzag sections cut with a sharp scalpel. Fine silk threads are then bound to the extremities for connection to the transducer; resting load is usually adjusted to 2.5-3.0 g. The isolated tracheal tube of the guinea pig can also be set up intact, by sealing the lower end (linked to a glass hook) and by connecting the upper end to a pressure transducer. The system is filled with buffer to obtain a resting intraluminal pressure of 1 mm Hg and the preparation can be stimulated intra- and extraluminally via platinum wire electrodes with square wave pulses. 6 All the preparations of the guinea pig airways described above can be easily set up using standardized isolated organ baths, thermostatted at 37°, and oxygenated with a mixture of 95% 02-5% CO2. Tracheal rings and zigzag preparations, as well as the lung parenchymal strips, can also be superfused at constant flow of 8 to 10 ml/min or arranged according to the laminar flow technique. Guinea pig lung parenchymal strips are also widely used preparations for the study of eicosanoids and related drugs. After sacrifice by bleeding, the lung are excised and care should be taken to save approximately 1 cm of trachea. After a gentle wash with Krebs-Henseleit buffer, the lungs are slightly inflated with air and strips about 3 mm wide, 3 mm thick, and 2-3 cm long are cut along the outer curvature of the lobe rim. The extremities are then connected using a silk thread to an isotonic force transducer under a resting tension of 1 to 1.5 g, at 37° and gassed with a mixture of 95% 02-5% CO2; the tissues are equilibrated in approximately 30-60 min. Usually a contractile dose response curve to LTC4 and LTD4 ranges from 0.1 to 1/xM and involves, at least in part, cyclooxygenase-derived constrictor products of arachidonic acid, namely TxA2. LTB4 is equiactive with sulfidopeptide LT and significant induction of tachyphylaxis might take place. It is important to keep in mind that both guinea pig trachea and lung parenchyma are capable of metabolizing the peptide LT and therefore their functional response might change according to the tissue content of transpeptidase or aminopeptidase. 7 Guinea pig lungs can also be set up intact, isolated, perfused, and ventilated. For this study guinea pigs are anesthetized with pentobarbital (30 mg/kg, ip) and the trachea cannulated for artificial ventilation using a respiratory pump (50-60 cpm, 1.5 ml tidal volume/100 g body weight and approx. 2 cm water end-expiratory pressure). In order to stop spontaneous breathing, the animals are paralyzed with pancuronium bromide (4 mg/kg, iv). Before getting ready to transfer the lungs from the chest cavity to a thermostatted glass chamber, a thoracotomy is performed and the pulmonary artery dissected; the right ventri6 R. A. Coleman and G. P. Levy, Br. J. Pharmacol. 52, 167 (1974). 7 D. W. Snyder and R. D. Krell, J. Pharmacol. Exp. Ther. 231, 616 (1984).
[67]
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cle is cut open and the pulmonary artery cannulated with the cannula used to deliver the perfusing buffer (Krebs-Henseleit) by means of a peristaltic pump. Once cannulation is completed, the lungs are freed from the left atria and other mediastinal structures and suspended in an appropriate chamber; perfusion is carried out at 8 to 12 ml/min. The trachea is then connected via a "1"piece to the respiratory pump and to a pressure transducer for continuous recording of ventilation pressure. A second pressure transducer inserted between the peristaltic pump and the pulmonary artery allows recording of pulmonary arterial pressure. The perfusion buffer is collected from the bottom of the glass chamber and recirculated using the same peristaltic pump for several hours (3-4), if necessary. Alternatively, the lung effluent can superfuse a number of selective bioassay tissues for detection of prostaglandin or SRS-A-Iike activity. Challenge can be performed by bolus injections of different agonists into the cannula inserted in the left atrium. By using this experimental approach the lungs can be challenged repeatedly and maintain their capacity to release (and metabolize, e.g., PGE2) eicosanoids depending on the type and intensity of the stimulus. Usually no tachyphylaxis occurs. It is also possible to study the profile of eicosanoid release that takes place after an anaphylactic shock in oitro. For this purpose guinea pigs are sensitized to allergens; the one most widely used is ovalbumin. A variety of protocols for inducing sensitization can be followed and all seem to induce antibody production successfully. Two major sensitization procedures 8 have been proposed for (a) production of high titer specific IgE- and moderate quantities of specific IgG-like antibodies or (b) production of IgG-like antibodies. (1) Guinea pigs of either sex are given one intraperitoneal injection of 0.5 ml saline containing 1 /~g ovalbumin and 100 mg AI(OH)3. One milliliter of adjuvant is added to the antigen solution 1 hr before injection. The animals can be challenged in oioo with 5 /xg/kg ovalbumin 2 months later. (2) Guinea pigs are injected ip with 5 mg ovalbumin on day 0 and 10 mg on day 2 (injection volume 0.1 ml). The animals are ready for in vivo challenge with 120/zg/kg ovalbumin 2 months later. A modification of procedure (1) has been reported 9 as successful in sensitizing guinea pigs: two subcutaneous injections of 0.5 ml saline containing 10/xg of ovalbumin dispersed in 1 mg of AI(OH)3 are given with an interval of 2 weeks. The animals are then used 10 days after the second injection. Another protocol is based on intraperitoneal (100 mg) and subcutaneous (I00 mg) injections of ovalbumin; the animals are killed 3 weeks 8 p. Andersson and H. Bergstrand, Br. J. Pharmacol. 74, 601 (1981). 9 j. Randon, J. Lefort, and B. B. Vargaftig, Br. J. Pharmacol. 92, 683 (1987).
628
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[67]
later. When the anaphylactic shock is to be carried out in vitro, it is possible to modulate its severity by using different amounts of antigen. For a massive release of eicosanoids, a bolus injection of 2-5 mg ovalbumin into the pulmonary artery (or in case of the isolated tracheal spiral or parenchymal strip a concentration of 0. I-0.5 mg/ml ovalbumin in the organ bath) is advised. However, amounts as small as 0.1-0.2/zg have been reported to induce TxB2 release from sensitized lung strips. These sensitization procedures are successful in about 90% of the treated animals.
EICOSANOID
STUDIES
USING HUMAN/ANIMAL
LUNG
621
Acknowledgment Supported by N I H grants H L 34215 and H L 18828 to R L and H L 31591 to KM.
[67] Preparation of Human and Animal Lung Tissue for Eicosanoid Research By T. V1GAN~), M. T. CRIVELLARI,M. MEZZETTI, and G. C. FOLCO
Eicosanoids are fatty acids of great biological significance because of their ubiquity, high potency, and multitude of other effects. Among all the organs that have been investigated, the lungs are the most active in forming and metabolizing eicosanoids; human and animal lung tissue can oxidize arachidonic acid (AA), generating cyclooxygenase as well as lipoxygenase-derived products that appear to play a role in the development of asthma, allergic reactions, and inflammation. In fact, leukotrienes (LT) have potent spasmogenic actions on human lung tissue in vitro, affect mucus production and clearance, and may be important factors in the pathogenesis of airway hyperresponsiveness. A sufficient number of in vitro methods has been developed utilizing human and animal lung tissue for eicosanoid research; this chapter will describe techniques that are useful in understanding the physiopathological role of eicosanoids in the pulmonary system as well as their functional effects. Preparation of Human Lung Parenchyma to Produce Eicosanoids Normal human lung parenchyma is a preferential tissue to use and probably represents the richest source of sulfidopeptide LT and of PGD2. The tissue is usually available following surgery for pulmonary carcinoma or bronchiectasis; the lung parenchyma should be immediately collected in a buffered solution such as Tyrode's [composition (in g/liter): NaC1, 8.0, KC1, 0.25, CaC1 0.15 MgCI2-6 H20, 0.01, NaH2PO4"H20, 0.066, glucose, 1.0, NaHCO3, 1.0] or Krebs-Henseleit [composition (in g/liter): NaCI, 6.9, KCI, 0.35, KHzPO4, 0.16, MgSO4.7 H20, 0.29, CaC12, 0.28, glucose, 1.0, NaHCO3,2.1], pH 7.4, kept at 4° and gassed with a mixture of 95% 02-5% CO2. It is imperative that the tissue not be frozen as it loses its capacity to respond to different stimuli. METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
622
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[67]
Small biopsies of approximately 100 mg each are sampled at random from the excised lung portion and used for histological examination to exclude the possibility of neoplastic infiltrations. Usually the available peripheral tissue is free of macroscopically evident bronchi or vessels and is chopped manually to fragments of approximately I00 mg each. The fragments are washed again using the same buffer and can now be utilized for release experiments. It is possible to follow at least two different experimental protocols in order to mimic a release of lipid mediators that is likely to occur during immediate hypersensitivity reactions. The fragments can be passively sensitized using diluted reaginic serum from hyperimmune patients (1500-5000 ng/ml of specific IgE). The tissue is incubated overnight (1 g tissue/5 ml diluted serum) at room temperature in a Dubnoff shaking incubator without need for direct oxygenation; alternatively, the passive sensitization can be carried out using a shorter incubation step (3 hr) at 37°. Lung parenchyma is then washed extensively using the same buffer and divided into portions of about 1 g resuspended in 10 ml of fresh Tyrode's or Krebs buffer. The parenchymal fragments are preincubated for 10 min under gentle agitation and challenged with the appropriate antigen (usually 500-1000 ng/ml), agonists or vehicle. At the end of the incubation, an aliquot (max. 0.5 ml) is collected for prostaglandin assay using RIA I or EIA 2 while another is immediately tested for the presence of eicosanoid-like activity using appropriate bioassay systems. 3 The remaining volume (approximately 8 ml) can be added to the required internal standards (PGB2 500 ng or tracer amounts of radiolabeled LT, 20,000 dpm) and frozen or further processed for quantitative assays of prostanoids using physicochemical methods. It is also possible to trigger the immunological release of different autacoids from pulmonary tissue using specific anti-human IgE antibodies which are usually raised in sheep or goat. Our personal experience, accumulated in about 40 experiments with an equal number of human specimens, indicates that the mediator release obtained in this experimental condition does not require the time-consuming step of passive sensitization. Usually after mincing and careful washing of the lung fragments, the tissue is preconditioned in Tyrode's buffer for approximately 30-60 min at 37° (1 g/5 ml), rewashed, and subjected to final incubation in Tyrode's buffer (1 g/10 ml). The challenge is performed using a final anti-human IgE 1 E. Granstrom and H. Kindahl, Adv. Prostaglandin Thromboxane Res. 5, 119 (1978). 2 p. Pradelles, J. Grassi, and J. Maclouf, Anal. Chem. 57, 1170 (1985). 3 S. Moncada, S. H. Ferreira, and J. R. Vane, Adv. Prostaglandin Thromboxane Res. 5, 211 (1978).
[67]
EICOSANOID STUDIES USING HUMAN/ANIMAL LUNG
623
antibody dilution varying between 1:300 to 1:1000. Preparation and storage of aliquots is carried out as described above. As the availability of good hyperimmune sera as well as that of antihuman IgE antibodies can present a problem, a preliminary check of the tissue releasability should be performed with the calcium ionophore A23187 (I-5/~M), stopping the reaction 20-30 min after challenge. By the concomitant use of a bioassay setup which utilizes a strip of longitudinal smooth muscle of guinea pig ileum, it is possible to follow rather carefully the development of the experiment. It is advisable to carry on the immunological challenge only in tissues that have responded successfully to A23187. Experience with approximately 200 human lung parenchymal specimens, indicates that while good releasability after A23187 treatment occurs in about 80-90% of the cases, a successful immunological challenge takes place in about 50% of the tissues. Preparation of Human Bronchial Tissue Both large and small human airways are suitable models to assess the pharmacological activity of eicosanoids as well as their role in pathophysiology. Bronchi of different caliber, from primary bronchi to terminal bronchioles, are utilized for such studies, however the latter, with an internal caliber of 1 to 2 mm, are better suited because they have no cartilage plates. These can be identified and differentiated from pulmonary arteries and veins of similar caliber. The vascular lumen can be pinched with small forceps and with gentle pulling can be detached from the surrounding parenchyma, whereas, the bronchioles cannot be freed. In addition, particularly following surgery for bronchiectasis, the bronchial lumen shows presence of mucous plugs. A fine polyethylene catheter is gently slipped through the full length of the bronchus in order to facilitate its localization. Further confirmation that one is dealing with a bronchiole comes from the fact that a gentle administration of air into the catheter leads to tissue inflation. The cannula is left in place and the bronchioles are carefully dissected free of parenchyma and blood vessels; it is advisable to leave a minimum layer of parenchyma to avoid inadvertent damage of the bronchiole smooth muscle. During this, as well as the following preparation steps, the tissue is kept moist with lukewarm saline. The polyethylene tubing is now fixed in a vertical position and the bronchiole spiralized to obtain a strip 2-3 mm wide and of varying length (usually 20-30 mm). Great care is taken to avoid the stretching of the preparation as this may damage its contractile function; a silk surgical thread is tied to both ends of the strip.
624
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[67]
The smooth muscle strips are suspended in a 10-ml organ bath, under an isometric resting tension of 200 to 500 mg, at 37°, in a Krebs-Henseleit solution oxygenated with a mixture of 95% 02-5% CO2. The time required to attain a steady baseline tone may vary considerably. Whenever possible the tissues should be used the day of surgery or stored overnight at 4 ° under a slow constant flow of oxygenated buffer and then set up the following day, without appreciable loss of sensitivity or contractility. Transducers are coupled to a multichannel pen recorder and during the stabilization period (I-2 hr) it is advisable to change the buffer every I015 min. Pharmacological studies can be performed by adding agonist eicosanoids as well as their potential antagonists in small volumes (0.05-0.1 ml). In this way, cumulative dose-response curves (for sulfidopeptide LT, 0.1-100 riM) can be constructed as tachyphylaxis for LT or bronchoactive prostaglandins (e.g., PGD2) has not been described in human bronchi. By using a similar technique, Dahlen e t al. 4 w e r e able to demonstrate that bronchi from allergic asthmatics respond to antigen exposure with a contraction that correlates with the release of sulfidopeptide LT. Human bronchiolar strips represent a relatively homogeneous structure and their functional changes are not affected by vascular smooth muscles that lie in close proximity, as is the case of the parenchyma. They can be used as detector organs for eicosanoids and, in general, for bronchoactive substances, such as histamine or PAF. The classical nonflow organ bath technique and also the perifusion technique (i.e., the tissue is set up in a tissue chamber where the buffer is constantly changed by means of a pump) require that the active compound be diluted in a volume that rarely is smaller than 5 ml. On the other hand, superfusion techniques involve only a very transient contact of the tissue with the substances added as bolus, but also require a rather high flow, 5-10 ml/min, once again leading to high dilution of the samples being tested. A technique that we have used successfully is laminar flow superfusion, first suggested by Ferreira 5 in 1976. The isolated assay tissue is immersed in mineral oil thermostatted at 37° and superfused with a drip of Tyrode's solution at a very low rate of flow (0.1-0.2 ml/min). Each drop of aqueous buffer floats slowly downward at the interface between the tissue and the oil, allowing a contact time that is approx. 1 sec/1 cm of strip length. It is important to ensure a close contact between the catheter delivering the buffer and the thread which connects the tissue with the 4 S. E. Dahlen, G. Hansson, P. Hedqvist, T. Bjorck, E. Granstrom, and B. Dahlen, Proc. Natl. Acad. Sci. U.S.A. 80, 1712 (1983). 5 S. H. Ferreira and F. De Souza Costa, Eur. J. Pharmacol. 39, 379 (1976).
[67]
EICOSANOID STUDIES USING HUMAN/ANIMAL LUNG
625
transducer, because lateral drops may be formed. This particular assay method should be used only if the partition coefficient between the water and oil phases of the compound under study guarantees full retention in the buffer. Spiral strips of human bronchioles are easily adjusted to the laminar flow technique and amounts as small as 0.3-1 pmol of sulfidopeptide leukotrienes can be detected. This method is preferred when only very minute amounts of different eicosanoids or drugs are available. Preparation of Guinea Pig Isolated Trachea, Parenchyma, and Lung The airway tree of the guinea pig has been extensively studied, particularly for eicosanoid research. The first evidence of the existence of thromboxane (Tx) A2 and sulfidopeptide LT came from experiments using this particular organ. The tracheopulmonary system of the guinea pig can be easily excised and the trachea and peripheral parenchymal strips can be isolated. These isolated preparations represent suitable models of the central and peripheral airways, respectively. Guinea pig lungs may also be used whole, perfused through the pulmonary artery, and occasionally even ventilated with a respiratory pump. It is, therefore, possible to perform functional studies at different airway levels as well as to investigate the capacity of the organ to respond to different stimuli with a secondary formation of eicosanoids. The tracheal smooth muscle of the guinea pig is well known for its outstanding responsiveness to multiple agonists; a contractile doseresponse curve to sulfidopeptide LT ranges from 0.03 to 100 nM and no tachyphylaxis occurs. Moreover, this response is likely to be modulated by the concomitant formation of other dilator prostanoids (perhaps PGE2) as the maximal response is potentiated by pretreatment of the tissue with indomethacin or meclofenamic acid (1/zM). Several preparations have been described using the isolated guinea pig trachea and with careful dissection the muscle fibers can be arranged to run longitudinally. A chain of tracheal sections can be made by cutting individual rings of cartilage and tying them together. At least three rings are required to obtain contractions that are adequately recorded: resting tension 1-2 g and isotonic tone recording. Tyrode's or Krebs physiological solutions are normally used. Alternatively, the trachea can be set up as a spiral or a zigzag section and this technique brings the muscle sections close to each other; the smooth muscle fibers are located dorsally and usually the cartilaginous rings are cut ventrally along the middle, from the larynx to the first bifurcation. The organ is opened, fixed on a cork disc
626
CELL MODELS OF LIPID MEDIATOR PRODUCTION
[67]
with fine pins at its comers and zigzag sections cut with a sharp scalpel. Fine silk threads are then bound to the extremities for connection to the transducer; resting load is usually adjusted to 2.5-3.0 g. The isolated tracheal tube of the guinea pig can also be set up intact, by sealing the lower end (linked to a glass hook) and by connecting the upper end to a pressure transducer. The system is filled with buffer to obtain a resting intraluminal pressure of 1 mm Hg and the preparation can be stimulated intra- and extraluminally via platinum wire electrodes with square wave pulses. 6 All the preparations of the guinea pig airways described above can be easily set up using standardized isolated organ baths, thermostatted at 37°, and oxygenated with a mixture of 95% 02-5% CO2. Tracheal rings and zigzag preparations, as well as the lung parenchymal strips, can also be superfused at constant flow of 8 to 10 ml/min or arranged according to the laminar flow technique. Guinea pig lung parenchymal strips are also widely used preparations for the study of eicosanoids and related drugs. After sacrifice by bleeding, the lung are excised and care should be taken to save approximately 1 cm of trachea. After a gentle wash with Krebs-Henseleit buffer, the lungs are slightly inflated with air and strips about 3 mm wide, 3 mm thick, and 2-3 cm long are cut along the outer curvature of the lobe rim. The extremities are then connected using a silk thread to an isotonic force transducer under a resting tension of 1 to 1.5 g, at 37° and gassed with a mixture of 95% 02-5% CO2; the tissues are equilibrated in approximately 30-60 min. Usually a contractile dose response curve to LTC4 and LTD4 ranges from 0.1 to 1/xM and involves, at least in part, cyclooxygenase-derived constrictor products of arachidonic acid, namely TxA2. LTB4 is equiactive with sulfidopeptide LT and significant induction of tachyphylaxis might take place. It is important to keep in mind that both guinea pig trachea and lung parenchyma are capable of metabolizing the peptide LT and therefore their functional response might change according to the tissue content of transpeptidase or aminopeptidase. 7 Guinea pig lungs can also be set up intact, isolated, perfused, and ventilated. For this study guinea pigs are anesthetized with pentobarbital (30 mg/kg, ip) and the trachea cannulated for artificial ventilation using a respiratory pump (50-60 cpm, 1.5 ml tidal volume/100 g body weight and approx. 2 cm water end-expiratory pressure). In order to stop spontaneous breathing, the animals are paralyzed with pancuronium bromide (4 mg/kg, iv). Before getting ready to transfer the lungs from the chest cavity to a thermostatted glass chamber, a thoracotomy is performed and the pulmonary artery dissected; the right ventri6 R. A. Coleman and G. P. Levy, Br. J. Pharmacol. 52, 167 (1974). 7 D. W. Snyder and R. D. Krell, J. Pharmacol. Exp. Ther. 231, 616 (1984).
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cle is cut open and the pulmonary artery cannulated with the cannula used to deliver the perfusing buffer (Krebs-Henseleit) by means of a peristaltic pump. Once cannulation is completed, the lungs are freed from the left atria and other mediastinal structures and suspended in an appropriate chamber; perfusion is carried out at 8 to 12 ml/min. The trachea is then connected via a "1"piece to the respiratory pump and to a pressure transducer for continuous recording of ventilation pressure. A second pressure transducer inserted between the peristaltic pump and the pulmonary artery allows recording of pulmonary arterial pressure. The perfusion buffer is collected from the bottom of the glass chamber and recirculated using the same peristaltic pump for several hours (3-4), if necessary. Alternatively, the lung effluent can superfuse a number of selective bioassay tissues for detection of prostaglandin or SRS-A-Iike activity. Challenge can be performed by bolus injections of different agonists into the cannula inserted in the left atrium. By using this experimental approach the lungs can be challenged repeatedly and maintain their capacity to release (and metabolize, e.g., PGE2) eicosanoids depending on the type and intensity of the stimulus. Usually no tachyphylaxis occurs. It is also possible to study the profile of eicosanoid release that takes place after an anaphylactic shock in oitro. For this purpose guinea pigs are sensitized to allergens; the one most widely used is ovalbumin. A variety of protocols for inducing sensitization can be followed and all seem to induce antibody production successfully. Two major sensitization procedures 8 have been proposed for (a) production of high titer specific IgE- and moderate quantities of specific IgG-like antibodies or (b) production of IgG-like antibodies. (1) Guinea pigs of either sex are given one intraperitoneal injection of 0.5 ml saline containing 1 /~g ovalbumin and 100 mg AI(OH)3. One milliliter of adjuvant is added to the antigen solution 1 hr before injection. The animals can be challenged in oioo with 5 /xg/kg ovalbumin 2 months later. (2) Guinea pigs are injected ip with 5 mg ovalbumin on day 0 and 10 mg on day 2 (injection volume 0.1 ml). The animals are ready for in vivo challenge with 120/zg/kg ovalbumin 2 months later. A modification of procedure (1) has been reported 9 as successful in sensitizing guinea pigs: two subcutaneous injections of 0.5 ml saline containing 10/xg of ovalbumin dispersed in 1 mg of AI(OH)3 are given with an interval of 2 weeks. The animals are then used 10 days after the second injection. Another protocol is based on intraperitoneal (100 mg) and subcutaneous (I00 mg) injections of ovalbumin; the animals are killed 3 weeks 8 p. Andersson and H. Bergstrand, Br. J. Pharmacol. 74, 601 (1981). 9 j. Randon, J. Lefort, and B. B. Vargaftig, Br. J. Pharmacol. 92, 683 (1987).
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later. When the anaphylactic shock is to be carried out in vitro, it is possible to modulate its severity by using different amounts of antigen. For a massive release of eicosanoids, a bolus injection of 2-5 mg ovalbumin into the pulmonary artery (or in case of the isolated tracheal spiral or parenchymal strip a concentration of 0. I-0.5 mg/ml ovalbumin in the organ bath) is advised. However, amounts as small as 0.1-0.2/zg have been reported to induce TxB2 release from sensitized lung strips. These sensitization procedures are successful in about 90% of the treated animals.
