44
p
Medical Research Society
15.6) and synovial fluid 49.2 mg/L (SD 15.5)
(Pharmacia B2M RIA, normal < 3mg/L, dstection
limit 0.1 mg/L). Samples of both serum and
synovial fluid were chromatographed on a Superose
12 column using Fast Protein Liquid
Chromatography. Eluted fractions were analysed
for B2M using the Pharmacia B2M RIA. B2M was
detected in a single peak, (nominal M.W.10000
Daltons) for all samples. B2M containing
fractions were then analysed using SDSPAGE (8-25%)
on a PHAST system followed by Western blotting.
Results showed that B2M in the synovial fluid and
serum of these patients was present in the
monomeric form with a molecular weight of 12000
Daltons. There was no evidence for the presence
of dimers or higher polymers.
Thus, polymerisation of B2M does not appear to
occur in the synovial fluid or serum of patients
with dialysis amyloid. Other factors need to be
considered to explain amyloidogenesis, such as the
polymerisation of B2M at the cellular level.
peripheral leucocytes from normal subjects to 3
chemotactic factors, f -meu-Leu-phe (fl1LP), casein and CsA
in vitro using a microchemotaxis chamber. Lymphocyte
chemotaxis to CsA (n=6, median cell count = 26.0) was
inhibited by cyclosporin at concentrations of 200Ong/ml
(median cell count = 1.20; p<0.02 Wilcoxon signed rank
test), 100Ong/ml (median = 3.85; p<0.02) and sOOng/ml
(median = 17.8; p<0.02). Chemotaxis to casein (n=4,
median cell count 27.0) was inhibited by cyclosporin at
concentrations of 200Ong/ml (median = 16.6; p<O.Os) and
100Ong/ml (median = 24.5, p<O.Os). Lymphocyte chemotaxis
to fl1LP (n=6, median = 24.8) was inhibited by cyclosporin
in doses as low as 62.sng/ml (median = 11.6; p<0.02).
Cyclosporin (2000ng/ml) failed to inhibit chemotaxis of
neutrophils and monocytes to the same chemoattractants.
Cyclosporin had no adverse effect on cell viability.
These results suggest that cyclosporin inhibits
lymphocyte chemotaxis in vitro. This ability may be
important in the prevention of lymphocyte recruitment to
sites of allograft rejection in vivo.
168
166 LEUCOCYTE RESPONSE FOLLOWING ENDURANCE EXERCISE
J D ROBERTSON, R J L DAVIDSON* AND R J MAUGHAN
Department of Environmental & Occupational Medicine and
*Haematology Unit, University Medical School, Aberdeen
AB9 2ZD
The time course of the leucocyte response has been
studied in young healthy males followin~ two endurance
runnin~ events. Blood was drawn from an antecubital vein
before and at intervals after (a) a 21.1km road race
(n=9) and (b) a 25.6km road race (n=7). The leucocyte
count was measured by a Coulter Counter S Plus IV with
differential counting performed microscopically on a
stained blood film. All counts wer g ~Ipressed in absolute terms and ~iven as units x 10 1 •
In the first event the peripheral leucocyte count was
studied in days followin~ the race. The exercise induced
a maior increase in leucocyte count (6.2+1.2 pre-race,
13.5+4.9 immediately post-race, p(O.OOl)-which had returned to basal count within 24h after the race and did
not differ thereafter. The change was due to increases
in neutrophil (3.8+1.7 to 9.6+4.0, p(O.OOl) and lymphocyte counts (1.9+0~6 to 3.2+0~9, p(O.Ol) without any
si~nificant alterations in ;onocyte, basophil Or eosinophil numbers. There were no changes in cell numbers over
the subsequent 3 days except that in the 72h post-race
sample lymphocyte number was lower (to 1.3~ 0.2, p<O.Ol)
and basophil number was elevated (0.02~0.04 at 48h prerace to 0.05+0.02, p<0.05).
In the second event the leucocyte count was followed
in the hours post-race. The leucocyte number changed in
a similar fashion to the first event over the period of
the race. The lymphocyte number had returned to normal
by Ih after the race whereas the neutrophil number continued to rise (pre-race 3.2+1.0, 5min after the race
9.3+3.1 p<O.Ol, Ih after 11.7+4.6 p(O.Ol, 2h after
12.0+4.2 p<O.OOl and 6h after-8.4+2.0 p<O.OOl).
In-conclusion a significant leucocytosis follows endurance running lar~ely as a result of the increase in
neutrophil number reaching a maximum 2h following the
exercise.
INTERACTION OF SENESCENT NEUTROPHILS WITH
FIBROBLASTS IN VITRO
*C. HASLETT, *S. HALL AND +P.M. HENSON
*Department of Medicine, Royal Postgraduate Medical
School, Hammersmith Hospital, Du Cane Road, London.
+National Jewish Center For Immunology and Respiratory
Medicine, Denver, USA.
Neutrophil
infi ltration
is cornnon in chronic
inflammation, which may be complicated by fibroblast
proliferation, but it is not known whether neutrophils
(PMN) can interact directly with fibroblasts (FIBS).
Human PMN (>98% pure) were isolated from the blood of
normal donors, aged in vitro for 24 hours such that
>g8%6 of PMN excluded trypan blue, suspended at
5x10 ml in medium, and 1ml added to well~ of cell
monolayers and incubated for 1 hour at 37 C after which
the non-adherent PMNs were washed away with cold
saline. In a microscopic assay, the number of "aged"
PMN interacting with human cell monolayers per randomly
selected high power field (PMN/HPF)was:
PMN/HPF(meantSD) (n=)
Epidermal keratinocytes
5t 5
(55)
3t 4
(55)
Microvascular endothelial cells
Flow 2000 (foetal lung line FIBS)
20t 5 * (45)
Flow 4000 (foetal kidney line FIBS)
12t 5 * (45)
Flow 2002 (embryonic lung line FIBS)
34t15 * (50)
Flow 5000 (whole embryo line FIBS)
41t10 *
(50)
Neonatal foreskin (primary culture FIBS) 82t 7 * (60)
Foetal lung (primary culture FIBS)
15t 5 * (35)
Foetal skin (primary culture FIBS)
34t22 * (50)
* p(O.OOI relative to the extent of adherence between
fresh PMN and the respective cell monolayer. Fresh PMN
adherence for all cell types was <3 PMN/HPF. These
data show that "aged", but not freshly isolated PMN
interact with monolayers of FIBS from a variety of
sources but not endothelial or epithelial monolayers.
Electron microscopic and trypsinisation studies suggest
that some of the "aged" PMN had been phagocytosed by
the FIBS. Further study of the mechanism underlying
this observation may lead to a greater understanding of
the link between inflammation and tissue scarring.
MACROPHAGE
PHAGOCYTOSIS OF AGED NEUTROPHILS
REPRESENTS A NOVEL FUNCTION FOR THE ARGININE-GLYCINEASPARTATE-SERINE (RGDS) ADHESION SIGNAL OF THE INTEGRIN
FAMILY OF CELL-SURFACE RECEPTORS.
169
INHIBITION OF LYMPHOCYTE CHEMOTAXIS IN VITRO BY
CYCLOSPORIN
L WANG, DH ADAMS1, D BURNEIT, RA STOCKLEY2, E ELIAS,
AND J NEUBERGER1
1Liver Unit, Queen Elizabeth Hospital, Edgbaston,
Birmingham and 2The Lung Irrrnunobiochemical Research Group,
General Hospital, Birmingham.
Lymphocytes play an important role in instigating
rejection of organ allografts and are probably recruited
to the site of rejection by locally produced chemotactic
factors. The irrrnunosuppressant drug cyclosporin is
effective in preventing allograft rejection and is thought
to work by inhibiting the production of lymphokines by
host lymphocytes. The effect of cyclosporin on lymphocyte
chemotaxis has not previously been studied. We assessed
the effect of cyclosporin on the chemotactic responses of
167
J. SAVILL and C. HASLETT
Department of Medicine, Royal Postgraduate Medical
School, Hammersmith Hospital, Du Cane Road, London, W12
Human neutrophils (PMN) "aged" in vitro for 24 hrs
undergo apoptosis which leads to recognition and
phagocytosis by monocyte-derived macrophages (M~), while
the PMN are still >98% viable, by a mechanism inhibitable
by aminosugars and basic aminoacids (Clin Sci 1987: 73,
Suppl 17; 52p and Clin Sci 1988: 74, Suppl 18; Ip),
molecules which also inhibit some adhesive functions of
the RGD-:bearing glycoprotein fibronectin (FN). We sought
evidence that the RGDS adhesion signal plays a role in M0
recognition of aged PMN.
Medical Research Society
In a 30 min serum-free assay at 37°C, FN in solution
showed
concentration-dependent
inhibition
of
M~
recognition of aged PMN (at 100~g/ml; 14.~1.8%, n=8;
mean±SE%
of
control),
which was specific to
FN
(fibrinogen 100~g/ml - 107.5~4.8% n=13:albumin 100~g/ml 96.4~3.5%,n=8)
and did not affect M~ recognition of IgG
opsonised ox erythrocytes (ElgG). Pre-incubations for 30
min with FN at 50~g/ml at 4°C, followed by washing prior
to assay "localised" this effect to the MIS (35.3+5.816,
n=6: cf FN present - 31.6~1.8%, n=6), not the PMN
(97.8~6.5%,
n=6) and adherence of MIS to surfaces coated
with FN 100~g/ml inhibited subsequent PMN uptake (49.2
~2.9%, n=lO cf albumin-coated wells:
93.2~2.5%, n=lO).
These results imply that a FN-binding structure on the MIS
participates in recognition of aged PMN.
Since the
tetrapeptide RGDS, but not its close analogue RGES,
inhibits
aged PMN recognition in a
characteristic
concentration-related fashion (at ImM RGDS: 33.1+2.4%,
at ImM RGES: 93.2~5.2 n=5 - E=glutamine), but not
n=8
ElgG uptake, these data suggest that a member of the
integrin family of FN-binding, RGD-dependent cell-surface
adhesion receptors participates in MIS recognition of aged
PMN, a process which may limit tissue injury at inflamed
sites.
170 MONOCYTE ADHERENCE IN BRONCHIECTASIS AND THE
EFFECT OF BACTERIAL LIPOPOLYSACCHARIDE
C. A. Owen, S. C. Afford, S. L. Hill, D. Burnett
and R. A. Stockley
The Lung Immunobiochemical Research Group, The
General Hospital, Steel house Lane, Birmingham
Monocyte/macrophages have adherent
properties which may be essential for recruitment and activity at sites of inflammation.
We
have compared the adherent properties of monocytes from healthy subjects and patients with
bronchiectasis.
Monocytes were isolated using Nycodenz
osmotic and density gradients from 6 healthy
controls and 12 patients with bronchiectasis, 6
producing mucopurulent (MP) and 6 producing
purulent (P) secretions. The cells were greater
than 90% pure (morphology and positive staining
for esterase).
The average yield from control subjects was
5.42 x 10 7 ; SD ± 1.9/1 of blood.
19.8%, ± 7.0,
were adherent to fibronectin coated tissue culture flasks.
The proportion of adherent cells
was increased by 0.1 pg/ml bacterial lipopolysaccharide (LPS) to 45.8%; ~ 12.2; (p < 0.025)
and 1.0 pg/ml LPS to 55.8%; ± 7.5; (p < 0.025).
More monocytes were isolated from the
patient groups (MP = 1.32 x 10-; ± 6.7 /1 of
blood; p < 0.005 : P = 9.38 x 10 7 ; ± 4.2; p <
0.025).
A greater proportion of the MP cells
were adherent (50% ± 7.39) compared to control
cells (p < 0.005).
Cells from the P patients
showed more adherence (65% ± 12.4) compared to
both the controls and the MP patients (p <
0.025).
LPS (0.1 pg/ml) significantly increased
the proportion of adherent cells to 64.8%; ±
3.6; (p < 0.025) MP group and to 73.25; ± 15.6;
(p < 0.05) P group.
The addition of 1.0 pg/ml
of LPS produced no further increase although the
results were still higher than for normal
subjects (p < 0.01).
The results suggest a greater proportion of
monocytes from subjects with bronchiectasis
adhere spontaneously suggesting a degree of in
vivo activation.
171
CLINICAL STUDIES 01." THE POST-VIRAL FATIGtIE
SYNDROME (PVI."S) WITH SPECIAL REFERENCE TO
SlCELE'l'AL MlJSCLE l"tJNC'l'ION
It, Teahan, V. R. Preedy. D. G. SlIlith and T. J •
Peters
Divi.ion of Clinical CBll Biology, NRC Clinical
Research CBntze, HarroW, MiCldlesex
Little is IaIoWn of the pathogenesis of the
sympt:cIaa in patient. with PVPS although
~le
45 p
a r:eoent pze11JlinlU:Y nport (Byrne et al ..
1985, AUBt. W.W. J. M8d., 15, 305) suggested
that type II fibre atrophy is an gportant
featuze of the synd~. we thenfoze
undertook a llYn-tic study to Characterise
the featuns of ~le ))~try and
anpholoqy in PVPS. A total of 18 ~
patients, aged 18-6Oy, with cl.a88ical clinical
&yIIptOll8 attr1llut;a))le to PVPS of acute onset
wsze studied. guadr1c8pe ~le ))1opey showed
no~l histoloqy in all Su))ject., 2 patients
had aiM type II f1))ze atrophy (atrophy factors
282, 280, contE018 < 180) and 2 patient. showed
aiM type I f1))ze hypertrophy (hypertrophy
factors - 792, lOll, WR < 400). serlD cr:eat1ne
kinase activit1lts wsze no~l 70 ~ J.8 ( _ ~
BE" (range 27 - J.66), contE018 20 - 205 IO/l)
!:Nt 2 pat1ltnts had %educed sexua carnos1nalle
activities (85, 65, contE018 100-200 ~l).
Mlluu~nt of total 1:lody potuSiI.B (
It), an
index of __cle . . . . , .howed nozmaJ. values (85
- 105" contxolB) in the 6 patients studied.
MAY of 1llU8C1e lllG. content (1Ilg/1IIg OIIA) showed
Significantly ~ l_lB, P < 0.001 ( _ .t:
BE) in the patients (0.517 ~ 0.011, n - 5)
ClClIIIPS%ed to contEOls, (0.605 .t: 0.03, n - 6).
It is concluded that, although EOUt1ne DlUscle
function tests includinq histoa:lJ:Ptte-try, do
not xel1a))ly identify patients with PVPS,
muscle lllG. content is %educed suggesting an
.iJIlpa1%ed IllU8Cle pxotein synthetic capllCity in
these patients.
172 THE EFFECTS OF DRUG THERAPY UPON HUMAN RESISTANCE
ARTERIOLE MORPHOLOGY IN ESSENTIAL HYPERTENSION
AM HEAGERTY, SJ BUND AND C AALKJAER*
Department of Medicine, Leicester, UK and * .Department of
Biophysics, Aarhus, Denmark
Essential hypertension is characterized by an
increase in peripheral vascular resistance. We have
previously demonstrated that in the established disease,
human resistance arterioles display an increase in
media/lumen ratio, decreased sensitivity to calcium but
no increased sensitivity to vasoconstrictor agonists
such as noradrenaline (Circ Res 1987; 61: 181-186). It
remains unclear whether the alteration in vascular
structure is a consequence of the pressure load or
intimately linked to the generation of the raised blood
pressure. In order to investigate this further,
resistance vessels were studied from 9 essential hypertensive patients (7 male, mean age 48+5.1 years) before
and after drug control of their blood-pressure. Eight
patients received at least 12 months treatment and one
patient had 4 months therapy. Blood pressure was significantly reduced by medication (178+8.3/108+5.4 vs
150+7.8188+2.4 mmHg, p<:O.Ol). Resistance arterioles
were obtained from biopsies of skin before and after
treatment, taken under local anaesthesia, cleaned and
mounted in a myograph. After reduction of.blood
pressure there was a significant fall in media/lumen
ratio (11.8~1.14 vs 7.3~0.4%, p=0.016), indicating that
the vascular hypertrophy had regressed. Sensitivity to
calcium increased but did not attain statistical significance (pD2 Ca before 4.2~0.05 vs pD2 Ca after 4.6~
0.25 log units NS, n=5).
These data suggest that effective antihypertensive
medication regresses the medial thickening observed in
arterioles of patients with untreated essential hypertension. Furthermore the decreased sensitivity to
calcium appears to be a phenomenon inherent to the
resistance vessel unaffected by therapy.