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Medical Research Society 60 TUMOUR NECROSIS FACTOR (TNF ALPHA) PRODUCTION BY PERIPHERAL BLOOD MONOCYTES AND ALVEOLAR MACROPHAGES FROM PATIENTS WITH HIV-RELATED LUNG DISEASE A B MILLAR', R F MILLER', AND S J G SEMPLE' N M FOLEY', G A w ROOK' 1 7 ~ sarcoid-related differences may play a central role in determining the fate of the granulomata (their resolution or progression to fibrosis) in the lungs of sarcoid patients. In support proportions of these suppressor macrophages in sarcoid BAL correlate well to clinical activity (Anslie el a11989), and are modified by efficacious therapeutic regimes (Spiteri el at, 1989). Departments of Medicine' and Medical Microbiology', University College and Middlesex School of Medicine, Mortimer Street, London, W1N 8AA Tumour necrosis factor (TNF alpha) is a peptide excreted by mononuclear phagocytic cells which causes fever, weight loss and effects the function of a number of cell types. It has been suggested that this cytokine may have a role in the manifestation of HIV infection (Lahdervirta.J et We have specifically al. Am J Med 1988;85:28). investigated HIV-related lung disease. Ten HIV positive patients with respiratory symptoms underwent fibreoptic bronchoscopy (FOB) and bronchoalveolar lavage. Five had pneumocystis carinii pneumonia (PCP), three had bacterial pneumonia ( B e ) and two had normal bronchoscopic findings (NB). Peripheral blood monocytes were obtained by venepuncture before FOB and monocytes and macrophages were harvested by adherence to plastic. The cells were cultured in RPMI (plus 10% serum) and with sequential dilutions of LPS. The same procedure was followed in four HIV negative patients with normal fibreoptic bronchoscopies. The spontaneous production of TNF from alveolar macrophages for the four groups was (mean (f SD) iu/ml) 194 f 53 (PCP), 169 f 30 (BP), 109 f 49 (NB), 10 f 9 (control) and for peripheral blood monocytes was 120 f 24 (PCP), 109 f 43 (BPI, 87 f 36 (negative FOB) and 12 f 7 (control). The maximally stimulated levels of TNF production from alveolar macrophages were (mean (f SD) iu/ml) 257 f 62 (PCP), 238 f 41 ( B P ) , 220 f 53 (negative FOB) and 59 f 13 (control) and those from peripheral blood monocytes were 198 f 51 (PCP), 174 f 62 (BP), 68 f 29 (negative FOB), We conclude that alveolar 63 f 13 (control). macrophages and peripheral blood monocytes from HIV infected individuals secrete TNF more readily than those from normal controls. 61 . ALVEOLAR MACROPHAGESTHAT SUPPRESS T-CELLS MAY BE CRUCIAL TO THE PATHOGENIC OUTCOME OF PULMONARY sARcoIDosIs. M A SPITERI,S CLARKE' and L W POULTER Departments of Thoracic Medicine B Immunology. Royal Free Hospital &Schoolof Medicine, London NW3 ZPG, ENGLAND Distinct alveolar macrophage (AM) subpopulations have been identified in sarcoid bronchoalveolar lavage (BAL), (Spiteri el al, 1988). Proportional differences in these subsets are found in sarcoid patients, when compared to normal. In particular a specific AM subpopulation emerges in active sarcoid BAL (27.2 & 6.1% compared to 7% in normals). Macrophages within this subset appear to react with both monoclonal antibody probes, that have previously identified both dendritic cells and classic macrophages (Poulter et al, 1986). When isolated from normal BAL, this AM subpopulation exhibits distinctive physiological and functional features: the cells adhere to glass, are phagocytic, with Fc and c3b receptor expression, and contain fibronectin. Functional analysis shows that although such AM express HLA-DR molecules on their surface, they are capable of actively down-regulating, by as much a s 40%. the induction of T-cell responses set up by other stimulator macrophages present in BAL. This study aims to identify changes within these suppressor A M specific to sarcoidosis, and that may relate to the clinical outcome of the disease. This AM subpopulation was isolated from BAL of 10 active sarcoid patients, and compared to that from 10 healthy volunteers. Sarcoid-related differences in both phenotype and function were found. A greater proportion of suppressor macrophages were phagocytic, had increased lyzosomal enzyme activity and Fc receptor expression, a s well as high fibronectin content. Over 45% of them expressed a separate antigen RFD9 (identifies epithelioid cells). Fupctional tests revealed that T-cell responsiveness was completely abolished by this AM subpopulation. We postulate that these suppressor macrophages modulate T-cell action in human immune responses: a s such,their increased proportion in sarcoid BAL, and 62 FIBROBLAST RECOGNITION OF AGED NEUTROPHILS IS MEDIATED BY THE RGD ADHESION SIGNAL AND IS MODULATED BY CHARGED PARTICLES S.E.HALL, J.S. SAVILL, and C.HASLETT Department of Medicine, Royal Postgraduate Medical School, DuCane Rd,London, W12 OHS Neutrophils (PMN) contain a variety of histotoxic agents and they have been implicated in the pathogenesis of a variety of diseases, yet their fate in tissues is poorly understood. We have recently described a mechanism whereby aging PMN undergo programmed cell death, a process determining the recognition of intact senescent PMN by macrophages (Savill et al.,1989 J. Clin. Invest. 81 (3) 865-875). Fibroblasts (FIB) also have the ability to ingest aged PMN (Haslett et al., 1988 Clinical Science p 44)).The present study concerns the mechanism of this recognition process. FIB recognition of aged PMN was measured as described previously (Hall et al., 1989 Faseb J. 1547) and inhibitory effects of various agents expressed as a % of this interaction (Mean fSE). Amino sugars which were cationic at physiological pH (glucosamine and galactosamine) inhibited the interaction (27.5*3% and 44.5&7%, respectively), an effect not seen with other amino sugars (N-acetyl glucosamine; 96.5*6.5%). A similar effect was seen with cationic amino acids (L-argine; 69.1*8.6%), but not neutral or acidic amino acids (L-glutamine; 109.4*5%, L-glutamic acid; 97.8*8.6%, n=9) Therefore, the presence of cationic molecules at inflamed sites may interfere with this recognition process. The interaction was cation dependent, with Mg2+ being more potent on a molar basis than Ca2+. This pattern of cation dependency suggested a role for the RGD (arginine-glycine-aspanate)adhesion signal, and the peptides RGDS and GRGDSP, inhibited FIB recognition of aged PMN (at 1mM RGDS; 28.%3%, GRGDSP; 25.152.8%) However, the close analogue GRGESP where Glu is substituted for Asp did not inhibit; (1 13.6*8.9%, n=9).The involvement of the RGD signal indicates that a molecule of the integrin family on the FIB surface is involved in this recognition process. Thus FIB may provide an alternative pathway for PMN removal at inflamed sites, using similar recognition mechanisms to the macrophage. a 63 MODULATION OF NEUTROPHIL APOPTOSIS (PROGRAMMED CELL DEATH) BY INFLAMMATORY MEDIATORS A LEE and C HASLEm Department of Medicine, Royal Postgraduate Medical School, Ducane Road, London W12 OHS England Apoptosis (programmed cell death) in intact aging neutrophils (PMN) leads to their uptake by macrophages (Savill et al. 1989. J. Clin. Invest. 865-875) and may represent an injury-limiting PMN disposal mechanism, which promotes the resolution of inflammation. Factors modulating apoptosis may influence neutrophil longevity and subsequent removal at inflamed sites. Therefore human PMN (>98% pure) were isolated from peripheral blood and aged in culture in the presence and absence of inflammatory mediators including lipopolysaccaride (LPS from E. Coli 01 ll.B4), Complement factor 5a (human recombinant C5a), the synthetic chemotactic peptide N-formyl-norleucyl-leucylphenylanaline (FNLP) and granulocyte/macrophage colonystimulating factor (GMCSF). Programmed cell death was assessed by light microscopical features of apoptosis (confirmed by electron microscopy) and expressed as % PMN with features of apoptosis. LPS, C5a, FNLP and GMCSF inhibited the rate of PMN apoptosis 18P Medical Research Society over 24 hours without inducing cell aggregation, loss of cells or their membrane integrity (trypan blue exclusion >98%). LPS lOOng - lOug/ml inhibited the rate of apoptosis. Using a 13hour time point as an index, LPS inhibition was dose dependent : Controls; 41.3 f 4.8% (n=7 mean f SEM), LPS 100ng/ml; 25.9 f 4.5%, LPS lug/ml; 12.9 f 2.9%. LPS lOug/ml; 6.1 f 0.9%. C5a inhibited apoptosis at lO-7M and 10-6M at 18 hours :Controls; 55.1 f 4.3% (N=5), C5a; 24.3 f 6.0% (lO-7M). C5a; 13.0 f 1.4 % (10-6M) (N=3). GMCSF inhibited at 500U/ml : Controls; 59.7 f 4.1% (N=6), GMCSF 500U/ml; 30.3 f 2.9%. FNLP caused a significant inhibition of P M N apoptosis at 9 hours in culture : Controls; 26.7 f 6.6%, FNLP; 12.7 f 3.3% (10-6M). These data suggest that P M N longevity may be prolonged at inflamed sites where high concentrations of inflammatory mediators and bacterial products are present. G4 A DOUBLE-BLIND STUDY OF THE RESPONSE TO CHANGES I N DIETARY S O D I U M INTAKE I N ORTHOTOPIC CARDIAC TRANSPLANT RECIPIENTS D.R.J. SINGER, N.D. MARKANDU, M.G. MACGREGOR, *M. YACOUB. c o e x i s t i n g ischaemic h e a r t d i s e a s e ( S t e w a r t e t a l , Br. Heart 3. 1985, 54, 290-297). We t h e r e f o r e performed a follow-up s t u d y i n t e n normal s u b j e c t s ( 2 8 i 2 y r ) who r e c e i v e d 7 d a y s o f randomised t r e a t m e n t s w i t h placebo, b e n d r o f l u a z i d e 5mg (B) , B + t r i a m t e r e n e 50mg ( T 5 0 ) , B+T200, and B+spironolactone lOOmg (Sp) Following e a c h t r e a t m e n t p e r i o d , r e s p o n s e s (K, Mg, ECG) t o i n h a l e d s a l b u t a m o l 2mg (S) were measured. R e s u l t s are p r e s e n t e d as means and 95% C I . N e i t h e r B nor S had any e f f e c t on plasma magnesium. The hypokalaemic response t o B+S [ 2.92( 2.70-3.1 Z)mnol/l] was a t t e n u a t e d by t h e a d d i t i o n of T200 [3.43(3.22-3.64)mmol/l; p<O.OI ANOVA] and Sp [3.53(3.32-3.74)mmol/l; p<O.OOl], b u t n o t by T50 [3.10(2.90-3.3l)mmol/1]. T wave f l a t t e n i n g i n r e s p o n s e t o B+S [0.24(0.19-0.29)mV] was b l u n t e d by t h e a d d i t i o n o f TZOO [0.33(0.28-0.37)mV; p<O.O5] and Sp [0.42(0.370.47)mV; p<O.O11, b u t n o t by T50 [0.25(0.20-0.30mV). Sp and T200 a l s o diminished t h e frequency and a m p l i t u d e o f U waves and S-T depression.Thus, t h e a d d i t i o n of h i g h d o s e t r i a m t e r e n e (200mg) and s p i r o n o l a c t o n e t o bendrof l u a z i d e a t t e n u a t e d K and ECG r e s p o n s e s f o l l o w i n g b e t a a d r e n o c e p t o r s t i m u l a t i o n by i n h a l e d s a l b u t a m o l , whereas a c o n v e n t i o n a l d o s e o f t r i a m t e r e n e (5Omg) had no e f f e c t . . BUCKLEY, G . A . Blood P r e s s u r e U n i t , Department o f Medicine, Charing Cross h Westminster Medical School, London W6 8RF h *Cardiothoracic Unit, Harefield Hospital, Harefield, Middx. Plasma ANP l e v e l s a r e i n c r e a s e d f o l l o w i n g o r t h o t o p i c c a r d i a c t r a n s p l a n t a t i o n , which r e s u l t s i n s u s t a i n e d d e n e r v a t i o n o f t h e donor h e a r t . However, t h e r e s p o n s e t o changes i n d i e t a r y sodium i n t a k e and, i n p a r t i c u l a r , t h e e f f e c t s of t h i s on plasma ANP, have n o t been s t u d i e d i n orthotopic cardiac transplant recipients. We s t u d i e d 8 h e a l t h y male o r t h o t o p i c c a r d i a c t r a n s p l a n t r e c i p i e n t s ( a g e r a n g e 26-61 y r s ) , on t h e i r u s u a l d i e t and on t h e 5 t h day of low and h i g h sodium d i e t s . I n t h e double-blind p a r t o f t h e s t u d y s u b j e c t s were a d v i s e d how t o reduce t h e i r d i e t a r y sodium i n t a k e t o 10 mmolslday. They t h e n r e c e i v e d i n random o r d e r an a d d i t i o n a l 340 mmols of sodium (Slow Sodium, C i b a ) f o r 5 days and Slow Sodium placebo ( C i b a ) f o r 5 days. Measurements were o b t a i n e d 24 h r s a f t e r t h e i r l a s t dose o f immunosuppressi v e t r e a t m e n t . On t h e low sodium d i e t s u p i n e blood p r e s s u r e was 12718821014 (mean ZSEM) mmHg, 24 h r u r i n a r y sodium e x c r e t i o n 31.325.3 mmolsl24 h r s and plasma ANP 56.326.3 pglml. On t h e h i g h sodium d i e t s u p i n e blood p r e s s u r e was 1411932413 mmHg, 24 h r u r i n a r y sodium e x c r e t i o n 335.0233.1 mmolsl24 h r s and plasma ANP 90.6: 1 1 . 3 pg/ml (PCO.01). These r e s u l t s showed t h a t d e n e r v a t i o n of t h e h e a r t i n man does n o t r e s u l t i n any major a b n o r m a l i t y i n t h e r e g u l a t i o n of l a r g e changes i n sodium i n t a k e ; i n p a r t i c u l a r s u b j e c t s were a b l e t o c o n s e r v e sodium. Furthermore, t h e i n c r e a s e s i n plasma ANP were s i m i l a r t o t h o s e s e e n i n normal s u b j e c t s changing from a low t o high sodium i n t a k e , s u g g e s t i n g t h a t i n n e r v a t i o n o f t h e h e a r t i s n o t r e q u i r e d f o r t h e plasma ANP response t o changes i n sodium i n t a k e . 65 THE EFFECTS OF TRIAHTERENE AND SPIRONOLACTONE ON THE HYPOKALAEHIC AND ECG SEQUELAE OF BENDROFLUAZIDE AND INHALED SALBUTAMOL 83 LIPWORTH, DG McDEVITT, RA CLARK and AD STRUTHERS Departments of R e s p i r a t o r y Medicine and Pharmacology, Ninewells H o s p i t a l , Oundee. Clinical B e t a - a g o n i s t s and d i u r e t i c s are o f t e n p r e s c r i b e d together i n patients with airflow obstruction. W e have p r e v i o u s l y shown (Lipworth e t a l , Am. 3. Med. 1989, 8 6 , 653-657) t h a t p r i o r t r e a t m e n t w i t h t h e potassium-losing d i u r e t i c b e n d r o f l u a z i d e (5mg) p o t e n t i a t e s t h e hypokalaemic (K) and ECC e f f e c t s of high-dose i n h a l e d s a l b u t a m o l (2mg). T h i s may p r e d i s p o s e t o c a r d i a c arrhthymias, p a r t i c u l a r l y i n s u s c e p t i b l e p a t i e n t s with 66 CAPTOPRIL THERAPY IN CHRONIC HEART FAILURE: DOES THE SIZE O F THE FIRST DOSE MATTER? J McLAY, J McMURRAY, A BRIDGES a n d AD STRUTHERS D e p a r t m e n t s of Clinical Pharmacology a n d Cardiology, Ninewells Hospital a n d Medical School, Dundee DDI 9SY I t i s still common practice to i n i t i a t e captopril in a low d o s e (i.e. 6.25mg) in case of f i r s t dose hypotension. The a i m of t h e s t u d y was to e x a m i n e whether t h i s low s t a r t i n g d o s e i s really necessary by measuring t h e blood pressure response to e i t h e r a high initial dose of captopril (25mg 6 hourly for 4 doses) o r a low i n i t i a l dose of captopril (sequentially 6.25mg, 6.25mg, 12.5mg, 25mg) in chronic h e a r t f a i l u r e patients. Forty p a t i e n t s with s t a b l e chronic d i u r e t i c controlled h e a r t f a i l u r e were recruited i n t o a double blind placebo controlled study. Hyponatraemic or hypovolaemic p a t i e n t s were excluded. The p a t i e n t s randomly received e i t h e r t h e low d o s e regime or t h e high d o s e r e g i m e shown above. Supine BP and pulse were measured at 15 min intervals for 2112 hours, p a t i e n t s w e r e t h e n allowed to walk around b e f o r e answering a questionnaire re: symptoms of dizziness or light headedness. No p a t i e n t s withdrew from t h e t r i a l through hypotension, none complained of symptoms of hypotension and none recorded a n increase in dizziness in t h e visual a n a l o g u e scale. Mean supine blood pressure fell a f t e r t h e initiation of captopril b u t t h e magnitude a n d t h e d u r a t i o n of t h i s BP f a l l was n o t d i f f e r e n t in those who began with a n initial dose of 25mg a s opposed to those who began with an initial dose of 6.25mg. In t h e low dose group, t h e f a l l in mean BP was -7 * 4mmHg a f t e r placebo a n d -16 i 4mmHg a f t e r 6.25mg CPT. In t h e high dose group, t h e f a l l in mean BP was -10 i 4mmHg a f t e r placebo a n d -13 i 4mmHg a f t e r 25mg CPT. These results suggest t h a t symptomatic hypotension is unlikely to occur in p a t i e n t s who a r e not hyponatraemic a n d not hypovolaemic. Furthermore, t h e f a l l in BP is of t h e s a m e magnitude whether t h e s t a r t i n g dose is 6.25mg or 25mg. In addition t h e r e is n o d i f f e r e n c e between t h e s e doses in t h e duration of t h e hypotensive effect.