Download Modulation of Neutrophil Apoptosis (Programmed Cell Death) by

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.