close

Вход

Забыли?

вход по аккаунту

?

Low density neutrophils in patients with systemic lupus erythematosus rheumatoid arthritis and acute rheumatic fever.

код для вставкиСкачать
1334
LOW DENSITY NEUTROPHILS IN PATIENTS WITH
SYSTEMIC LUPUS ERYTHEMATOSUS, RHEUMATOID
ARTHRITIS, AND ACUTE RHEUMATIC FEVER
ELISA HACBARTH and ANDRE KAJDACSY-BALLA
Ficoll-Hypaque density gradient preparations of
peripheral blood mononuclear cells from patients with
systemic lupus erythematosus, rheumatoid arthritis,
and acute rheumatic fever were highly “contaminated”
with low buoyant density neutrophils. Plasma from
these patients could induce an in vitro decrease of
buoyancy in neutrophils with normal buoyant density.
Similar change could be induced by complementactivated sera and aggregated gamma globulin. These
data suggest that activated neutrophils are a common
finding in the peripheral blood of these patients and may
influence the interpretation of any studies with these
cells. Functional studies of lymphocytes separated by
Ficoll-Hypaque gradients should also take into account
the higher degree of impurity of the cell preparations in
patients with rheumatic diseases.
Researchers have reported wide variations in
the composition of mononuclear cell preparations obtained by Ficoll-Hypaque gradient preparations (1-3).
The percentage of lymphocytes in the gradient-derived
cell suspension is significantly decreased in patients
with various forms of cancer (4-7). This decrease has
been attributed to decreased buoyant density of the
nonlymphoid cells in the peripheral blood of these
patients, which causes a disproportionate number of
~_____
From the Division of Rheumatology and the Laboratory of
Immunopathology, Department of Medicine, Universidade Federal
do Parana, Curitiba, Parana, Brazil.
Dr. Hacbarth’s work was supported by a grant from Syntex
do Brasil.
Elisa Hacbarth, MD; Andre Kajdacsy-Balla, MD, PhD.
Address reprint requests to Andre Kajdacsy-Balla, MD,
PhD, Department of Pathology, Hahnemann University Hospital,
Broad and Vine, Philadelphia, PA 19102.
Submitted for publication April 3, 1985; accepted in revised
form June 18, 1986.
Arthritis and Rheuaatism, Vol. 29, No. 11 (November 1986)
cells to be retained at the gradient interfaces. The
mechanisms of this change have not been elucidated,
however. Such changes have also been observed in
patients with chronic renal failure and in those who
have recently had anesthesia and surgery (8,9).
Similar findings in cell preparations from the
blood of some arthritis patients led us to pursue a
study in which we observed the percentages of the
different cell types in the Ficoll-Hypaque gradients.
We also tested in vitro some factors that could influence the buoyant density of peripheral blood cells.
PATIENTS AND METHODS
Patients. Blood was obtained by venipuncture from 9
patients with rheumatoid arthritis (RA), 9 patients with
systemic lupus erythematosus (SLE), and 13 patients with
acute rheumatic fever (ARF). The RA and SLE patients’
disease fulfilled the diagnostic and classification criteria of
the American Rheumatism Association (10,l l), and the ARF
patients’ disease fulfilled the Jones criteria (12). Patients
taking corticosteroids or immunosuppressive drugs were
excluded. The normal control group consisted of 23 healthy
blood donors who had not used any drugs during the week
prior to venipuncture. For some of the in vitro experiments,
blood was obtained by venipuncture from patients with
lepromatous leprosy and from patients with metastatic gastric carcinoma.
Blood samples from patients and normal individuals
were analyzed simultaneously, with the observer having no
knowledge of the diagnosis.
Peripheral blood cell separation and enumeration. Ten
milliliters of venous blood was collected and mixed immediately with 20 mg of disodium EDTA. Total white blood cell
counts were performed with a model Ssr Coulter counter
(Coulter, Hialeah, FL). Differential counts were done manually. Leukocyte separation was performed on Ficoll
(Pharmacia, Uppsala, Sweden) and sodium diatrizoate
(Hypaque; Winthrop Laboratories, Rio de Janeiro, Brazil)
LOW DENSITY NEUTROPHILS
gradients, density 1.076, according to the method of Aiuti et
al (2), with minor modifications. All cell separations were
done within 2 hours after blood collection. No differences in
results were found among separations done at 15, 30, 60, or
120 minutes after collection.
Following centrifugation, the cells that had been
removed from the gradient, together with one-half of the
Ficoll-Hypaque above the erythrocyte pellet, were washed 3
times in 0.1M phosphate buffered saline (PBS), pH 7.4. Cells
were then resuspended in 1 ml of PBS and counted on an
improved Neubauer hemacytometer. Cell viability was evaluated by trypan blue dye exclusion. Cell preparations with
<96% viability were discarded. Cell concentration was
adjusted to 5 x lo5 cells/ml.
Cytologic preparations were made in duplicate, with
0.2 ml of cell suspension and 0.1 ml of 22% bovine serum
albumin placed in a cytocentrifuge (Cytospin 2; Shandon
Southern Products, Asmoor, UK) at 1,000 revolutions per
minute (700g) for 5 minutes. The slides were stained, by
previously described techniques, for peroxidase, nonspecific
esterase, and with Giemsa-Wright (13,14). Counts included
1,500 cells in the Giemsa-Wright-stained slides and 500 cells
in the enzyme cytochemical studies. The results were quite
consistent among these different staining methods; therefore, in the in vitro studies, only the Giemsa-Wright stain
was used. In some experiments, we also enumerated cells in
the pellet sedimented below the Ficoll-Hypaque zone.
Addition of corticosteroid. Venous blood from 3 normal individuals was collected in disodium EDTA (2 mg/ml),
divided into 2-ml aliquots, and incubated with hydrocortisone (Sigma, St. Louis, MO) in concentrations varying from
& w 4 M at 37°C for 30,60,90, or 120 minutes. At the end of
the incubation, the leukocytes were separated and enumerated as described above.
Addition of plasma to normal cells. Venous blood
from 3 normal individuals was collected in disodium EDTA
(2 mg/ml) and centrifuged at 500g for 5 minutes at room
temperature. The cell pellets were separated and divided
into various aliquots, which were resuspended with equal
volumes of either (a) autologous plasma, (b) plasma from
other normal individuals, or (c) plasma from patients with
abnormal leukocyte differential counts on Ficoll-Hypaque
gradients (diagnoses of lepromatous leprosy, metastatic gastric carcinoma, and RA). These mixtures were incubated at
37°C for 15, 30, or 60 minutes, and then subjected to
leukocyte separation and enumeration as described above.
Addition of aggregated human gamma globulin in the
presence of complement. Human gamma globulin (Cohn
fraction 11; Sigma) was diluted in 0.02M PBS, pH 7.2, to a
final concentration of 16 mg/ml, incubated in a water bath at
63°C for 20 minutes, and centrifuged at 1,500g for 10 minutes
at room temperature. The supernatant was used without
further treatment. Venous blood samples from 7 normal
individuals were collected and defibrinated with glass beads.
Preliminary studies showed that neither blood collection
with EDTA nor defibrination altered relative cell counts in
Ficoll-Hypaque gradients. Aliquots of 2 ml of defibrinated
blood were mixed with 0.1 ml of aggregated human gamma
globulin (AHG) and incubated at 37°C for 30 minutes before
Ficoll-Hypaque cell separation and enumeration as de-
1335
scribed above. Control aliquots were incubated with 0.1 mi
of PBS.
Complement activation. The alternative pathway of
complement was activated by incubation of normal human
serum with 5 mg/ml of inulin for 30 minutes at 37°C. Inulin
was removed by centrifugation at 1,200g for 10 minutes at
room temperature. Venous blood from 4 normal individuals
was collected in disodium EDTA (2 mg/ml) and incubated for
30 minutes at 37°C with equal volumes of either (a) autologous inulin-activated serum o r (b) autologous heatinactivated serum. The mixtures were then subjected to cell
separation and enumeration as described above.
Addition of aggregated human gamma globulin in the
absence of complement. Human gamma globulin (Cohn fraction 11; Sigma) was diluted in 0.02M PBS, pH 7.2, and
filtered through a Sephadex G-200 column to obtain a
preparation that was free of aggregates and of most impurities. The fraction of eluate with the highest concentration of
immunoglobulins was retrieved and divided into 2 aliquots.
One of the aliquots was stored at 4°C for a few hours until
use. In the other aliquot, the immunoglobulins were heataggregated for 20 minutes at 63"C, then centrifuged at 1,OOOg
for 10 minutes to remove large aggregates. Normal venous
blood samples were collected with higher final concentrations of EDTA (0.02M) to assure inhibition of complement,
and divided into 3 aliquots of 2 ml. To each aliquot, 0.1 ml of
1oc
9c
sc
-
-
i
7c
00
6C
v)
W
c
> 50
V
0
a
I
2
3a
20
10
0
NORMAL
AR F
S LE
RA
Figure 1. Percentage of lymphocytes at the interface of the FicollHypaque gradients in patients with acute rheumatic fever (ARF),
systemic lupus erythematosus (SLE), and rheumatoid arthritis
(RA), and in normal controls.
HACBARTH AND KAJDACSY-BALLA
1336
either PBS, nonaggregated gamma globulin, or heat-aggregated gamma globulin was added. Each aliquot was incubabed for 30 minutes at 37"C, immediately placed onto FicollHypaque gradients, and enumerated as described above.
Addition of plasma to normal granulocytes. Ten milliliters of venous blood was collected and mixed immediately
with 20 mg of disodium EDTA. The tube was allowed to
stand vertically for 1 hour at 37°C. The leukocyte-rich
plasma and buffy coat were aspirated with a Pasteur pipette.
Each aliquot of this suspension was incubated at 37°C for 30
minutes with equal volumes of either (a) plasma from SLE
patients, (b) autologous plasma, or (c) normal serum activated by inulin. After incubation, each cell suspension was
carefully layered on a Ficoll-Hypaque gradient (density
1.110). This density was chosen during preliminary experiments because it gave the best separation between
leukocytes and red blood cells. It was obtained by mixing
Ficoll-Paque (Pharmacia), density 1.077, with Hypaque
(Winthrop-Breon Laboratories, New York, NY). The cells
at the interface were removed gently and were immediately
assessed for changes in volume and formation of cellular
aggregates, by use of a Coulter counter (Model ZBI)
equipped with a 100-channel volume channelyzer.
Statistical analysis. Data obtained from the different
groups of patients and normal controls were compared by
nonparametric statistical methods (15,16): analysis of variance for posts of Kruskal-Wallis, complemented by the
Dunn test of multiple comparisons. Paired samples in the in
vitro studies were compared by the Wilcoxon test. The
Mann-Whitney U-test was used to test subgroups of acute
rheumatic fever patients for clinical differences. The Spearman rank sum test was used for the determination of
correlation coefficients.
RESULTS
Lymphocyte and neutrophil content in FicollHypaque gradients. Figures 1 and 2 show the percentages of lymphocytes and neutrophils, respectively, at
the interface of the Ficoll-Hypaque gradients, in the
different subject groups. All 3 groups of patients had
significantly fewer lymphocytes and more neutrophils
than did the control group (P < 0.05). The RA, SLE,
and ARF groups did not differ from each other in these
percentages. We did not detect any effect of sex, age,
or use of antiinflammatory drugs on the percentages of
A 141%
a
b
!
:
a
a
a
. a
a
b
i
:
a
a
a
a
b
a
b
H
a
a
m
a
b
a
m
:
a
ma
:
.
a
a
a
a
NORMAL
Figure 2. Percentage of neutrophils at the interface of the FicollHypaque gradients in patients with acute rheumatic fever (ARF),
systemic lupus erythematosus (SLE), and rheumatoid arthritis
(RA), and in normal controls.
b
a
a
a
ARF
SLE
Rh
Figure 3. Ratio between lymphocytes and neutrophils at the interface of the Ficoll-Hypaque gradients in patients with acute rheumatic fever (ARF), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA), and in normal controls.
LOW DENSITY NEUTROPHILS
1337
lymphocytes or neutrophils in these gradients (data
not shown).
Differences between patients and normal subjects became even more evident when we studied the
ratio between lymphocytes and neutrophils (L :N) in
the gradients (Figure 3). Upon arbitrarily choosing an
L :N ratio of 240, it can be seen that 19 of 23 normal
controls had values in this range, while only 3 of 13
ARF patients, 1 of 9 SLE patients, and none of 9 RA
patients reached this level.
In order to exclude cell aggregation as the
explanation for our findings, we used a phase-contrast
microscope to look for aggregated neutrophils in the
Ficoll-Hypaque interface, before washing the cells.
This was done with the in vitro studies also. Only a
few neutrophils (<3%) were found in clumps. The
number of clumped lymphocytes in such preparations
was similar.
In the 13 samples in which we studied the
leukocyte composition in the pellet that was sedimented below the Ficoll-Hypaque zone, we observed that
very few lymphocytes (<5% in each case) were found
there, even in cases in which a low percentage of
lymphocytes was found at the interface above the
Ficoll-H ypaque gradient.
In some experiments, following a procedure
commonly used in many laboratories, we centrifuged
the blood, removed the plasma, and resuspended the
cells with buffer prior to layering them onto FicollHypaque gradients. The L :N ratios were very similar
to the ratios obtained when the blood was layered onto
Table 1. Effect of in vitro addition of plasma on the buoyant density of normal cells*
~~~
Differential cell counts
%
Cells/plasma
Experiment 1
Normal/autologous
Normalflepros y
Experiment 2
Normal/autologous
NormallRA
Normalheoplasia
Normalhormal t
Exneriment 3
Normal/autologous
NormaUleprosy
Normalheoplasia
lymphocytes
%
%
mono- neutrocytes
phils
%
basophils
L:N
93.6
72.3
3.2
4.4
2.4
22.5
0.8
0.8
39.3
3.2
97.6
87.5
91.0
95.7
1.0
2.0
1.4
2.7
1.1
10.0
7.2
0.9
0.3
0.5
0.4
0.7
88.7
8.7
12.6
107.0
92.0
71.5
58.0
2.4
1.4
0.4
5.6
26.8
41.2
0.2
0.4
16.4
2.6
1.4
* In each experiment, all normal cells came from the same individual.
L : N = lymphocyte :neutrophil ratio; RA = rheumatoid arthritis.
t Homologous normal plasma.
Figure 4. Effect of incubation of normal cells with aggregated
human gamma globulin (AHG) and complement. The lines link the 2
findings of the lymphocyte: neutrophil ratio (L: N) from each normal
individual. PBS = phosphate buffered saline.
Ficoll-Hypaque without this intermediate step. Thus,
we later bypassed this step in order to avoid unnecessary manipulation of the cells and a small loss of
neutrophils in the discarded plasma.
Effect of in vitro treatment with hydrocortisone.
We did not detect any difference in the cell counts
between samples incubated with hydrocortisone, at
any concentration tested, and those not treated with
hydrocortisone.
Effect of in vitro addition of patient plasma on
the density of normal cells. The results obtained for the
differential cell counts of normal cells after 30 minutes
of incubation with patient plasma are shown in Table
1. The L:N ratio was about 10 times lower when
abnormal plasma was added to normal cells than when
normal autologous or homologous plasma was used.
Results obtained with 15- and 60-minute incubations
were similar.
Effect of in vitro addition of aggregated human
gamma globulin, in the presence of complement, on the
density of normal cells. In these experiments, aggregated gamma globulin was allowed to interact with
normal serum, activating complement in the presence
of normal cells (Figure 4). Shifts in the proportion
of lymphocytes and neutrophils were similar to
those obtained with serum from patients with inflammatory diseases.
1338
HACBARTH AND KAJDACSY-BALLA
Table 2. Effect of complement activation on the density of normal cells*
Differential cell counts
lymphocytes
%
monocytes
neutrophils
%
basophils
96.4
54.4
2.2
2.4
1.4
42.8
0.4
69.0
1.2
94.2
15.9
2.2
0.6
3.4
83.3
0.2
0.2
21.1
0.2
95.5
35.9
3.6
0.1
-
63.1
0.3
95.4
0.5
89.5
66.4
10.0
2.2
0.4
31.2
0.2
%
Serum added
for incubation
Experiment 1
Inactivated
Activated
Experiment 2
Inactivated
Activated
Experiment 3
Inactivated
Activated
Experiment 4
Inactivated
Activated
%
1.o
-
-
L:N
235.0
2.1
* Inactivated serum was autologous heat-inactivated serum; activated serum was autologous inulin-activated serum. L : N =
lymphocyte :neutrophil ratio.
Effect of complement activation on the density of
normal cells. Table 2 shows the profound effect of
inulin-activated serum on the distribution of cells in
the Ficoll-Hypaque gradient. The percentage of
neutrophils had an average increase of 48 times the
original count, and the L : N ratio decreased to
an average of v124 the values found with inactivated serum.
Effect of in vitro addition of aggregated human
gamma globulin on the density of normal cells. The
samples from all 7 normal subjects studied had smaller
L: N ratios when they were incubated with aggregated
human gamma globulin than when they were incubated with nonaggregated human gamma globulin (Figure 5 ) . The presence of 0.02M EDTA blocked activation of complement in these experiments. Incubation
of the samples with nonaggregated gamma globulin did
not induce significant alterations, compared with incubation with PBS (data not shown).
Effect of in vitro addition of patient plasma on
the volume of normal granulocytes. Aliquots of normal
leukocytes were incubated with each of 6 SLE plasma
samples, with autologous plasma, or with serum activatad by inulin. An increase in cell volume occurred
after incubation with 2 of 6 patient samples and with
serum activated by inulin. Only the 2 plasma samples
that were capable of inducing a change in the L : N
ratios of normal cells could induce an increase in
neutrophil volume. The median corpuscular volume of
neutrophils incubated with autologous plasma was
264 pm3. The median neutrophil volumes from the
same normal individual, after incubation with the 2
SLE plasmas, were 312 pm3 and 316 pm3, respectively. Neutrophils incubated with inulin-activated serum had a median volume of 317 pm3.
Figure 6 compares the volumes of cells incubated with autologous plasma versus cells incubated
with 1 of the SLE plasma samples. Unlike the
neutrophils, the volume of the lymphocytes did not
change during incubation with any of the plasma
samples or with inulin-activated serum. There were
no cellular aggregates detectable in the high volume
channels.
Correlation of clinical activity of acute rheumatic
fever with L:N values. A group of 10 patients with
acute rheumatic fever was studied for possible correlations between clinical activity and the L :N ratio. For
this purpose, the 5 patients with the highest L :N ratios
(>lo) were compared with the 5 patients with the
lowest ratios (<lo), with regard to various parameters
of disease activity (see Table 3). Antistreptolysin
0 titers were included since they correlate with severity of disease. In 11 of 12 parameters, the patients
with lower L : N ratios had indications of more severe
disease activity (P < 0.005). The differences were
statistically significant in only 4 of these comparisons, however.
500)
NAHG
AH G
Figure 5. Effect of incubation of normal cells with aggregated
human gamma globulin (AHG) versus nonaggregated human gamma
globulin (NAHG). The lines link the 2 findings of the lymphocyte: neutrophil ratio (L:N) from each normal individual.
LOW DENSITY NEUTROPHILS
1339
lo00
C
800
.-c
0
-
Q
3
P
0
n
-
600
400
Q,
0
200
0
50
200
400
600
1000
800
3
Cell Volume ( pm 1
Figure 6. Mean corpuscular volume of normal cells after incubation with autologous plasma (solid line) or
lupus plasma (broken line). The first peaks correspond mainly to lymphocytes and the second peaks to
neutrophils.
DISCUSSION
Our results show that peripheral blood samples
obtained from patients with SLE, RA, and ARF have
a high number of neutrophils at the interface of FicollHypaque gradients. This increase in the proportions of
neutrophils could be due to either a decrease in
lymphocytes (the predominant cell at the gradient
interface), an increase in neutrophils, or a combination
of these 2 factors. If it were due to a decrease in
lymphocytes, we would have found a decreased number of lymphocytes in the whole blood count before
centrifugation, or we would have found a population of
lymphocytes with greater buoyant density in the pellet
below the Ficoll-Hypaque gradient. Normal blood
lymphocyte counts in the majority of our patients, and
the absence of significant numbers of lymphocytes in
the pellet, provide evidence that variations in lymphocyte numbers do not explain the profound alterations
in the proportion of neutrophils.
There was no correlation between numbers of
lymphocytes in the unfractionated blood and in the
gradient (r, = 0.04); neither was there a significant
correlation between neutrophil counts before and after
gradient separation (r, = 0.20). The only valid explanation for the lower percentages of lymphocytes,
therefore, is that they are chiefly a consequence of
contamination with lower buoyant density neutrophils.
Currie et a1 (4) qnd Check et a1 (5-7) found
similar alterations in the Ficoll-Hypaque gradients of
cells obtained from patients with cancer. Their data
were explained by variations in the absolute lymphoTable 3. Correlation between clinical activity of acute rheumatic
fever and lymphocyte :neutrophil ratios (L: N)
Clinical
parameter*
Joint count
Signs of carditis
Previous attack
Temperature, "C
Heart rate, beats per minute
kSR (Westergren), mdhour
Seromucoid (Winzler), mg%
White blood cell count,
x 103/4
Antistreptolysin 0 titer, Todd
units
Jones criteria
Major
Minor
Higher L:N
(n = 5)
2.6
f
2.3
Lower L:N
(n = 5)
5.4
f
3.1
3
2
4
3
37.1 2 0.7
92 12
58 ? 28
7.9 2 4.3
6.3 f 0.8
*
37.6 +. 0.9
92 f 15
71 f 24
9.8 2 1.9
1 1 .o ? 3.4t
345 +. 317
950 f 960
*
3.8 2.3
1.4 f 0.5
2.4 f 1.5
5.6 f 1.5$
1.6 f 0 3
4.0 1.3$
*
* Values for signs of carditis and previous attack are the numbers of
patients; all other values are the mean 1 SD. ESR = erythrocyte
sedimentation rate.
t P < 0.004 versus higher L : N group, by Mann-Whitney U-test.
$ P < 0.03 versus higher L : N group, by Mann-Whitney U-test.
*
1340
cyte counts before gradient separation, changes in
lymphocyte recovery, and increased retention of immature myeloid cells. In patients with SLE, RA, and
ARF the explanation must be different, since there is a
lack of important lymphocyte alterations and there are
high numbers of mature. neutrophils in the gradient
interface. Decreased buoyant density of neutrophils is
a better explanation for our findings.
Experiments in which normal cells were incubated with patient plasma showed that changes in
neutrophil buoyant density can be induced by humoral
factors. These changes are accompanied by an increase in cellular volume, as demonstrated by the
experiments with the cell channelyzer. The absence of
neutrophil aggregates on direct microscopic examination, and in the higher cell volume channels of the
ZBI-Coulter counter, provide evidence against the
hypothesis that neutrophil aggregation would explain
high L:N ratios. Chemotactic factors in the serum
can induce aggregation of neutrophils, but these
changes are reversible within 8 minutes (17) and would
not be expected to influence our results.
We have attempted to characterize plasma factors that could induce change in neutrophil buoyant
density in vitro. Endogenous corticosteroids are unlikely candidates, since hydrocortisone did not induce
any change. We looked for factors that activate
newtrophils and are known to be present in the plasma
of patients with rheumatic diseases. The first candidates for such studies were circulating immune complexes, since immune complexes have been reported
to be a common finding in these patients (18). Normal
human serum incubated with aggregated gamma globulin induced detectable changes in the L:N ratio in
7 of 7 samples of normal cells studied (Figure 4).
These changes were similar to those obtained with
patient plasma, and could be multifactorial. Therefore,
in one group of experiments we studied the effect of
isolated AHG (in the absence of complement activation), and in another group we investigated the activation of complement by a different mechanism, the
alternative pathway.
Figure 5 shows that AHG can induce a shift in
the L:N ratio even with EDTA concentratiops that
completely block the activation of both the classical
and alternative pathways of complement. Previous
passage through the Sephadex column would exclude
most contaminants that could influence cell buoyant
density. In addition, nonaggregated gamma globulin
did not influence the L:N ratios of normal cells (data
not shown). We conclude that neutrophils undergo a
HACBARTH AND KAJDACSY-BALLA
decrease in cell buoyant density after interaction with
aggregated gamma globulin. Whether this is the effect
on a small subpopulation or on the majority of the
neutrophils is not known, and we are conducting
studies to investigate this further. Many effects of
immune complexes and AHG on neutrophils have
been described, including release of enzymes (19-23),
adherence to surface (19,20,22), phagocytosis (21,23),
and stimulation of the hexose monophosphate shunt
(23). This is the first report of a decrease in neutrophil
buoyant density induced by antibody molecules.
A more dramatic shift in the L:N ratio was
detected when normal cells were incubated with
autologous serum activated by inulin (Table 2).
Goldstein et a1 (24) have shown that activation of the
alternative pathway of complement can induce
lysosomal enzyme release from human leukocytes.
Activation of complement can also induce a change in
the sedimentation behavior of responding granulocytes, a finding that may depend on factors such as cell
volume, density, shape, and deformability (25).
Pember et a1 (26) have shown that decreases in the
density of polymorphonuclear leukocytes of murine
peritoneal exudate and of human peripheral blood
could be induced in vitro by exposure of the cells to
endotoxin-activated serum. A decrease in the buoyant
density of polymorphonuclear leukocytes was also
demonstrated in vitro by those authors, using the
synthetic chemotactic peptide N-formyl-methionylleucyl-phenylalanine .
Considering that activation of complement has
been documented in SLE, RA, and ARF (27-30), the
abnormal numbers of low density neutrophils found in
the peripheral blood of our patients could have been,
at least in part, a consequence of complement activation. Lewis et a1 (31) have recently shown that
hemodialysis may induce similar neutrophil density
changes; this finding was ascribed to complement
activation by hemodialysis membranes.
We hypothesize that the neutrophils in the
peripheral blood of patients with the diseases we
studied are activated, by immune reactants such as
immune complexes and complement, to degranulate,
increase their cell volume, and decrease their buoyant
density. Loss of buoyant density has been associated
with degradation when neutrophils are exposed to
synthetic chemotactic agents. Experiments such as
those shown in Figure 6 suggest that these changes do
not occur only in a small subpopulation of neutrophils,
but that a major shift in the average volume of
neutrophils is seen.
LOW DENSITY NEUTROPHILS
Neutrophil buoyant density may be correlated
with disease activity in acute rheumatic fever (Table
3). We have not studied this correlation in other
diseases, but values such as L :N ratios may prove to
be useful as parameters of disease activity in rheumatic diseases. This may be expected in situations in
which complement activation, immune complexes, and
neutrophil activation play a role in disease mechanisms.
Granulocyte contamination of Ficoll-Hypaque
gradients for separation of mononuclear cells in blood
samples from patients with SLE, RA, and ARF is not
always taken into consideration when studies of lymphocyte function are done in such patients. High levels
d contamination may invalidate the results of many
studies that have been conducted without taking into
account differences in the purity of cell separation
between patients and normal controls. Even some
of our samples from normal individuals contained
large numbers of neutrophils. Neutrophil function
studies in these patients may also need to be reexamined: neutrophils isolated from the lower portion of
the gradient are not altered as much as the neutrophils found in the mononuclear cell layer, which are
usually discarded by investigators interested in neutrophil responses.
We report here, for the first time, that neutrophils of low buoyant density may be found in patients
with SLE, RA, and ARF. These neutrophils have been
stimulated by humoral factors present in the plasma of
these patients. Aggregated gamma globulin and activated complement have been shown to be possible
factors, but other factors may be present also. Recognition that stimulated cells are present in circulation,
and possibly present in tissue, will help in the interpretation of studies of neutrophil function in patients
with these diseases.
ACKNOWLEDGMENTS
The authors wish to thank Dr. Neil F. Novo and
Yara Juliano for statistical assistance, Drs. Emilio Granato
and Paul Horan for methodologic advice and assistance, Dr.
Aparecido B. Pereira and Doreen Lunberg for reviewing the
manuscript, and Paulina Buleck, Arlene Porter, and Patricia
Cromartie for secretarial assistance.
REFERENCES
1. Boyum A: Isolation of mononuclear cells and granulo-
cytes from human blood. Scand J Clin Lab Invest
[Suppl] 21 :7-89, 1968
2. Aiuti F, Cerottini JC, Coombs RA, Cooper M, Dickler
1341
HB, Froland S, Funderberg HH, Greaves MF, Grey
HM, Kunkel HG, Natvig J, Preud’Homme JL,
Rabellino E, Ritis RE, Rowe DS, Seligman M, Siegal
FP, Stjernsward J, Terry WD, Wybran J: Identification,
enumeration and isolation of B and T lymphocytes from
human peripheral blood. Clin Immunol Immunopathol
3584-597, 1975
3. Zucker-Franklin D: The percentage of monocytes
among “mononuclear” cell fractions obtained from normal human blood. J Immunol 112:234-240, 1974
4. Currie GA, Hedley DW, Nyholm RE, Taylor SA: Contamination of mononuclear cell suspensions obtained
from cancer patients by the Boyum method. Br J Cancer
3835-556, 1978
5. Check IJ, Hunter RL, Lounsbury B, Rosenberg K,
Matzk G: Prediction of survival in head and neck cancer
based on leukocyte sedimentation in Ficoll-Hypaque
gradients. Laryngoscope 90: 1281-1290, 1980
6. Check IJ, Hunter RL, Rosenberg KD, Herbst AL:
Prediction of survival in gynecological cancer based on
immunological tests. Cancer Res 40:4612-4616, 1980
7. Check IJ, Hunter RL, Karrison T, DeMeester TR,
Golomb HM, Vardiman J: Prognostic significance of
immunological tests in lung cancer. Clin Exp Immunol
43:362-369, 1981
8. Robertson AJ, Gibbs JH, Potts RC, Brown RA, Milne
MK, Lawson JIM, Beck JS: Effect of anaesthesia and
surgery of the pre-S-phase cell cycle kinetics of mitogenstimulated lymphocytes of previously healthy people. Br
J Anaesth 55:339-347, 1983
9. Gibbs JH, Robertson AJ, Brown RA, Potts RC, Murdoch JC, Steward WK, Becks JS: Mitogen-stimulated
lymphocyte growth and chronic uraemia. J Clin Lab
Immunol9:19-25, 1982
10. Tan EM, Cohen AS, Fries J, Masi AT, McShane D,
Rothfield NF, Schaller J, Tala1 N, Winchester R: Criteria for the classification of systemic lupus erythematosus (proposed 1982 revision) (abstract). Arthritis Rheum
(suppl) 25:S3, 1982
11. Blumberg BS, Bunim JJ, Calkins E, Pirani CL, Zvaifler
NJ: ARA nomenclature and classificationof arthritis and
rheumatism (tentative). Arthritis Rheum 7:93-97, 1964
12. Jones Criteria (Revised) for Guidance in the Diagnosis of
Rheumatic Fever. New York, American Heart Association, 1965, p 8
13. Wintrobe MM: Clinical Hematology. Eighth edition.
Philadelphia, Lea & Febiger, 1981, pp 22-23
14. Yam LT, Li CY, Crosby WH: Cytochemical identification of monocytes and granulocytes. Am J Clin Pathol
55:283-290, 1971
15. Siege1 S: Nonparametric Statistics for the Behavioral
Sciences. New York, McGraw-Hill, 1956
16. Blum JR, Fattu NA: Nonparametric methods. Rev Educ
Res 24:467487, 1954
17. O’Flaherty JT, Kreutzer DL, Ward PA: Neutrophil
1342
18.
19.
20.
21.
22.
23.
24.
aggregation and swelling induced by chemotactic agents.
J Immunol 119:232-239, 1977
Theofilopoulos AN, Dixon FJ: Immune complexes in
human diseases: a review. Am J Pathol 100529-594,
1980
Henson PM: The immunologic release of constituents
from neutrophil leukocytes. I. The role of antibody and
complement on nonphagocytosable surfaces or phagocytosable particles. J Immunol 107:1535-1546, 1971
Henson PM: The immunologic release of constituents
from neutrophil leukocytes. 11. Mechanisms of release
during phagocytosis, and adherence to nonphagocytosable surfaces. J Immunol 107:1547-1557, 1971
Leffel MS, Spitznagel JK: Intracellular and extracellular
degranulation of human polymorphonuclear azurophil
and specific granules induced by immune complexes.
Infect Immun 10:1241-1249, 1974
Henson PM, Johnson HB, Spiegelberg HL: The release
of granule enzymes from human neutrophils stimulated
by aggregated immunoglobulins of different classes and
subclasses. J Immunol 109:1182-1 191, 1972
Henson PM. Oades ZG: Stimulation of human neutrophils by soluble and insoluble immunoglobulin aggregates. J Clin Invest 56:1053-1061, 1975
Goldstein IM, Brai M, Osler AG, Weissmann 0:
Lysosomal enzyme release from human leukocytes:
mediation by the alternative pathway of complement
activation. J Immunol 1 1 1:33-37, 1973
HACBARTH AND KAJDACSY-BALLA
25. Catsimpoolas N, Kurts SR, Skrabut EM, Griffith AL,
Valeri CR: Cytotaxins alter the sedimentation behavior
of human granulocytes. Science 205;93&937, 1979
26. Pember SO, Barnes KC, Brandt SJ, Kinkade JM: Density heterogeneity of neutrophilic polymorphonuclear
leukocytes: gradient fractionation and relationship to
chemotactic stimulation. Blood 61:1105-1115, 1983
27. Zvaifler NJ: Breakdown products of C’3 in human
synovial fluids. J Clin Invest 48: 1532-1542, 1969
28. Perrin LH, Lambert PH, Miescher PA: Complement
breakdown products in plasma from patiehts with systemic lupus erythematosus and patients with membrane
proliferative or other glomerulonephritis. J Clin Invest
56: 165-170, 1975
29. Svartman M, Potter EV, Poon-King T , Earle DP: Immunoglobulins and complement components in synovial
fluid of patients with acute rheumatic fever. J Clin Invest
56~111-117, 1975
30. Kaplan RA, Curd JG, DeHeer DH, Carson DA,
Pangburn MK, Miiller-Eberhard HJ, Vaughan JH: Metabolism of C4 and factor B in rheumatoid arthritis:
relation to rheumatoid factor. Arthritis Rheum 23:
911-920, 1980
31. Lewis SL, van EPPs DE, ( h ~ ~ o w e t hDE: Density
changes in leukocytes following hemodialysis or exposure to chemotactic factors. Am J Nephrol 6:232-239,
1986
Документ
Категория
Без категории
Просмотров
0
Размер файла
781 Кб
Теги
lupus, neutrophils, patients, low, feve, rheumatic, systemic, arthritis, erythematosus, acute, density, rheumatoid
1/--страниц
Пожаловаться на содержимое документа