Respiratory Medicine (1990) 84, 419--433
Lung inflammation and injury: models and mechanisms Abstracts of the 9th summer meeting of The British Association for Lung Research, held in Edinburgh
3-4 September, 1990
420 Abstracts
Preface to Abstracts
In this issue of the Journal are published for the first time the proceedings of the British Association for Lung Research (BALR). BALR was founded in 1981 by a group of medical and non-medical scientists in medicine, research establishments and drug companies, who identified the need for a society which brought together workers from different scientific disciplines who had in common the experimental study of lung disease. The aims of the society were to 'promote interest and encourage studies in the field of experimental research related to the elucidation and treatment of lung disease'.
We hold one day workshops usually in or around London in the Winter and Spring, often in a research establishment. There have previously been meetings of the society at Aldermaston, Huntingdon and at Porton Down, where BALR was the first outside society allowed to hold a meeting. The meetings are organized by the Meetings Secretary with the local organizer and there are invited speakers and free communications. The topics covered have included immunology and ultrastructure of the lung, the effects of viruses, drugs, irradiation and toxins on the lung, and animal models and in vitro techniques in lung research. The annual meeting is over two days usually in a University department and has been held at Bath, Cardiff, London, Surrey and the present meeting Edinburgh. On the first day there is a symposium with invited experts often from overseas particularly the USA. The symposia have included the cellular and biochemical mechanisms in emphysema, pulmonary carcinogenesis, nonrespiratory functions of the lung and the present meeting on the models and mechanisms of pulmonary inflammation. On the second day there are free communications and a poster session. One of the most enjoyable aspects of the Summer Meeting is the Young Scientist of the Year Award which is made to the best paper presented in the open session. There have often been discussions as to who is young in this context but the society is small enough for people to agree as to who is eligible, although one of the winners did raise some eyebrows! The standard of presentation and the work presented is of a very high order and this competition has proved to be a highlight ofthe meeting and has been admired by our visiting experts who are on the judging panel. Although the society has only about 200 members the workshops usually attract at least 50 people and the annual meeting around 100.
BALR is most grateful to the Editorial Board of Respiratory Medicine which has agreed to publish abstracts of the Annual Meeting in the future. The vitality of the society has been mainly due to the nonmedical scientists-- biochemists, cell and molecular biologists and toxicologists--but they have always valued and sought the input of the medical scientist and clinician so that they could clearly relate their research to the patient. Although there has always been a hard core of clinicians in the society we have always felt that there was a need for more clinical participation and hopefully the publicity of our meetings by the journal will stimulate more clinicians to join. I am sure that they will find it a most friendly and stimulating society, as I have done during the past 8 years.
BERNARD FOX
Chairman of BALR 1985--89 Department of Histopathology
CharhTg Cross and Westminster Medical School London. U.K.
�9 1990 Bailli~re Tindail 0954-6111/90/050419+ 15 $03:00/0
Abstracts 421
K.F.Chung MODELS OF BRONCHIAL ASTHMA
Department of Thoracic Medicine, National Heart & Lung Institute, London SW3 6LY
Bronchial asthma is characterised by intermittent bronchospasm and bronchial hyperactivity (BHR) resulting from a chronic airway inflammatory process. There are no satisfactory models of bronchial asthma and there is no disease in animals that resemble closely human asthma. Transient BHR can be provoked in man and animals and this has formed the basis of much work linking inflammatory cell damage and mediators to the development of BHR. Aspects of the inflammatory process such as eosinophil infiltration and airway vascular changes may be mimicked by airway challenge with allergen and mediators. These models provide some insight into possible inflammatory mechanisms involved in altering lung function, mechanisms which can later be studied in human asthma. What is urgently needed is a convincing model of persistent BHR.
Models of f~ronicLung Infection Peter Cole. Host Defence Unit, Dept. of Thoracic Medicine, NHLI, LOhKDN ~;3 6LY.
The pathogenesis of chronic lung infection has received little attention until comparatively recently.
Chronic lung infection in man is usefully studied in the model of bronchiec- tasis - the final common pathway of several conditions predisposing to persistent lung infectioru In this condition primary disorders of muoociliaryclearanee allow those bacteria whlchproduce cilio-inhibitorysubstances to colonise the airways, mainly in mucus. Here the presence of such microorganisms stimulates a host inflammatory response which becomes chronic because it fails to eliminate the colonists. This causes damage to "innocent bystander" lung and further colonis- ation - in a vicious circle of events leading to death or lung transplantatioru
An experimental model of bronchiectasis allows the various factors implicated in pathogenesis of the human disease to be manipulated. It provides basic information on host-microbial interrelations at the mucosal surface and enables logical therapy to be tested - promising results being explored in clinical trials in human disease.
Mechanisms of Lung Inflammation in COPD W MacNee, Unit of Respiratory Medicine, Department of Medicine (RIE), City Hospital, Greenbank Drive, EDINBURGH EHI0 5SB.
The major cause of COPD is cigarette smoking, although inhaled irritants such as ozone also contribute. The airway and alveolar injury which occurs in this condition is thought to occur as a result of an imbalance between proteases/antiproteases and oxidants/anti-oxidants. An in vitro model to study smoke-induced injury to airspace cells using A549 type cells will be discussed. Leucocytes are present in greater numbers in the distal airspaces of smokers. Neutrophil sequestration in the lungs can be studied by measuring neutrophil kinetics in the lungs of both man and animals and the mechanism of this sequestration can be determined by in vitro models of the pulmonary micro-circulation. Using these techniques we have found that neutrophils are delayed in the pulmonary circulation during acute cigarette smoke exposure. This delay in neutrophil traffic may result from a smoke-induced alteration in cell deform- ability. Although activated leucocytes are also delayed in transit in the pulmonary circulation in animals, there is little evidence of smoke-induced activation of leuco- cytes in vitro, rather both leucocytes and epithelial cells appear to sustain oxygen- induced damage when exposed to cigarette smoke. Such a combination of in vivo/in vitro techniques in both man and animals may help to elucidate the complex inflam- matory response in COPD.
422 Abstracts
Experimental Bronchitis: The Effects of Anti-lnflammatory Drugs PK Jeffery, DF Rogers, MM Ayers & R Godfrey. Department of Lung Pathology, National Heart & Lung Institute, Brompton Hospital, LONDON SW3 6HP.
Chronic bronchitis (chronic hypersecretion) and chronic bronchiolitis (small airways disease) both contribute to airflow obstruction in man, the latter associated with progressive deterioration in lung function. Mucous metaplasia and hyperplasia are characteristic histological changes. Experimentally, cigarette smoke (CS) given daily for 2 weeks induces similar histological changes in the airways of specific pathogen-free rats, providing a suitable animal model for study. The CS-induced secretory cell hyperplasia is inhibited completely or to varying degrees by prophylactic administration (intraperitoneal injection) of either indomethacin, flurbiprofen, dexamethssone, prednisolone, hydrocortisone (each at 2 or 4 mg/kg body weight) or a mucolytic drug, N-acetylcysteine (Nat), given orally as a 1% solution of the drinking water. Nac also inhibits the associated mucus-hypersecretion, alters the kinetics of the proliferative response and affects airways permeability. It takes between 21 and 84 days, depending on airway level, for the increase in secretory cell number to return to control values (ie recover). Indomethacin and flurbiprofen (4 mg/kg, by ip injection) shorten recovery to between 4 and 9 days in intrapulmonary airways but have no effect on recovery time in the rat trachea. Nac has a similar recovery effect in 6 of 7 airway levels which showed cigarette smoke-induced mucous cell hyperplasia.
~e Use of Animal _~v~1, in the S ~ of i ~ Fibrosis Geoffrey J Laurent. Biochemistry Unit, Department of Thoracic Medicine, University of London, National Heart and Lung Institute, LOI~3ON SW3 6LR.
Studies of fibrotic lung disorders using experimental animals are performed to give us information on pathogenesis of human disease. They also allow us to test agents directed at preventing these diseases when they develop in man. Both sorts of studies are aimed at improving therapy in a group of respiratory disorders which at present are treated inadequately. The theoretical advantage of animal models is clear - they allow us to study the development of disease from its earliest stages and are amenable for studies using new drugs. However, if the model is inappropriate, information obtained could be misleading. With that in mind, it is useful to have criteria for developing animal models of pulmonary fibrosis. Three such criteria are: (i) the agent (or procedure) is known to cause pulmonary fibrosis in man; (2) the sequence of morphological and biochemical changes mimic, as far as is known, those in man; and (3) the lung pathology at death is similar to that of human disease. There are currently several experimen- tal models which in large part fulfill these criteriau This paper suggests that, combined with other approaches, the continued use of animal models of pulmonary fibrosis is warranted so as to better understand pathogenesis and develop new drugs aimed at preventing fibrotic lung disorders.
Abstracts 423
TheEffect Of Dexamethasone On Oxygen-InducedPulmona~ InflammationIn The Preterm Guinea Pig. G Phillips, I Town, M Landrsau, J Louden, F Kelly. Departments of Human Nutrition and Medicine I, University of Southampton, Southampton SO9 3TU, U.K.
Dexamethasone (DEX), is widely used in the treatment of Bronchopulmonary dysplasia, a chronic lung disease of preterm infants, although its precise mode of action remains unclear. We have assessed the effects of DEX in the preterm guinea pig model of oxygen-induced pulmonary inflammation. Preterm guinea pigs, delivered by Caesarian section three days prior to normal term (68 days), were randomly exposed to either 95% O^ or 21% O_ for 72h and then room air for up to 96h. One half
�9 z z of the pups in each group were given a daily injection of either DEX (lOmg/kg) or an equivalent volume of saline. Bronchoalveolar lavage (BAL) was performed at 3, 5 and 7 days after delivery. The results for the oxygen exposed animals are shown below.
Time (d) Neutrophil count (X 104/mi) Saline DEX
3 15.1~12.3 9.1• 5 51.3• 4.5• 7 31.7• 26.2• Mean data • std dev * p<0.05; DEX v Saline.
Total BAL protein (mg/ml) Saline DEX
0.7• 1.5• 0.4• 0.5• 0.2• 0.2• (N = 7-9 animals/group)
DEX reduced the neutrophilia associated with oxygen-lnduced injury of the immature lung, but had no effect on tissue injury as measured by the influx of plasma protein.
Charaeterisation Of Cell Aggregates in Bronchoalveolar i~vage Obtained From ~ts After Intratraeheal Instillation Of Organic Dust DM Brown, K Donaldson. Institute of Occupational Medicine Ltd, Roxburgh Pl~e, EDINBURGH EH8 9SU
Intratraoheal instillation of dust collected from the air of wool mills into rat lungs resulted in the production of an acute inflammatory response as assessed by increased n~nbers of neutrophils in bronchoalveolar lavage. This effect had returned to control values by 3 days post exposure. Aggregates of mononuclear cells which did not coincide with the peak of inflammation were present from day 3 and lasted for at least 14 days. The time course of this observation suggested involvement of the immune system and we therefore set out to characterise the phenotypes of cells making up the aggregates. Using monoclonal antibodies to rat macrophages and T-lymphoeytes, and subsequent immunostaining, we demonstrated that the aggregates were predominantly comprised of alveolar macrophages and neutrophils, with only very occasional T-lymphooytes, The aggregation of cells suggests that there may be lectin-like activity in wool dust which could result in increased proliferative responses in lymph nodes which we have detected.
The Metabolism Of 3-Trif luoromethylpyridine By Isolated Olfactory Epithelial C~lls . , W A Evans#~ C G Curtis ~ P M Hext ~, R J Rlchards . Department of Blochemlstry , Unlversity of Wales College ~[ Cardiff, Cardiff CFI IST and Central Toxicology Laboratory ~' ICI plc, Alderley Edge, Cheshire.
The olfactory epithelium of mammals is recognised as an important site for the metabolic transformation of xenobiotics. Studies in vivo have revealed that the accumulation of 3- trifluoromethylpyridine (3FMP), or a metabolite thereof, produces rapid and selective damage to the rat olfactory epithelium. In the present study, an isolate of olfactory cells, enriched for NADPH reductase, has been identified a~4the target for 3FMP. When the cells are incubated with IX C]- 3FMP, HPLC analysis reveals the presence of several metabolites released into the medium and the major product is 3FMP N-oxide. This N-oxide is selectively toxic to the olfactory cells in vitro. The same metabolite is not toxic to isolates of lower respiratory tract ( Clara and epithelial type II) cells.
424 Abstracts
TBe Role of the Cytochrome P450 System in the Activation of Systemic Lung Toxins R.D. Verschoyle. M.R.C. Toxicology Unit, Woodmansterne Road~ CARSHALTON, Surrey, SM5 4EF
A number of systemic lung toxins are activated by the pulmonary cytochrome P450 system.
We have investigated the involvement of individual P450 isozymes in the activation of three toxins that damage the alveolar epithelium; 0,0,S-trimethylphosphorothiolate and (methylcyclopentadienyl) manganese tricarbonyl, in rats, and also butylated hydroxytoluene, in mice.
Compounds modifying the activity of specific P450 isozymes have been used to decrease the acute toxicity of these pneumotoxins. Changes in lung weight and also in enzyme concentrations in broncho- alveolar lavage fluid show that this decrease in toxicity is related to decreased lung damage.
Specific substrates have been used to assess the induction and inhibition of particular isozymes in lung and changes in the pulmonary concentration of these isozymes, following chemical manipulation, have been measured by immunoblotting.
Abstracts 425
Clara Cells, Reduced G~utathione And Cigarette Smoke R J Richards, F Fahim , L Masek. Department of Biochemistry, University of Wales Colleg~ of Cardiff, Cardiff CFI IST, UK and Department of Biochemistry , Ain Shams University, Cairo, Eygpt
Human airways contain high levels (0.4mM) of the antioxidant and phase II metabolism component, reduced glutathione (GSH) the amount of which increases in cigarette smokers (0.8mM). The source of this GSH is unknown but the bronchiolar Clara cell (3.2mM) is one likely provider. An in vitro assay system, based on the attachment of functionally competent mouse Clara cells to an extracellular matrix, is used to investigate the interaction between soluble cigarette smoke components (SCSC), the cells and GSH. The toxic dose for 50% of the Clara cells (TDs0 value) for water SCSC is equivalent to 0.0014 of a cigarette. When GSH (0.31-2.50mM) is also incubated with the cells in the presence of SCSC it provides a dose dependant protective effect. However, when the cigarette smoke components are collected through a solution of 0.4mM GSH prior to addition to Clara cells then the toxicity of the mixture is higher than the SCSC collected in water.
Morphine Inhibition of Cigarette Smoke ( CS ) -Induced Goblet Cell Secretion in Guinea Pig Trachea In Vivo H-P Kuo, JAL Rohde, PJ Barnes, DF Rogers. Department of Thoracic Medicine, National Heart & Lung Institute, LONDON SW3 6LY.
Opioid drugs inhibit a number of neurally-mediated responses in the airways (TIPS 1990:11,185-189). Using a semi-quantitative morphometric measure, the mucus score (MS) which is inversely related to the degree of mucus discharge, we have shown that neural mechanisms contribute to the control of goblet cell discharge in guinea pig airways (Am J Physiol 1990:259(3), in press). We have now assessed the effect of morphine on CS-induced tracheal goblet cell secretion in vivo in anaes-
thetised, artificially-ventilated guinea pigs. Mean MS of 642 (SEM 57, n=6) in air controls was reduced (p<0.01) to 343 (SEM 55,n=8) after i0 tidal volumes of CS (diluted i:I0 in air) blown into the inspiratory tubing. The response was blocked by filtering the smoke or by ganglion- ic blockade. Morphine (img/Kg,iv) had no effect on MS in air controls but blocked the reduction in MS in response to CS (MS=729 SEM 84, n=4). The inhibition by morphine was reversed by the specific opioid receptor antagonist naloxone (MS=317, SEM I0, n=4). We conclude that the part- iculate phase component of CS-induced goblet cell secretion which is mediated via the ganglion is inhibited by opioid receptor activation.
A Possible Role For Cholesterol In The Enhanced Lymphoproliferation Responses Observed In The Lungs Of Patients With Extrinsic Allergic Alveolitis (EAA). DA Hughes, PJ Townsend, PL Haslam. Cell Biology Group, Department of Cardiothoracic Surgery, National Heart & Lung Institute, LONDON SW3 6LY
We have previously observed close correlations between levels of cholesterol, lipid-laden macrophages and lymphocytes in bronchoalveolar lavage samples from patients with EAA. We, and others, have also reported that the density of HLA-DR, DP and DQ expression on macrophages is increased in EAA. These findings suggested that cholesterol may enhance the antigen-presenting function of mononuclear phagocytes by modulating the expression of HLA-D region products. We have, therefore, examined the in vitro effect of exogenous cholesterol on HLA-D expression and antigen-presenting function of normal blood monocytes (BM). Purified BM were incubated with or without cholesterol (0.8 mg/ml) in serum-free medium for 24 hrs at 37 ~ The cholesterol induced a significant increase in both the percent of BM expressing HLA-DP and DQ antigens and the intensity of expression of all three D-region products, quantified by immunofluorescent staining and flow cytometry. Antigen-presenting function was assessed by pulsing BM with tetanus toxoid and adding them to purified autologous lymphocytes. Lymphoproliferation, following antigen-presentation, was assessed by 3H-thymidine uptake after 5 days in serum-free medium. Cholesterol pre-incubated BM significantly increased lymphocyte proliferation. These findings indicate that cholesterot may augment antigen-presentation by modulating the expression of HLA-D region products, and may, therefore, play a role in the enhanced local lymphoproliferative responses observed in EAA.
426 Abstracts
Clinical Assessment of the C~ents of Airflow Obstruction in Asthma I Gregg, University of Southampton, 23 Monks Wood Close, Southampton SO2 3~
Due to the heterogeneous nature of asthma, its proper management depends upon selection of the most appropriate therapy for every individual patient, h~th to relieve acute attacks and, in the long-term, to suppress it in those patients in whom it is persistent. The most important single factor in achieving this end is to identify the mechanisms causing bronchial narrowing and to assess their relative importance whenever a given patient presents with asthma. The mechanisms that cause airflow obstruction can be categorised as - a) muscle constriction and acute mucosal inflammation, generally reversible by adrenoeeptor agonists, b) chronic mucosal inflammation and intraluminal plugging, which is refractory to adrenoceptor agonists, and c) structural da~e of the airways, which is irreversible and is either caused by exogenous factors (eg infection or smoking) or is incurred due to inadequate treatment of the inflan~,atory component of asthma itself. By making measurements of peak expiratory flow (PEF), the clinician can infer which of the above components are present and the contributions made by each to the total amount of airflow obstruction. The aim of this paper is to discuss the use of PEF measurements by the doctor and by patients themselves in the management of asthma, based upon the personal experience of the author over the course of some 30 years.
Effect Of Mepacrine (Mep) Pretreatment In Experimental Endotoxic Shock BA Briggs, SE Jukes, A Lazaar, A Guz, SF Smith. Department of Medicine, Charing Cross & Westminster Medical School, LONDON, W6 8RF
Endotoxin, a lipopolysaecharide (LPS) is implicated in the pathogen- esis of septic shock. Raised phospholipase A2 activity (PLA2) may be involved in the subsequent development of the condition. If so, a PLAz-inhibitor, Mep, may block the response. 5 rabbits were pretreated with 25mg Mep/ kg ip and 5 with vehicle (PBS) at 18 and 16 hour before injection of LPS, 10ug/kg iv. Blood pressure, heart rate and temperat- ure were recorded and blood taken pre-LPS (Oh) and at intervals to 3h.
Time(h) BP(mmHg) HR(bpm) T(~ PLAz (nmol produet/ml) PBS+LPS 0 88(7S-94) 235(211-261) 38.6(36.7-40.0) 27(19-42) Mep+LPS 0 83(75-123) 284(228-500) 39.0(38.8-40.0)* 29(17-55)
Time(h) ABP(% Oh) ~HR(% Oh) Z~T(% 0h) ~PLAz (% Oh) PBS+LPS 1 +6(-15-+13) +32( +3-+83)* +i(+<i-+2)* +9(-14- +84) Mep+LPS 1 +6(-26-+12) +23(-35-+44) +4(+i-+4) ~ +5(-12- +62) PBS+LPS 3 -7(-17-+13) +49(+10-+113)* +3(-i-+5) +75(-13-+129) Mep+LPS 3 +4( -9-+13) +38(+8-+I08) ~ +6(+4-+6) ~ +134(+108-+184) ~ *>PBS/LPS,0h; ~>Mep/LPS,0hr; 2p<0.1; 2-tailed test; Median(Range)
This study indicates that the role of PLA2 in LPS-induced injury is unclear, and that Mep is pyrogenic in the rabbit.
Growth Factors For Human Lung Fibroblasts In The Soluble Products Of Clot Formation. AJ Gray+,JT Reeves*,NK Harrison, JE Bishop, P Winlove**, GJ Laurent. Biochemistry Unit, Department of Thoracic Medicine, National Heart and Lung Institute, London SW3. *University of Colorado, Denver, USA. **Imperial College, University of London, UK.
Damage to the endothelium with subsequent oedema is an early manifestation of interstitial lung disease. This may give rise to an influx of proteins and formation of a fibrin clot which acts as a matrix to which fibroblasts adhere. Subsequens fibroblast replication is believed to be stimulated by entrapped mitogens. We hypothesised that the soluble proteins generated by the action of thrombin on fibrinogen may play a role in stimulation of fibroblast replication. To examine this we expressed liquid from a fibrin clot and measured its mitogenic potential on human lung fibroblasts using a colormetric assay (J Cell Sci 1989; 92: 513-8). The clot supernatant caused a mitogenic response (51+_6%, above control),which was equiv- alent to about half that elicited by an optimal stimulus such as 10% new born calf serum. Gel filtration chromatography of clot supernatant indicated two peaks of mitogenic activity containing proteins of 50-70 kDa and >200kDa respectively. These observations indicate the generation of mitogens during haemostasis and suggest a novel mechanism for the initiation of fibrotic disease.
Supported by a grant from the British Heart Foundation.
Abstracts 427
Collagen Metabolism Of Systemic Sclerosis Lung Fibroblasts: Effects Of TGF-B. NK Harrison, AC Argent, RJ McAnulty, AD Cambrey, JS Campa, RM du Bois, CM Black', GJ Laurent. Biochemistry Unit, National Heart and Lung Institute, LONDON SW3 6LR. "Royal Free Hospital, LONDON NW3.
Patients with systemic sclerosis (SSc) frequently develop pulmonary fibrosis although the mechanisms by which this occurs remain unknown. The aim of this study was to compare rates of collagen synthesis and degradation by lung fibroblasts from patients with sac (n=6) and control cell lines (n=6), and to assess the effects of TGF-S on these processes. Cells were grown to confluence and incubated for 24 hours in culture medium containing ascorate, L-proline and S-aminoproprionitrile supplemented with various concentrations of calf serum and TGF-S. Collagen synthesis and degradation of newly-synthesised collagen were measured by estimating protein-bound and free hydroxyproline respectively, in the culture medium, by high pressure liquid chromatography. Mean (range) rates of collagen synthesis expressed as pmol hydroxyproline.~gDNA-1.h -I for sac cells was 16 (3-38) and did not differ from controls 17 (4-39). Greater than half the collagen was degraded rapidly, although again groups did not differ (mean 55% and 62%). Increased synthesis was observed in response to increasing serum concentrations and to TGF-B, but no significant differences were were apparent between the groups. Results suggest that if sac lung fibroblasts are synthesizing excess collage in vivo this phenotype is not maintained in vitro.
ThePretermGuineaPig: A ModelForTheStudyofO~gen-inducedLungl~u~ In ThePreterm ln~nt. F.J. Kelly, G. Phillips, A.D. Postle, S.T. Holgate and G.I. Town, Departments of Human Nutrition, Immunopharmacology and Child Health, University of Southampton, Southampton, U.K. Research into the pathogenesis of acute and chronic neonatal lung disease has been hampered by the lack of a suitable small animal model of prematurity. The principal limitation of current models in the rat, mouse and rabbit is that due to their relatively short gestation periods, pups are very immature at birth and do not survive following preterm delivery. In this study we have demonstrated that viable, preterm guinea pigs can be delivered by Caesarian section up to 8 days prior to normal term (68 days). No animal survived delivery before 60 d gestation. Those delivered at 63d had a 50% survival rate at 24 h (n=94), while 98% of those delivered at 65d survived (n=48). There was a progressive rise in lung PC concentration from 0.93 (Std dev 0.18) @mol/mg DNA at 55 days, rising to 3.86 (0.5) at term. Preterm pups were found to satisfy a number of important criteria; including development of respiratory distress following delivery, subsequent evidence of hyaline membrane formation and presence of an inflammatory response in the lung following oxygen treatment. Furthermore, and most importantly, we have determined that preterm pups are more susceptible to oxygen-lnduced pulmonary injury than term counterparts.
A Study of Human Lung Epithelial Type II Cell Cluster Formation in vitro Using Time Lapse Video Microscopy L Bingle, N Shah, B Fox*, TA Partridge*, D Kaplan, A Guz, TD Tetley. Department of Medicine and Department of Histopathology*, Charing Cross & Westminster Medical School, LONDON W6 8RF.
In previous studies, human alveolar epithelial type II cells, co- cultured with fibroblastic cells and macrophages from the same lung, were found to form clusters around a central core of connective tissue. Over 8 days in vitro, type II cells retained their cuboidal morphology and increased in cluster size but not cluster number, suggesting type II cell division, a rare phenomenon in vitro. Subsequent studies (using 3H-thymidine incorporation and specific antibody staining for dividing cells) showed that type II cell division did occur, but did not entirely account for the observed increase in cluster size. Thus time lapse video microscopy was set up to examine the relative importance of cell aggregation to cluster formation; there was rapid aggregation of the type II cells in vitro - within 3h - that continued for another 21h, but was rare after this time. These combined studies suggest that both cell division and cell aggregation is involved in type II cell cluster formation in vitro.
428 Abstracts
Kinetic~Studies Of Pentamidin% Uptake By Rat Lung Slices. H Jones"~ G Blundell, S Farr , R J Richards. Welsh School of Pharmacy and Department of Biochemistry, University of Wales College of Cardiff~ P O Box 78~ Cardiff CFI IXL.
Aerosol inhalation of pentamidine isethionate~ an aromatic diamine~ is the major treatment for Pneumocystis carinii infections. This organism resides in the alveolar space and induces pneumonia in approximately 80% of patients with AIDS. The cellular site of lung uptake and the response of the organ to single / multiple dose regimes has not been reported. The present investigation~ which forms part of a study of the sequestration and toxicity of pentamidine~ is concerned with the uptake of the drug by rat lung slices in vitro. Using [JH]- pentamidine~ Km and Vmax values for the drug are calculated at 1012~M and 9520nmols/g lung/h. The uptake of putrescine~ which is considered as a marker of epithelial cell integrity~ is inhibited in a non competitive manner by concentrations of drug in excess of 250~M. These data indicate that putrescine and pentamidine do not share the same uptake system and that the drug may induce epithelial cell damage.
The Induction Of Tolerance To Porfluoro Isobutene (PFIB)-Induced Lung Oedema
N Robinson, B Mankelow, D Swaneton, CDE Porton Down, SALISBURY, SP4 0JQ. The inhalation of PFIB, one of the pyrolysis products of PTFE, can cause damage
within the lungs leading to lung oedema. To-date there is still no effective, specif- ic therapy for such chemically-lnduced lung oedema. Prophylaxis of PFIB-induced oedema can be considerably more effective with a number of thiols, N-acetylcysteine for example, giving good protection. However, the use of thiols to give prophylactic protection is limited; a single dose of thiol will only protect for a few hours.
Exposure of rats to a low dose of PFIBwill confer a significant degree of protection for up to two weeks to the same rats subsequently exposed to a higher oedemagenic dose of PFIH. The mechanism of tolerance induction is unknown for PFIB, there being no correlation between tolerance and the light microscopy picture within the lungs, respiratory parameters, the initially induced oedema or to the level of thiols within the lung fluid. The only correlation found so far is with the numbers of alveolar macrophages present.
Pre-exposure to a low dose of PFIB can induce a degree of protection to a subsequent exposure which is of considerably more benefit than that given by current- ly available prophylactics. An understanding of the mechanism of tolerance may sug- gest a pathway by which long lasting tolerance to PFIB can be induced without the necessity of pre-exposure to this osdemagen.
Abstracts 429
Tissue And Serum Responses To Pulmonary Deposition Of Fluorescein Isothiocyanate (FITC) - A Possible Model For Immunologieally-mediated Lung Injury. DM Brown i , K Donaldson i , D Lamb 2 , Elizabeth Ramage 2 and Sarah Howie 2 . I Institute of Occupational Medicine Ltd. Roxburgh Place, EDINBURGH. 2Department of Pathology, University of Edinburgh, EDINBURGH.
Autoimmune cell-mediated immune responses have been implicated in various interstitial lung diseases and CMI against connective tissue components has been demonstrated in the Bleomyein model of pulmonary fibrosis. In the modern environment small highly reactive molecules with the potential to be haptens are found. To study the effect of such chemicals on the lung we examined the effect of a single instillation of the hapten fluoreseein isothiocyanate (FIT(I) on the rat lung. A single instillation of FITC caused a severe mononuclear cell infiltrate into FITC-positive areas of lung where the FITC was associated with interstitial fibres, possibly elastin. There was progressive, severe fibrosis in these areas up to three months during which time increased lymph node germinal centre activity and elevated antibody to FITC-haptenated protein could be demonstrated. A single FITC instillation provides a model of lung injury and fibrosis in the presence of a sustained immune response.
The Immunohistology Of Sarcoidosis SB Fazel, SEM Howie, AS Krajewski, D Lamb. Department of Patholo- gy, Edinburgh University, Teviot Place EDINBURGH EH8 9AG Studies on frozen sections and cells recovered from broncho- alveolar lavage (BAL) have implicated cell-mediated immunity in the pathogenesis of sarcoidosis. The aims of this study were to study the B and T lymphocyte distribution in sarcoid lymph node and open lung biopsies using paraffin-reactive monoclonal anti- bodies, including markers for CD45 isoforms. Single and double- staining was performed on 29 sarcoid biopsies, using the avidin- biotin complex method. Two main results emerged. First, observa- tions in lung lesions revealed the unexpected presence of large numbers of B lymphocytes. This finding contrasts greatly to pub- lished studies of B cell numbers recovered from BAL. The other result involved the analysis of CD45 isoform expression in sar- cold lymph nodes. The ratio of 18:1 for CD45RO+:CD45RA+ T lympho- cytes in the sarcoid inter-granulomatous regions was significant- ly greater than the i:I ratio reported for paracortex of normal lymph nodes. These findings may aid in the explanation of the pathogenesis of sarcoidosis.
Reduction of Bronchial Wall Elastin in Experimental Bronchiectasis (Bx). D Guerreiro, J Robde, H Todd, J Lapa e Silva, M Sheppard and PJ Cole. Host Defence Unit, Dept.Thoracic Med. and Dept.of Lung Pathology, NHLI, LONDON SW3 6LY
Elastase, released from neutrophils attracted by bacteria to the bronfhial lumen in Bx, is postulated to be responsible for destroying bronchial elastin. We have measured stained elastin in an experimental model of Bx.
Experimental Bx was derived in rats by partial ligation of the apical axial bronchus and distal intrabronchial injection of live Pse~Q~ru~iKIQ~A (Pa+LIG group). A test group (PaL+LIG) and 6 control groups were used: aged-matched normal rats, sham-operated rats, LIG alone, live ~[~qiDosa alone, heat-killed ~aeruqinos~ alone and also with LIG. The rats were killed at 4,8 and 12 wk., lungs inflated, apical lobes orientated and sectioned uniformly, and sections stained with Verhoeff's elastin counterstained with Van Gieson. The percentage of elastic fibres was assessed blindly in each group at each time point using a point counting method.
There was a significant (p<0.001) decrease in the percentage of bronchial wall elastin fibres in the Pa+LIG group compared with controls. There was no significant difference between control groups.
This experimental medel of bronchiectasis mimics human disease in its reduced bronchial wall stainable elastin content and promises to be useful in study of the pathogenesis of this inflammatory bronchial wall destructiora
430 Abstracts
Growth and Metabolic Characteristics of Fibroblasts Derived from Control and Fibrotic Human Lung Tissue. C McShsrry. University Department of Immunology, Western Infirmary, GLASDOW, Gli 6NT
Primary fibroblast cell lines were derived from explants of "fibrotic" and "control" normal human lung tissue. Fibrotic fibroblasts proliferated faster in vitro than controls mainly due to
rapidly growing clones of cells within the fibrotic cell population. These cells also produced more collagen in vitroo, measured by increased
gene expression of procollagen types I and III. The difference with controls was most marked at confluence where control expression was largely switched off, but fibrotic cells continued to express these genes at significant levels. These fibroblasts in culture synthesised more PGE2 than the control cells and this difference was greatly accentuated after exposure to exogenous interleukin-l. Under these conditions fibrotic cells could produce enough PGE2 to inhibit the growth of normal fibroblasts without affecting their own growth. These observations suggest that idiopathic pulmonary fibrosis may be due to the emergence of clones of cells which can outgrow normal fibroblasts, and which produce excessive amounts of collagen.
Injury to Alveolar Epithelial Cells in vitro by Vapour and Particulate Phase Cigarette Smoke S M Lannan, I D M Brown, 2 K Donaldson, 2 W MacNee I , Unit of Respiratory Medicine I , Department of Medicine (RIE), City Hospital, and Institute of Occupational Medicine 2, City Hospital, EDINBURGH EHI0 5SB.
Cigarette smokers have been shown to have an increase in airway and alveolar epithelial permeability. We studied the effect of both vapour and particulate phase cigarette smoke on an alveolar cell line, A549. The 51Cr-labelled epithelial cells were exposed to cigarette smoke for a set number of puffs and then plated out and the loss of ability to attach was taken as a measure of cell injury. The percentage of cells that could attach within 24 hours was less in cells exposed to vapour or particulate phase cigarette smoke than control cells (10.07+7.67% and 10.56+3.83% versus 35.90+98%). This injurious effect of cigarette smoke was found to be dose dependent.
Research funded by the Normal Salvesen Emphysema Research Trust.
What Is The Relationship Between Emphysema And The Intra Alveolar Macrophage Population? WAH Wallace, A McLean, D Lamb. Department of Pathology, University of Edinburgh Med School, Teviot Place, EDINBURGH. EH8 9AG.
We have counted the number of intraalveolar macrophages (IAM) and measured the alveolar surface area/unit volume of lung (AWUV) in a series of 16 resected lung lobes with small peripheral lesions. There were 12 smokers, 2 non-smokers, and 2 ex-smokers > 6 months;age range 54-72.
The number of IAM varied from 608 to 1485 per cubic mm of fixed lung tissue There was no correlation between the number of IAM per mm 3 and the mean AWUV for the lobe. When the macrophage number was expressed as number IAM/mm 2 of alveolar surface area there was a relationship between increased macrophage number and a greater degree of microscopic emphysema i.e. low AWUV values (r - 0.62, p < 0.05). We suggest that when assessing intraalveolar macrophage numbers the values need to be expressed both in terms of lung volume and alveolar surface area.
Abstracts 431
Purification and Partial Characterization of Sheep Bronchial Inhibitor of Polymorphonuclear Neutrophil Elastase R Mistry, PD Snashall, A Guz and TD Tetley. Department of Medicine, CharinE Cross & Westminster Medical School, LONDON W6 8RF
Migration of polymorphonuolear leukocytes into the lunzinterstitiumduring inflammation is thought to involve the release of neutrophil elastase (NE). Inadequate inhibition of NEby endogenous alpha-l-proteinase inhibitor and low ~,lunEepithelial cell-derived bronchial mucus proteirmse inhibitor(BI) may lead to proteolytic lung da~e in emphysema and acute lung injury. To study this, we ar6 usin~ the sheep model of endotoxin-induced acute lung injury because it is possible to sample, in parallel, three compartments of the lung - plasma, interstitium (lung lymph) and epithelial lining liquid (bronchoalveolar lavage). In order to understand the role of BI in this sheep model, sheep BI has been purified from sheep bronchoalveolar lavage. It was purified by the modified method of SmithCEand Johnson DA, Biochem. J.(1988) 225, 463-472. The protein migrated as two hands parallel to htm~an BI on 20~ SDS-PAGE, with a median MW of 14,000. Like human BI, sheep BI inhibits htmlan NEon a I:I molar basis. A~qimmunoassay to detect this protein will enable us to study the role BI may have in vivo.
Development of Flow Cytometric procedure for the purification of Clara Cells Isolated from Mouse Lung. A Taya, J P Kellington. Biomedical Research Department, /LEA Environment and Energy, Harwell Laboratory, Oxfordshire OXII ORA.
The bronchiolar epithelial Clara cells is considered to be one of the progenitor cells of radiation - induced lung tumours and also it plays a major role in the metabolism of inhaled foreign compounds. Thus, pure isolates of Clara cells could provide a potential tool to study the role of this cell in the biology of lungs as well as in the pathogenesis of lung disease.
At our Laboratory, we have developed a flow cytometric method for the purification of Clara cells. Clara cells were isolated from mouse lungs by enzymatic digestion of the lungs with 0.25% trypsin solution. A crude fraction of Clara cells was obtained with purity of 60-70% as judged by nitro blue tetrazoli~m stain. Clara ceils were further purified using flow cytometry. The sorting is based on the ability of the dense Clara ceils to scatter light more than any other cell present in the crude fraction (the other major cell is the macrophage). The purity of the sorted Clara cells is over 90% as determined by nitro blue tetrazolium and rhodamine ]23 stains and electron microscopy. The sorted Clara cells can be used to study normal or abnormal cell metabolism, nuclear aberration and neoplastic changes in Clara cells isolated from animals exposed either to radiation or chemicals.
PAF-induced Immediate Hyperreactivity To Carbachol In Conscious Guinea- pigs. JR Thorne, KJ Broadley, Division of Pharmacology, Welsh School of Pharmacy, UWCC, PO Box 13, CARDIFF CFI 3XF
Specific airway conductance (sGaw) was measured in conscious guinea-pigs (male, Dunkin-Hartley) by whole body plethysmography. Exposure of the guinea-pigs to an aerosol containing PAF-acether (10}/g/ml, i0 i/rain flow rate) for 30 or 60s caused a decrease in sGaw indicative of bronchoconstriction. The peak reduction in sGaw (25+5%, n = 6) occurred after 5 rain with return to baseline after 1 hour. Exposure to an aerosol of carbachol (0.02 mg/ml) for 60s caused a bronchoconstriction, compared with the saline control, which was slightly reduced when repeated 7 days later. The carbachol-induced bronchoconstriction (peak reduction in sGaw of 10+9%) was significantly potentiated when the animals were exposed 7 days later at 1 hr after the 30s exposure to PAF (peak reduction of 28+4%). Therefore a single inhalation of PAY appears to induce an immediate hyperreactivity to the bronchoconstrictor effects of carbachol.
432 Ahstracts
INHIBITORY EFFECT OF PLEURAL LAVAGE FLUID ON TNF AND IL-1 PRODUCED BY PLEURAL LEUKOCYTES IN VITRO
Xiao Yang Li and Kenneth Donaldson Institute of Occupational Medicine, 8 Roxburgh Place, Edinburgh.
We have demonstrated that normal pleural leukocytes produce large quantities of TNF and IL-1 in culture which can be further increased by the addition of endotoxln. We questioned whether this was the natural situation in the pleural space so the aim of this study was to measure the levels of TNF and IL-1 in normal pleural space and determine how the levels may be regulated. Although pleural leukocytes secreted high quantities of TNF and IL-1 in culture, no detectable TNF or IL-1 was found in pleural lavage fluids. In contrast, the pleural lavage fluids inhibited TNF and IL-1 produced by pleural leukocytes in a concentration- related manner. This inhibition could be overcome by the addition of exogenous TNF and IL-1. The results indicate that pleural fluids contain inhibltors against these important Inflammatory mediators, which may play important role in maintaining normal pleural functions.
Tumour Necrosis Factor production by rat alveolar macrophages exposed to organic and inorganic dust. Kenneth Donaldson and David M. Brown, Instltute of Occupational Medicine,
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B Roxburgh Place, Edlnburgh,EH8 9SU. Alveolar macrophages are a potent source of the pro-inflammatory cytoklne Tumour Necrosis Factor (TNF). We have demonstrated previously that the inorganic mineral dust quartz, which causes silicosis, can stimulate the release of the cytoklne Interleukln-1 and produces pulmonary fibrosis. We recently also found that organic dust collected from the air of wool mills was capable of causing intense inflammatlon in rat lung and, in the long term, also caused severe fibrosis. We report here that both quartz and wool mill dust have the ability to stimulate the release of TNF from rat alveolar macrophages but that the non-pathogenlc dust Ti02 was virtually inactive in causing TNF release. Research Funded by the Health and Safety Executive and the Commission of the European Communities.
)4ethylene Chloride (I)~ Morphological, Imunohistochemical And Biochemical Effects On Mouse Lung Following Inhalation Exposure L L Smith, I Wyatt, T Green, R W Lewis, P M Hext and J R Foster. ICI PLC Central Toxicology Laboratory, Macclesfield, Cheshire, UK.
The long tern exposure of male B6C3FI mice to 4000ppm methylene chloride (MC) has previously been shown to cause lung tumours. As part of a programme to investigate the mechanism of tumorogenesis of MC, male B6C3FI mice were exposed to 4000ppm MC for 6 hours/day, five days/week for up to 13 weeks. Groups of mice were killed at intervals from one day to week 13, and the lungs were studied morphologically, biochemically and immunocytochemically for cytochrome P-450 PB, NADPH cytochrome P-450 reductase and glutathione S transferase E (GST). The major morphological effect was vacuolation of the Clara cells of the bronchiolar epithelium. The development of Clara cell vacuolation was closely correlated with the presence of P-450 PB for up to 7 weeks. However, after 13 weeks the Clara cells appeared to be insensitive to MC despite the presence of P-450 PB. At all times studied, whole lung microsomal act iv i ty was depressed with elevated cytosolic NPSH levels and sl ight increases in GST act iv i ty . I t appears that the in i t i a l Clara cell lesion results from the act iv i ty of the microsomal P-450 system and that with time (13 weeks) the Clara cell appears to develop tolerance to MC exposure.
Abstracts 433
Methylene Chloride (2)z Morphological And Biochemical Effects On Isolated Lung Clara Cells Following Inhalation Exp,)sure R W Lewis, I Wyatt, J R Foster, T Green, P M Hext, L L Smith. ICI PLC Central Toxicology Laboratory, Macclesfield, Cheshire, UK.
The long-term exposure of male B6C3F1 mice to methylene chloride (4000ppm) has resulted in the development of lung tumours. Shorter term studies demonstrated selective damage to the non-ctliated bronchiolar (Clara) cel l . In the present study mice were exposed to 4000ppm methylene chloride 6 hours/day, 5 days/week for up.to go days.
Clara cel ls isolated from treated mice were less functionally active than control cel ls, and had altered plating characteristics in short-term culture. The microsomal enzyme act iv i t ies measured in isolated Clara cells decreased to 17% of control values by week 7 of exposure. By week 13 levels were less severely decreased. These findings were in agreement with immunohistochemical assessments of Clara cell P-450 levels in lung sections~ there was less correlation between either of these parameters and enzyme levels measured in whole lung microsomes. These data demonstrate the advantages of target cell isolation and suggest a l ink between methylene chloride tox ic i ty and depressions in Clara cell P-450 activity.
The Role Of Thiols In The Pulmonary Toxicity Of Perfluoroisobutene
Alison F Lailey and David G Upshall, Biology Division, Chemical Defence Establishment, Porton Down, SALSIBURY SP4 0JQ.
Perfluorisobutene (PFIB) is an odourless, colourless gas with a boiling point of 6*C. It is produced when poyltetrafluorethylene (PTFE) is pyrolysed and when it is hot formed in an industrial environment. In man it produces 'polymer fume fever' a condition characterised by nausea, fever, sore throat, cough and pulmonary oedema. In rats its toxicity is sreater than that of phosgene with an LCt~o of 1250 mg min m -~ and damage to the alveolar type I and II cells and the pulmonary endothelium is evident.
Exposure to PFIB reduces the total lung thiol (NPSH), ghtathione (GSH) and Cysteine (CYSH) by 38-55%. Pretreatment with buthionine sulfoximine (BS0) and/or DEM increases the toxicity of PFIB whereas pretreatment with N-acetyl cysteine and esters of cysteine will reduce or prevent toxicity. The role of thiols in the toxic and protective mechanisms will be discussed.
The Effect of Pneumotoxins on the Distzibution of Gamma- Glutamyltransferase Activity in Rat Lung M.J. Lee, D. Dinsdale, M.R.C. Toxicology Unit, Woodmansterne Road, CARSHALTON, Surrey, SM5 4EF
Gamma-glutamyltransferase (GGT) is present in the alveoli and distal airways of the lung. It is probably involved in the uptake of glutathione from the epithelial lining fluid.
The activity of this enzyme, assayed colorimetrically in broncho- alveolar lavage fluid, is increased in rats dosed with pneumoto• which affect the Type I pneumocytes of the alveoli (eg O,O,S- trimethylphosphorothiolate) or the Clara cells of the bronchioles (eg l-nitronaphthalene).
The distribution of this activity has been demonstrated, by ultrastructural histochemistry, in the epithelium of the alveoli and of the bronchioles from control animals. The enzyme itself has been localised, by immunocytochemistry, using silver-enhanced gold probes.
These techniques have been used to demonstrate pneumotoxin-induced changes in the distribution of GGT in both the Clara cells and Type II pneumocytes of the rat.
