Clinical Science (2002) 102, 69–75 (Printed in Great Britain)
Abnormalities in primary granule exocytosis in
neutrophils from Type I diabetic
patients with nephropathy
Nicholas J. M. FARDON, Robert WILKINSON and Trevor H. THOMAS
Department of Medicine (Nephrology), Medical School, University of Newcastle-upon-Tyne, Framlington Place,
Newcastle-upon-Tyne NE2 4HH, U.K.
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Microalbuminuria in Type I diabetes involves a cell membrane abnormality and is associated with
a large increase in cardiovascular risk. The hypothesis that the membrane abnormality alters
granule exocytosis in neutrophils, which could contribute to the increased incidence of
cardiovascular disease, was investigated. PMA-stimulated expression of CD11b and CD69 on
neutrophils from normal controls (NC), long-term uncomplicated Type I diabetic control
patients (DC) and diabetic nephropathy patients (DN) was determined by fluorescence activated
cell scanning. Neutrophils from DN were faster than neutrophils from either NC or DC to
exocytose primary granules with CD69 following initial expression of the adhesion molecule
CD11b. However, a larger proportion of neutrophils from DN failed to withdraw CD11b from
the cell membrane after 90 min incubation. The protein kinase C (PKC) inhibitor, bisindolylmaleimide (BIM), showed that a larger proportion of neutrophils from DN, compared with
DC or NC, exocytosed primary granules independent of PKC. The calpain inhibitor, E64d,
showed that a larger proportion of neutrophils from both groups of diabetic patients, compared
with NC, exocytosed primary granules independent of calpain. Cytoskeletal disruption with
cytochalasin D had an effect on CD11b and CD69 exocytosis similar to that of BIM and E64d. The
pathways controlling granule exocytosis in neutrophils from diabetic patients are abnormal. A
change characteristic of DN causes rapid exocytosis of primary granules, and also causes the
adhesion molecule CD11b to persist on an increased proportion of neutrophils. This will make
an important contribution to increased vascular damage in these patients.
INTRODUCTION
About one-third of Type I diabetic patients develop
nephropathy [1,2]. This condition has a very poor
prognosis, mainly due to associated cardiovascular disease [3]. Indeed, diabetic nephropathy is sometimes
considered to be a microvascular disease [4]. There is
strong evidence that the susceptibility to nephropathy in
Type I diabetes is familial [5] and other associated familial
factors are hypertension, insulin resistance and dyslipidaemia [3,6,7], which are characteristics of the so-called
metabolic syndrome.
A cell membrane abnormality is believed to play a role
in diabetic nephropathy and the erythrocyte cell-membrane marker, Na+\Li+ countertransport, is abnormal in
patients with diabetic nephropathy and their first degree
relatives [8–10]. However, no membrane abnormality has
been shown in diabetic nephropathy that could contribute to either the renal pathology or the associated
cardiovascular disease. Evidence indicates that the protein
associated with the Na+\Li+ countertransport abnormality characteristic of diabetic nephropathy is tropomyosin, which has a role in stabilizing cytoskeletal actin
filaments [11]. These filaments are important for many
Key words: calpain, diabetes, diabetic nephropathy, exocytosis, neutrophil, protein kinase C.
Abbreviations: NC, normal controls ; DC, diabetic control patients ; DN, diabetic nephropathy patients ; PKC, protein kinase C ;
BIM, bisindolylmaleimide.
Correspondence: Dr T. H. Thomas (e-mail t.h.thomas!ncl.ac.uk).
# 2002 The Biochemical Society and the Medical Research Society
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N. J. M. Fardon, R. Wilkinson and T. H. Thomas
aspects of cell physiology, but their role in the expression
of integrins in neutrophils [12] and the intracellular
signalling from those integrins [13] is of particular interest
for the present study.
Neutrophils are an important contributor to vascular
damage [14] and attach at sites by interaction with their
cell surface β2-integrin, Mac-1 [15], which is a dimer
of CD11b and CD18 exocytosed from secondary
granules [16]. This is followed by exocytosis of primary
granules containing pro-inflammatory enzymes with the
appearance on the cell surface of markers such as CD69
[17]. Several observations show that inappropriate activation of this process causes vascular damage especially
in the myocardial circulation [18]. It has been suggested
that evidence for increased CD11b on leucocytes from
patients with ischaemic heart disease indicates a
smouldering inflammatory process in the vasculature
[19]. In addition, some beneficial effects of drugs may be
due to their effects on CD11b. Thus, the glycoprotein
IIb\IIIa receptor antagonist, abciximab, has been suggested to have benefit due to its blockade of CD11b [20],
and 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) also reduce CD11b expression [21].
We have shown previously [17], and this has been
confirmed by others [22], that neutrophils stimulated
with PMA first express and then down-regulate CD11b.
We have now tested the hypothesis that patients with
diabetic nephropathy have abnormal intracellular trafficking and membrane fusion of intracellular vesicles, and
that one effect is the abnormal expression of integrins on
neutrophils and their exocytosis of primary granules
containing inflammatory enzymes.
METHODS
Patients
Three groups of patients were studied : 11 patients with
Type I diabetes and evidence of diabetic nephropathy
(DN) for which the criterion was diabetes for less than 20
years with microalbuminuria of greater than 20 µg\min,
but serum creatinine less than 150 µmol\l, and serum
albumin greater than 35 g\l and with retinopathy ; a
control group of 11 patients with Type I diabetes (DC)
defined as diabetes for more than 25 years without
microalbuminuria and with serum creatinine less than
100 µmol\l ; 12 normal controls (NC) with no history of
diabetes, hypertension or renal impairment. The characteristics of each group are shown in Table 1.
Of the 11 DN, nine had received laser treatment for
retinopathy and seven were taking an angiotensinconverting-enzyme inhibitor. One of the seven was also
taking a calcium channel antagonist and another a
diuretic. Five patients in this group were taking lipidlowering medication. All DN had abnormal Na+\Li+
# 2002 The Biochemical Society and the Medical Research Society
countertransport. One patient in the DC group had
received laser treatment for retinopathy. One was taking
a beta-blocker and lipid-lowering medication, and one
other was taking a calcium channel antagonist. None of
the NC was taking regular medication. NC and DC
subjects had normal Na+\Li+ countertransport.
All subjects gave informed consent, and the Joint
Ethics Committee of the Newcastle Health Authority
and Newcastle University approved the study protocol.
Microalbuminuria
Each patient performed three successive overnight timed
urine collections. Urine albumin was measured by radioimmunoassay [23]. Urinary albumin excretion rate was
expressed in µg per min (normal 20 µg\min).
Neutrophil antigen expression
Venous blood was collected into a heparinized glass tube
and used within 2 h. Equal volumes of blood and PBS
were mixed in a glass tube and incubated for up to
90 min. PMA was added in the PBS to give a final
concentration of 10 or 100 ng\ml (16 or 162 nmol\l) in
the incubation mixtures. At various times 50 µl aliquots
were placed on ice with anti-human CD45 (common
leucocyte antigen) antibody conjugated to FITC together
with either anti-human CD11b or anti-human CD69
labelled with R. Phycoerythrin.
Appropriate isotype IgG controls were used to measure non-specific antibody binding. Red blood cells were
specifically lysed and fixed using a proprietary formaldehyde containing solution (Erythrolyse ; Serotec,
Abingdon, U.K.). White blood cells were washed twice
with PBS and analysed on a flow cytometer (FACScan ;
Becton Dickenson, Mountainverie, CA, U.S.A.).
The inhibitors bisindolylmaleimide GF 109203X
(BIM) and E64d were dissolved in DMSO and further
diluted 1 in 50 in PBS. When mixed with blood and PMA,
the BIM concentration was 10 µmol\l (500 times the in
vitro IC of isolated cells) [24] and E64d 100 µg\ml
&!
(292 µmol\l) [25]. The final concentration of DMSO was
1 in 400 (35 mmol\l), which in PBS alone or with PMA
had no effect on the expression of the surface antigens.
Cytochalasin D was also dissolved in DMSO and used at
a final concentration of 10 µmol\l.
FACS data were analysed using WinMDI software
(Scripps Institute, La Jolla, CA, U.S.A.). Neutrophils
were identified on the basis of their characteristic light
scattering properties and expression of CD45. The results
were expressed as the percentage of neutrophils positive
for CD69 or CD11b and the median fluorescence of the
population of positive cells. The median fluorescence was
converted into number of sites (molecules of equivalent
fluorochrome), using calibration beads (FluoroSpheres ;
Dako A\S, Glostrup, Denmark), and the non-specific
antibody binding was subtracted.
Abnormal neutrophil exocytosis in nephropathy
Table 1
Clinical characteristics
Values are meanspS.E.M. or median (range).
n
Sex (M/F)
Age (years)
Body mass index (kg/m2)
Mean blood pressure (mmHg)
Urinary albumin excretion
rate ( µg/min)
Creatinine ( µmol/l)
NC
DC
DN
12
6/6
47.7p4.4
23.8p0.8
84.4p3.6
4.4 (1.0–7.8)
11
6/5
44.7p4.7
25.1p0.8
87.1p3.4
4.6 (2.5–9.6)
11
5/6
36.2p3.4
30.5p1.6
99.9p3.5
217 (27–619)
81.6p2.8
86.5p4.2
88.5p6.0
Statistics
Normally distributed data are presented as meansp
S.E.M. and the probability of differences assessed using
the unpaired two-tailed Student’s t test. Non-parametric
data are expressed as medians with interquartile ranges,
and the Mann–Whitney U test used to calculate the
probability of differences.
RESULTS
Abnormal CD11b down-regulation and CD69
expression in DN neutrophils
The exocytosis of CD11b was similarly rapid with both
10 and 100 ng\ml PMA in neutrophils from all three
groups of subjects. Subsequently over a longer time
course there was down-regulation of CD11b in a
proportion of neutrophils closely allied to the exocytosis
of CD69. Therefore, the neutrophils that expressed CD69
can be taken as those that had down-regulated CD11b
[17] (Figure 1). There were two abnormalities in the
neutrophils from DN patients. Firstly, a much smaller
proportion of neutrophils from DN down-regulated
CD11b, with exocytosis of primary granules containing
CD69 after a 90 min incubation with 100 ng\ml PMA,
compared with neutrophils from NC or DC (44.3p4.2
versus 62.9p4.4 or 59.7p2.8%, P 0.006 ; Figure 2).
Secondly, the proportion of neutrophils that downregulated CD11b and exocytosed CD69 during the
90 min incubation did so faster in DN than in either of
the other groups [half time (t / ) : DN, 5.5p1.7 min ; DC,
"#
11.1p1.7 min ; NC, 17.5p2.7 min ; P l 0.024 and P
0.001 for DN versus DC and NC respectively ; Table 2
and Figure 2].
The abnormality in DN neutrophils, most clearly
revealed by stimulation with 10 ng\ml PMA, was the
rapid exocytosis of primary granules with CD69 (Figure
3). Since the response to the lower dose of PMA was
slower, longer incubation was needed to show the lower
total level of response in DN neutrophils. However, it
can be seen that whereas the maximum level of CD11b
Figure 1 The expression of the Mac-1 integrin subunit CD11b and the primary granule marker CD69 on neutrophils from NC
after 5, 15 and 30 min stimulation with 100 ng/ml PMA
The box regions show the same groups of neutrophils that have down-regulated CD11b and expressed CD69 with reduced CD45.
# 2002 The Biochemical Society and the Medical Research Society
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N. J. M. Fardon, R. Wilkinson and T. H. Thomas
Figure 2 CD11b down-regulation in neutrophils stimulated
with 100 ng/ml PMA
The rate of appearance of neutrophils that had down-regulated CD11b and
expressed CD69 on stimulation with 100 ng/ml PMA and the effect of BIM and
E64d in NC (#), DC ($) and DN (
). ¶, P 0.02 and ¶ ¶, P 0.001
versus NC ; N, P 0.006 and NN, P 0.001 versus NC and DC.
down-regulation and CD69 expression was achieved by
30 min in DN neutrophils, NC neutrophils continued to
respond in a linear fashion for up to 90 min (Figure 3).
The more rapid response of DN neutrophils was also
indicated by their expression of CD11b sites which was
maximal by 30 min whereas in NC neutrophils it
increased between 30 and 90 min (Table 3).
PKC inhibition impairs CD11b downregulation but DN neutrophils remain
abnormal
The PKC inhibitor BIM greatly reduced the proportion
of neutrophils that could down-regulate CD11b from
their membrane with expression of CD69. However,
BIM was much less effective in DN neutrophils and
caused only a 64 % inhibition of the CD11b downregulation\CD69 exocytosis response to 100 ng\ml
PMA in neutrophils from DN, compared with 96 % and
93 % inhibition in neutrophils from NC or DC respectively (Figure 2). BIM slowed the expression of
CD11b sites, which were lower after 30 min incubation
with PMA, but had little effect on the maximum levels
after 90 min incubation. The slowing effect of BIM on
CD11b expression was less in the neutrophils from
# 2002 The Biochemical Society and the Medical Research Society
Figure 3 CD11b down-regulation in neutrophils stimulated
with 10 ng/ml PMA
The rate of appearance of neutrophils that had down-regulated CD11b and
expressed CD69 on stimulation with 10 ng/ml PMA and the effect of BIM and E64d
in NC (#), DC ($) and DN (
). ¶, P 0.01 and §, P 0.02 versus NC.
diabetic patients and was most apparent with the lower
dose of PMA (Table 3).
Calpain inhibition impairs CD11b downregulation similarly with DN and DC
neutrophils
The calpain inhibitor E64d also reduced the proportion
of neutrophils that down-regulated CD11b and ex-
Table 2 Percentage of cells that had progressed to release
primary granules, indicated by CD69 expression, at various
times after stimulation with 100 ng/ml PMA
Values are meanspS.E.M. *, P 0.006 and **, P
P 0.006 and †† P 0.001 versus DC.
0.001 versus NC ; †,
Percentage of cells that exocytosed primary granules
Incubation time (min)
NC
DC
DN
5
15
30
90
10.5p2.1
30.1p3.6
44.0p2.4
62.9p4.4
22.8p1.4**
31.8p1.7
44.3p2.2
59.7p2.8
20.4p1.3**
28.5p2.1
33.1p2.1*††
44.3p4.2*†
Abnormal neutrophil exocytosis in nephropathy
Table 3 Level of expression in neutrophils retaining the CD11b component of the adherent integrin Mac-1 after mild
stimulation by incubation in PBS with either 10 ng/ml or 100 ng/ml PMA, in the absence or presence of BIM or E64d
Values are meanspS.E.M. *, P
0.005 and **, P
0.02 versus NC ; †, P
0.001, ††, P
0.01 and †††, P
0.05 versus 30 min incubation.
10−3iNumber of CD11b sites per cell
10 ng/ml PMA
100 ng/ml PMA
Incubation time (min)
NC
DC
DN
NC
DC
DN
PMA
30
90
26.0p2.9
36.3p3.8†††
33.0p3.6
24.4p4.4
31.8p3.3
32.4p3.7
40.8p3.1
40.4p2.1
37.0p3.9
35.2p3.8
39.7p3.9
31.9p2.5
PMAjBIM
30
90
4.8p1.1
32.7p1.7†
15.8p3.2*
29.4p3.0†
17.8p3.9*
29.2p3.5†††
19.9p0.9
31.1p2.1†
24.2p3.3
27.8p3.5
27.1p3.4
26.7p2.8
PMAjE64d
30
90
9.3p1.6
18.8p2.0†
18.6p3.3**
23.0p3.3††
18.3p2.7**
24.4p4.4†††
13.8p0.9
23.8p1.6†
23.2p6.0
25.5p3.9
18.8p2.8
22.3p3.6†††
pressed CD69. In the presence of E64d the proportion of
neutrophils that down-regulated CD11b on stimulation
with 100 ng\ml PMA was the same in DC and DN, so
that the difference between these groups was abolished
(Figure 2). However, they were both significantly different to NC. Unlike with BIM, in the presence of E64d
100 ng\ml PMA was more effective than 10 ng\ml in
causing neutrophils to down-regulate CD11b and express
CD69. The effect of E64d on CD11b sites expressed was
similar to the effect of BIM (Table 3).
Actin filament disruption inhibits CD11b
down-regulation
In the presence of cytochalasin D, 100 ng\ml PMA
caused all neutrophils from NC to exocytose CD11b
but at a slightly lower level than in untreated cells
(30.2p7.4i10$ versus 42.8p1.6i10$ sites\cell). However, the ability of these neutrophils to convert to the
exocytosis of primary granules with the expression of
CD69 was greatly impaired by cytochalasin D (9p3
versus 38p4 %, P l 0.004).
DISCUSSION
Two abnormalities are apparent on PMA stimulation of
neutrophils from DN. These are : (1) retention of CD11b
on a greater proportion of neutrophils, and (2) rapid
exocytosis of CD69 in those neutrophils that were
stimulated to express this marker.
The retention of CD11b was clearly associated with
nephropathy rather than diabetes since in NC and DC,
after stimulation for 90 min with 100 ng\ml PMA, only
37 % and 40 % of neutrophils respectively still expressed
CD11b, whereas in DN 56 % of neutrophils were CD11b
positive. CD11b is a component of the β2-integrin
adhesion molecule Mac-1 and its expression on neutrophils closely relates to neutrophil adhesion [22]. This
could lead in DN to more neutrophil adherence at sites of
activation and provide a focus for platelet aggregation
[26]. Blockade of Mac-1 in a model of lung injury, even
after injury is initiated, decreases neutrophil accumulation and inflammatory damage [27].
The rapid exocytosis of primary granules containing
CD69, in a proportion of DN neutrophils, suggests an
abnormality in intracellular trafficking, in addition to the
threshold effect that enables only a proportion of
neutrophils to respond. DC neutrophils also exocytosed
primary granules more rapidly than the NC neutrophils.
However, in contrast to DN, in DC the same proportion
of neutrophils down-regulated CD11b as in NC. Therefore the accumulation of neutrophils by adhesion will
have similar characteristics in DC and NC and this may
be an important characteristic for vascular damage.
The possible involvement of a PKC-independent
pathway and the PKC modifying protease calpain in the
abnormal neutrophil function in DN was examined
using the PKC inhibitor BIM and the calpain inhibitor
E64d, which has been widely used to establish a role for
calpain [28]. With both the low and high doses of PMA,
BIM at a concentration that blocks all known isoforms of
PKC [29] and E64d [30], both slowed, but only slightly
reduced, the appearance of CD11b sites, suggesting that
PKC does not play a major role in exocytosis of CD11b.
However, both BIM and E64d had a much greater
inhibitory effect on the down-regulation of CD11b and
expression of CD69.
Although DN neutrophils have an impaired ability to
down-regulate CD11b and express CD69, they are
abnormally resistant to inhibition of PKC with BIM and
inhibition of calpain with E64d. BIM virtually abolished
the down-regulation of CD11b and expression of CD69
in NC neutrophils. It is unlikely that BIM inhibits PKC
less effectively in DN than NC neutrophils, which
suggests that in DN neutrophils CD11b down-regulation
and CD69 expression have greater independence from
# 2002 The Biochemical Society and the Medical Research Society
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N. J. M. Fardon, R. Wilkinson and T. H. Thomas
PKC activation. This suggests either an alternative
pathway in DN that is much more active than in NC, or
that the target protein for PKC has a higher level of
phosphorylation in the resting-state in DN neutrophils.
One action of PKC is the inhibition of protein tyrosine
phosphatase activity [31,32], and the total blockade of
PKC by BIM may reveal an underlying difference in
protein phosphorylation in DN neutrophils. There is no
evidence that PMA stimulates processes that are not
dependent on PKC activation. However, we have recently shown that the PMA doses used in the present
experiments can activate calpain in neutrophils [33], and
PKC action may depend on calpain activity [28]. Thus
the relative effects of calpain inhibition with E64d in NC
and DN neutrophils are similar to those of BIM.
However, in DC neutrophils the effects are markedly
different; with BIM they are similar to NC neutrophils
and with E64d they are the same as DN neutrophils. This
indicates that a PKC-independent calpain effect is the
key difference between DN and DC neutrophils.
The most likely target for calpain is cytoskeletal
proteins that are known to be modified by calpain [30]. In
support of this hypothesis, disruption of cytoskeletal
remodelling by cytochalasin D had an effect very similar
to E64d on the expression of CD11b and CD69. It seems
possible that both PKC and calpain are involved in the
cytoskeletal remodelling that is required for CD11b
down-regulation and primary granule exocytosis, and
that the normal mechanism for this remodelling process
is impaired in DN. The resulting increased retention of
adhesion molecules on neutrophil, if occurring in vivo, is
likely to contribute to increased vascular damage. In this
case, an understanding of the altered neutrophil function
may lead to new targets for treatment to alleviate the high
levels of cardiovascular disease in DN. In fact, it seems
possible that the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor class of drugs may give some benefit by
reducing CD11b expression [21].
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13
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ACKNOWLEDGMENTS
This research was supported by grants from the British
Heart Foundation (Grant no. PG\97099) and the Northern Counties Kidney Research Fund.
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Received 29 May 2001/19 September 2001; accepted 23 October 2001
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