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Granulocyte-Macrophage
of the CDI lb Surface
By Mark
Colony-Stimulating
Adhesion
Molecule
A. Socinski,
Stephen
A. Cannistra,
Lowell
Richard
Schnipper,
and
The CD1 1 b (Mol) molecule
is a member
of a family
of
surface
glycoproteins
that
are
essential
for adhesiondependent
granulocyte
functions.
Brief exposure
of granulocytes
to human
granulocyte-macrophage
colony-stimulating
factor
(GM-CSF)
in vitro
increases
the
surface
expression
of CD1 1 b and increases
granulocyte
adhesiveness.
To assess
the possible
in vivo significance
of these
observations
we studied
the effect
of GM-CSF
on CD1 1 b.
CD1 1 a (LFA-1 ), and CD1 1 c (gp 1 50. 95) expression
on
granulocytes
from nine adult patients
with sarcoma
who
were
receiving
GM-CSF
as part of a phase
I trial. GM-CSF
was administered
as a continuous
infusion
at a dose of 32
or 64 g/kg/d.
Granulocyte
CD11b.
CD11a,
and
CD11c
expression
was
determined
by indirect
immunofluorescence
staining
of whole
blood.
thereby
minimizing
in vitro
manipulation.
A transient
leukopenia
developed
within
15
minutes
of initiation
of GM-CSF
treatment
that was associated
with
a marked
increase
in the surface
antigen
density
of CD1 1 b. A mean
1 .7-fold
increase
(P = .001 ) in
the percentage
of CD1 1 b-positive
granulocytes
and a mean
2.1 -fold
increase
(P = .002) in CD1 1 b surface
antigen
den-
H
UMAN
GRANULOCYTE-macrophage
colony-stimfactor
(GM-CSF)
is a glycoprotein
produced
T lymphocytes,
endothelial
cells,
and fibro-
ulating
activated
by
blasts.’
of the cDNA
production
Isolation
and the subsequent
CSF
(rGM-CSF)
factor
on human
was
hematopoietic
for
originally
marrow
progenitor
CSF
cyte
also augments
functions.
These
certain
effects
phil
phagocytosis,3
chemotaxis,4
of the effects
cells,
mature
include
it is clear
that
granulocyte
enhancement
been
shown
to rapidly
induce
for
tial
adhesion-dependent
of three
consists
and CDI lc
noncovalently
ited
of
proteins
these
of granulocyte
increase
the
impairment
that
minutes
CD1
been
has
lb
surface
shown
density
sion.’#{176}This
is accompanied
aggregation
in vitro.
GM-CSF
an
Blood.
agent
is currently
to
Vol 72,
(CDI
adhesion
surface
ameliorate
No 2 (August),
la
[LFA-l],
are
functions.
expression
little
CD1
by an increase
receiving
The results
effects
on the expression
From
the
evaluated
chemotherapy-induced
Department
1988:
pp 69 1-697
effects
adhesion
of
as part
GM-CSF
of CD1
in vivo
proteins
of
in vivo,
conse-
infusion
of
in sarcoma
a phase
I toxicity
in vivo has dramatic
lb in all patients.
AND
in
expres-
trials
myelosup-
Department
METHODS
Immunology
Division
Beth
ofMedicine,
reprint
and
Boston
1 1. 1988;
requests
“advertisement”
© I 988
This
article
in accordance
must
with
Dana
Hospital;
Department
of
the Section
Hematology
of
Medical
April
/9,
D. Griffin,
therefore
/8 U.S.C.
School.
1988.
MD,
Tumor Immunology,
Dana-Farber
Cancer Institute.
Boston, MA 02115.
The publication
costs ofthis article were defrayed
payment.
Medicine,
Hematology-Oncology.
University
accepted
to James
and
of
Israel
MedicalSchool;
January
Address
Tumor
Medicine.
Harvard
Submitted
charge
as
of
Institute,
of
Medicine,
the
on
in granulocyte
in clinical
GM-CSF
show that
Division
Cancer
indicate
being
the
function
adverse
Nine adult patients
with localized
unresectable
or
sarcoma
were studied.
None of the patients
had prior
chemotherapy
or bone marrow
involvement
as judged
by bone
marrow
aspirate
and biopsy specimens.
All patients
had normal
WBC counts (mean ± SEM, 7.6 ± I .4 x 103/,L) at the initiation
of
treatment.
No patient had clinical evidence
of infection
during the
study, and none was studied
within 2 weeks of surgery.
Informed
consent was obtained
from all patients
in accord with the Institutional
Review
Board for Human
Experimentation
of the DanaFarber
Cancer
Institute
and the Beth Israel Hospital.
Normal
Farber
In
as 15
increase
la
we evaluated
on granulocyte
patients
study.
lb.
with
adhesiveness.
for as little
on CD1
ofgranulocyte
could
have
as
‘ 2 Conversely,
of CD1 lb
in a 2.8-fold
effect
augmentation
aggregatability,
family
associated
granulocyte
to GM-CSF
to result
with
such
This
possess
distinct
a subunits
fi subunit (CDI8).
Inher-
molecules
mature
granulocytes’3
enhance
vitro exposure
of granulocytes
of a granulo-
functions
Antman,
Patients.
cell-
chemotaxis.”
[gp 150, 95])
that
linked
to a common
deficiencies
stimuli
surface
and
Karen
metastatic
(CD1 lb, Mol).’#{176}CDI lb is a
glycoproteins
that are essengranulocyte
aggregation,
phagocytosis,
expression
Elias,
MATERIALS
and monoof neutro-
antibody-dependent
surface
Since
GM-
mediated
cytotoxicity,5
degranulation
and superoxide
production
in response
to f-Met-Leu-Phe,6’7
as well as enhancement of monocyte
cytotoxicity
in vitro.8’9 GM-CSF
has also
cyte adhesion
protein
in vitro
member
of a family
of surface
Anthony
Expression
in vivo
sity was noted
after 1 2 hours of treatment.
No change
in
CD1 1 a or CD1 1 c expression
was observed
over the first 12
hours.
The level of CD1 1 b expression
was followed
in six
patients
for up to 5 days
of treatment
with
GM-CSF.
Compared
with the 1 2-hour
value.
three
of six patients
showed
a subsequent
decrease
in CD1 1 b expression.
two
remained
constant,
and one showed
a continued
increase
in CD1 1 b surface
density.
Fluorescence-activated
cell sorting of granulocytes
into highand low-density
CD1 1 bpositive
groups
revealed
a preponderance
of immature
myeloid
forms
in the low-density
CD1 1 b fraction.
which
suggests
that the late decrease
in CD1 1 b expression
in
some
patients
may be related
to a greater
proportion
of
circulating
immature
myeloid
forms
in the peripheral
blood.
This study suggests
that GM-CSF
administered
as a continuous infusion
rapidly
upregulates
the expression
of granulocyte
CD1 1 b in vivo. The influence
of this phenomenon
on
in vivo granulocyte
aggregation
may be clinically
relevant
with
regard
to the toxicity
of GM-CSF
and deserves
further
investigation.
S 1988
by Grune
& Stratton,
Inc.
quences,
GM-CSF
of this
Induces
the
Granulocytes
D. Griffin
particularly
cells.2 Although
this cytokine
proliferative
effect
on bone
its
named
hematopoietic
the study
Sullivan,
James
pression.
encoding
for human
GM-CSF
of purified
recombinant
GM-
has allowed
Factor
on Human
be
section
Division
of
44 Binney
St,
in part by page
hereby
marked
1 734 solely
to
this fact.
by Grune
& Stratton.
Inc.
0006-4971/88/7202-0033$3.00/0
691
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
692
volunteers
consent.
SOCINSKI
donating
blood
for some
experiments
also gave
informed
Administration
of GM-CSF.
Patients
received
purified
rGMCSF (Sandoz
Research
Institute,
East Hanover,
NJ) by continuous
infusion through a central venous catheter
on a protocol assessing
the
effects of GM-CSF
on chemotherapy-induced
myelosuppression.
The GM-CSF
was administered
in a preservative-free
normal saline
solution.
The specific activity of the material
used in this study was
8.0 x lO6U/mg
of protein
as determined
in vitro in a rapidproliferation
assay that measures
incorporation
of [3H] thymidine
by
GM-CSF-responsive
myeloid
cells obtained
from patients
with
chronic myelogenous
leukemia.’4
In the initial phase of this protocol
rGM-CSF
was administered
as the sole agent for up to 7 days to
assess dose response
and toxicity.
All patients
were studied during
this phase of continuous
infusion of GM-CSF
and were not exposed
to chemotherapy
prior to or during the study period. Five patients
(1
through
5) received
GM-CSF
at 32 ig/kg/d
and four patients
(6
through 9) received 64 tg/kg/d.
Immunofluorescence
analysis.
Peripheral
blood was collected
from patients
and normal controls
by venipuncture,
collected
into
heparinized
plastic
syringes,
and placed
immediately
at 4#{176}C
in
polystyrene
test tubes.
Cell surface
antigens
were detected
by
indirect
immunofluorescence
using a method to stain leukocytes
in
whole (unseparated)
blood (Coulter
Immunology,
Hialeah,
FL).
The whole blood technique
was used to avoid any in vitro manipulation that would activate
granulocytes.
Briefly, 100 zL of whole blood
was incubated
at 4#{176}C
with monoclonal
antibody
for ten minutes; this
was followed by two washes in phosphate-buffered
saline (PBS). The
cells were then further incubated
with fluoresceinated
antimouse
Ig
(Coulter
Immunology)
for five minutes
at 4#{176}C
followed
by two
additional
washes in PBS. Lysis of erythrocytes
was then accomplished using the Coulter
lysis reagent.
The leukocytes
were then
fixed by using a formaldehyde-methanol-based
fixative and subsequently
analyzed
by flow cytometry
in a Coulter
EPICS-C
flow
cytometer.
Granulocyte
“windows”
were set on the basis of 90#{176}
light
scatter, and only this fraction was analyzed
for surface fluorescence.
The cells in this window
were shown to be greater
than 95%
granulocytes
by sorting into collection
tubes and examining
cytocentnifuge smears following
Wnight-Giemsa
staining.
Also, the cells in
the granulocyte
window were shown to lack reactivity
(less than 5%)
with
monoclonal
antibodies
identifying
other
lineages.
These
included
anti-Mo2
(CDI4,
monocytes),’5
anti-T3
(CD3, T lymphocytes),”
and anti-BI
(CD2O, B lymphocytes).’7
CDI lb was detected
with monoclonal
antibody
904’s and CD1 Ia detected
with monoclonal antibody
2F12 (the latter kindly provided by Dr Jerome Ritz,
Dana-Farber
Cancer
Institute,
Boston).
CDI Ic was detected
by
monoclonal
anti-Leu-M5
(Becton Dickinson,
Mountain
View, CA).
Other
antibodies
tested included
anti-B73.l,’9
anti-MYl,#{176} antiMY4,2’ anti-MY7,2’
anti-MY8,2’
anti-MY9,
and
All antibodies
were used at saturating
concentrations
and were
obtained
from the original
investigators.
In one experiment
the
CDI 1 b-positive
granulocytes
were separated
into high- and lowdensity
CD1 1b fractions
by fluorescence-activated
cell sorting.
Cytospin
preparations
of the fractions
were subsequently
stained
(Wright
Giemsa),
and 200 cell differential
counts were performed
by three individuals.
Estimation
of change
in surface
antigen
density.
Fluorescence
intensity
measured
by flow cytometry
is proportional
to the number
of fluorescein
molecules
bound per cell. When a linear amplifier
is
used, fluorescence
intensity is linearly related to the channel number
on the flow cytometer.24
Estimation
of the amount
of increase
in
surface
antigen
expression
was performed
by comparing
the peak
fluorescence
intensity
of known monoclonal
antibodies
for which the
exact number
of binding
sites per cell had been previously
deter-
ET AL
mined.25 For this purpose,
purifed T cells were obtained
by standard
sheep cell rosetting
techniques.’4
A standard
curve relating
logarithmic to linear fluorescence
was constructed
by using anti-T4
(IgG2)
and anti-T8 (IgG2) monoclonal
antibodies.
For anti-T4 and anti-T8,
relative
linear fluorescence
intensities
of 3.0 and 6.0 (arbitrary
units),
respectively,
were assigned
on the basis of the known
1:2
relationship
of the number of binding
sites per cell.25 CDI lb peak
channel
fluorescence
measurements
were then converted
from a
logarithmic
scale to a linear scale, thereby allowing accurate
assessment of relative changes in surface antigen density.
In vitro
studies.
The in vitro effects of rGM-CSF
on human
granulocytes
were examined
by adding rGM-CSF
(obtained
as
media conditioned
by Chinese
hamster
ovary (CHO)
cells transfected with a full-length
cDNA clone of the human GM-CSF
gene,
kindly provided
by Drs Steven Clark and Gordon
Wong, Genetics
Institute,
Cambridge,
MA) to whole blood aliquots
at a dilution
of
1:500. Peripheral
blood was incubated
with GM-CSF
for 20 minutes
at 37#{176}C.
Controls
included peripheral
blood incubated
with “mock”
CSF (media harvested
from CHO cells transfected
with the same
vector
lacking the GM-CSF
cDNA)
or with media alone for 20
minutes at 37#{176}C.
Cell surface
staining
with monoclonal
antibodies
was then performed
as described
earlier.
RESULTS
Effects
CD!
of
lb
GM-CSF
on
expression
in
was
mock
individuals
rGM-CSF,
normal
granulocyte
Whole
vitro.
incubated
CSF, and
CDJ
blood
exposure
when
to GM-CSF.
compared
mock CSF
P = .013).
with
This
mature
expression
CSF-treated
whole
blood,
or
alone.
Importantly,
change
in overall
significantly
granulocytes
the surface
incubation
was
analyzed
by the
2.3 ± 0.17-fold
different
incubated
.013) or media
alone
there was no significant
(1.3 ± 0. 15,
In contrast,
of CD1
cell
and
normal
at 37#{176}Cfor 20 minutes
with
media
alone. The surface
expres-
sion of CD1 lb on mature
granulocytes
was
whole blood lysis technique
and increased
after
la
from
size
Ia in either
the
with
(1.3 ± 0. 12,
increase
in
CM-CSF,
mock
whole
blood
exposed
there
was
no significant
(estimated
the
by
light
to
scatter
profile)
after exposure
to GM-CSF,
mock
CSF,
or media
alone,
which
showed
that the increase
in CDI lb expression
was not due to an overall
increase
in cell surface
area.
Effects
of
CDllb
GM-CSF
in
on granulocytes
GM-CSF
in nine
significant
9% v 89%
CD11b
±
5%,
(mean
hours of treatment
overall
cell size
expression
in Table
1. There
lb-positive
.0001)
as well
as surface
0.22;
la or CD!
±
light
P
.002)
of CD1
la- or CD1
significantly
after
dose-response
surface
effect
expression
at 32 tg/kg/d
.tg/kg/d
(2.42
1 2 hours
There
with
when
did
12
of B73.l,
not change
of
treatment
appear
to be a significant
to fold
increase
regard
v the
=
lc-positive
not
comparing
(1 .76 ± 0.13)
± 0.42,
P
of
after
There
was no change
in
scatter).
The surface
In one patient,
the surface
expression
MY7,
MY8,
MY9,
and 80H.5
did
shown).
a
±
lc on mature
granulocytes
after
GM-CSF
did not significantly
with
nor did the percentage
not
was
expression
=
granulocytes.
MY1,
MY4,
(data
of
post-
(53%
granulocytes
2.05
with GM-CSF.
(estimated
by
of treatment
change,
is depicted
increase,
ofCDl
12 hours
=
P
expression
12 hours
in CD1
fold
surface
and
patients
increase
The
vivo.
pre-GM-CSF
,.l4).
with
GM-CSF
in CD I I b
the five patients
four patients
Since
the
treated
GM-CSF
treated
at 64
was
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GM-CSF
INDUCES
693
CD1 18
Table
1 . CD1 lb Surface
Expression
Relative
Case No.
Mean
Fluorescence
1.9
50
96
17
71
3
0.50
0.76
1.5
20
60
4
0.76
1.65
2.2
85
99
5*
0.60
1.04
1.7
56
95
6
0.90
2.00
2.2
80
98
7
0.50
0.82
1.6
28
88
8
0.84
1.90
2.3
87
99
9
0.63
2.30
3.6
50
98
SEM
0.64
relative
±
0.05
1.36
fluorescence
linear
granulocytes
in normal
by flow
saline,
it is unlikely
the
studies
did not show a significant
were exposed
to a normal
saline
the
the
patients,
count,
and
after
were analyzed
24 hours
after
In one patient
analyzed
at
(no.
initiation
at 15,
initiation
3) only
I 5, 60,
and
CD1
30,
effect
on
effect
(media
of GM-CSF
infusion,
±
9
89
expres-
The
the
count
value.
effect
total
WBC
la,
ter.
12
on
and
decreased
and
to less than
control
10% of the pretreat-
this
the total
phenomenon
WBC
began
and granulocyte
over the pretreatment
values.
Figure
in surface
antigen
density
of CD1 Ib,
CD1 Ic in patients
of CD1 lb increased
However,
the
surface
no. 4 and
maximally
expression
over the pretreatment
no. 3 a 1.5-fold
expression
and
and Methods.
5. The
at 60
surface
minutes
initiation
of GM-CSF
treatment
(mean
foldof 6.05 ± 0.8), with a subsequent
decrease
thereaf-
increased
In patient
at the
5
(isotype-identical
30 to 60 minutes
counts
had increased
lB depicts
the change
CDI
in the Materials
fluorescence
and by six hours
reverse,
±
.0001
treatment.
Within
to
as described
Background
ment
following
increase
as
of fluorescence
granulocyte
60, and 90
of GM-CSF
total WBC
count
and total granulocyte
count
in one of
patients
(no. 5) is shown
in Fig IA. Within
15 minutes
initiation
53
immunofluorescence.
expression
as well
treatment.
peak channel
count,
total
of CDI ib,
lb surface
90 minutes
of GM-CSF
0.22
±
after 24 hours of GM-CSF
of administra-
(nos. 4 and 5) the total WBC
and
surface
expression
CDllc
well as
as
after
vehicle
the
indirect
alone.
granulocyte
hours
of the
tht
The in vitro
granulocytes
was
from
using
tion.
when
solution
a result
obtained
cytometry
of CD 1 1 b measured
expression
2.05
P-
intensity
determined
0.20
±
.002
was
treatment.
Post-GM-cSF
1.5
lb expression
sion
()t
Granulocytes
Pre-GM-SF
1.00
CDI
CDI1a,
minutes
Fold Increase
0.76
represent
In two
CD1 lb-Positive
Post-GM-C5F
0.54
administered
alone)
Treatment
0.50
has been subtracted.
4 and 5 had the surface
Patients
12 Hour s Post-GM-CSF
1
positive
antibody)
and
lntensity
Pre-GM-CSF
PValues
Pr e-GM-CSF
2
±
tPcent
on Granulocytes
was observed
earlier
CD1
lc
GM-CSF
did
not
infusion.
changes
CD1 lc
at 12 hours,
points.
time
The
but no change
surface
change
during
Figure
2 shows
the
did increase
the
initial
the
the
of CD1
Ia
hours
of
24
pattern
of
surface
of CD1
percentage
9% pretreatment
from
was seen
expression
5. Although
in patient
no.
did not change,
granulocytes
of CD 1 1 b remained
level at 24 hours (Table
1).
increase
in CD1 lb surface
these
density
of
lc-positive
to 50% at 60
C)
minutes.
...
TOTAL
WEC
NEUOPHLS
I0
Surface
expression
ofCDl
lb and CDI la on granulocytes
was examined
for up to 5 days in six of the nine patients
studied.
Three
representative
the six patients,
200
day
MIN
3 (Fig
expression
3A),
two
days
1 to
expression
CDllb
B
the three
patterns
one showed
showed
showing
in Fig 3. Of
CD 1 1b expression
a constant
5 (Fig
occurred
patients
are shown
maximum
3C),
and
on day
1 (12
a decrease
on
increase
in CDI
in three
maximum
hours)
in CD1
(Fig
3E).
lb
Of
1b expression,
6
two
5
.
Ui-
CD11a
COlic
3
COlib
2
CSF
surface
15
30
60
90
24H
MIN
Fig 1 .
In vivo effect of GM-CSF
on total WBC and granulocyte
count (A) and on CD1 1 b, CD1 la, and CD1 ic surface
expression
(B)
during
the initial 24 hours of GM-CSF
infusion.
The points
in B
indicate
means
± SEM for two
separate
experiments.
surface
return
of
baseline
treatment.
CD 1 1b surface
expression
to the
36 hours after initiation
of GMother
patient,
a lesser decrease
in
observed
who showed
was
patients
expression
by
In the
expression
In the three
0
a
showed
pretreatment
ofCDl
lb after
ysis,
the CD 1 1b fluorescence
quent
days, thus suggesting
of cells. To investigate
tions
the high-density
CDI
centile)
was separated
(40%
either
the
histogram
reduction
constant
by day 5).
or increased
12-hour-interval
broadened
anal-
on subse-
the presence
of several
populathe nature
of these populations,
I b-positive
fraction
from the low-density
(upper
CDI
25th perI b-positive
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SOCINSKI
694
CD11b
CONTROL
CDI1a
CD11c
‘4
Hj
FIi ::1L::IL:i
FJ
0 MIN
15
MIN
30 MIN
60
MIN
90
MIN
B
Day
Totcl
Day
/1
13
\&7L
Neutrophils
,*.
C
IA 111I
L Iii U
LL
L
24 HRS
ET AL
. .
DENSITY
ANTiGEN
V1
Fig 2.
In vivo effect
of continuous
infusion
GM-CSF
on CD1 1 b.
CD1 1 a. and CD1 1 c surface
expression
on granulocytes.
Surface
antigen
density
is depicted
on the horizontal
axis. At 1 5 minutes,
the total WBC count was 0.7 x 1 O/isL
with
20% granulocytes.
which
made analysis
of large numbers
of granulocytes
difficult.
This
accounts
for the low
amplitude
of the histogram
at 15
minutes.
fraction
(lower
sorting
cell
analysis
Fig 4A
25th
percentile)
in patient
no.
by
7 on day
fluorescence-activated
5. The
one
of the presort
cell population
analyzed
along
with the windows
utilized
for
Figure
4B shows
populations
a comparison
of the
with
separation
postsort
clear
parameter
is shown
in
the cell sort.
CDI
low-density
of two
fraction
CD I 1 b-positive
a significantly
cells
lb-positive
higher
fraction
(metamyelocytes
and
(Table
band
forms)
was
fraction
compared
high-density
CD1
lb-positive
fraction.
These
and 3F, the number
of immature
the
of these
GM-CSF
35%
blood
CSF).
The
of three
with
surface
treated
There
expression
of
CD1
was
la
in four
treatment
patients
studied
to
expression
patients
examined.
of
0%
2%
patients
on subsequent
cells
CD1
the
granulocytes
suggest
circulating
with
to treatment
prior
5 days
with
la on mature
up to five days
no significant
on mature
the
in the
after
In one
there
in CDI
was
surface
that
Ia surface
expres-
be
activation,
by
critically
includadherence
to endothelial
receptor
nent
cells, and chemotaxis.26’27
CDI lb is also the
iC3b, an opsonic
fragment
of the third compocomplement
that can be fixed
to the surface
of
for
of
to act synergistically
IgG
to enhance
three
expressed
to
functions,
following
during
12 hours
protein
appears
adhesion-dependent
aggregation
also been shown
Fc portion
of
was
of the
in phagocyte
ing granulocyte
cell
monocytes
of
surface
a significant
is a unique
and
microorganisms
granulocytes
granulocytes
days,
increase
in
GM-
of GM-CSF
change
analyzed.
no major
lb (Mol)
involved
ultimately
accounting
for
of circulating
leukocytes
of CDI
with
data
treated
patients
increases
dramatically,
to 50% of the total number
(compared
in
with
of CD 1 1 b over
is partly
due
expression
of GM-CSF)
myeloid
other patients,
sion was seen.
on mature
In the two
DISCUSSION
immature
myeloid
cells poslb. As shown in Figs 3B, 3D,
of more
of CDI
the surface
expression
of CDI Ia
that occurred
on day 3 of treatment.
myeloid
seen
lb-positive
peripheral
the
increase
in
granulocytes
2). Conversely,
CD1
to increased
numbers
sessing
a lower density
with
immature
low-density
that the decrease
in in vivo surface
time (despite
continued
infusion
In vivo effect
of continuous-infusion
GM-CSF
adminisfive days on CD1 1 b surface
expression
on granulocytes
in three
patients
(A. C. and El. The day 1 value was measured
at 12
hours of GM-CSF
infusion,
day 2 at 36 hours. etc. The corresponding total
WBC
count
(Total)
and absolute
number
of mature
neutrophils
(Neutrophils)
and immature
peripheral
blood myeloid
forms
(Immature)
for each
patient
is shown
in B. D. and F.
Immature
myeloid
forms included
neutrophilic
bands, metamyelocytes.
myelocytes,
and promyelocytes.
populations
compared
of more
proportion
Fig 3.
over
tered
of the two
histograms
of cells
having
differing
relative
fluorescence
intensities.
Cytospin
preparations
of these
two populations
revealed
a
significantly
higher
proportion
of mature
granulocytes
in the
high-density
DAY
/N1ENS/TY
FLt5’E4CE
opsonized
complement
microorganisms.
autosomal
recessive
ence-related
depressed
functions
inflammatory
activation.”
Deficiency
disorder
and
iC3b
with receptors
phagocytosis
of
affecting
is characterized
responses
and
of CD1
granulocyte
lb
has
for the
serumis a rare
adher-
clinically
by
recurrent
infec-
tions.29
Given
the critical
role that
granulocyte
adherence-related
CD1 lb plays
in mediating
functions,
the factors
that
regulate
CD1 lb expression
may
granulocyte
function.
Peripheral
be important
in modulating
blood granulocytes
as well
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
GM-CSF
INDUCES
CD1
695
lB
decreases
in granulocyte
with
correlate
CD1
lb
on
on
in the
granulocytes
enhanced
adhesiveness
In addition,
to possess
mobilized
after
granulocyte
the
increase
activation.26
surface
f-Met-Leu-Phe,
inophore
A23187,
immediate
tude
effect
necrosis
surface
the
on
are
factor
lb
in vitro
include
C5A,
calcium
(TNF),
and
of CD1
of CD1
la.
lb
The
associated
with
could be inhibited
enhanced
aggregation
of granulocytes
by a monoclonal
antibody
directed
clinical
enhanced
increased
One
granulocyte
expression
such
GM-
is the
patients
dialysis
Table
2.
have
been
described
granulocytopenia
undergoing
membranes.’3
Differential
In
these
Cell Counts
Intensity
on
High density’
0.7
±
Lowdensity
‘High-density
tValues
3.3
and
represent
means
±
±
now
will
significantly
lb
on
mature
CD1
lb
was
seen
manly
SEM (%) of 200
cell differentials
Granulocytes
from
of
the damage
is produced
by treatment
it
of
directed
is
likely
that
±
30
forms
that
three
in
infusion
surface
vivo.
minutes
lung
of GM-
expression
The
of
of
increase
of
present
days,
blood
Previous
lb is not present
vasculature.
No
raised
33.0
5.7
45.3
by fluorescent-activated
12
the
during
forms
CD I I b
studies
have
on myeloid
with differentiation.34’35
induced
by GM-CSF
of sepsis.
observed
and
on the Basis
0.7
in
administration
in the peripheral
the authors
leukopenia
aggregation
investigators.
growth
receiving
I clinical
precur-
has been
previ-
Using
radionuclide-labeled
leukocytes,
this occurs secondary
to margination,
although
Sep arated
different
the
of this
in patients
of a phase
continuous
granulocytes.
CDI
±
0.6
±
Our
apparent
toxicity
the issue
data
may
be
margination
of Surface
P-.022
±
effects
These
immature
myeloid
lower
surface
density
of
Metamyelocytes/Bands
separated
the
in some patients,
and this phenomeattributable
to the dramatic
rise in
in the setting
transient
granulocyte
the
within
but is acquired
leukopenia
in the
toxicity
maximal
were
lb
animal
antibodies
observations,
granulocytes
mature
with
Transient
in
23.7
granulocytes
be reduced
increase
myeloid
P-.21
1.8
CD1
In
that
monoclonal
have
binding
in two of three patients
studied
and was
later in all nine patients.
On subsequent
ously reported.36
was shown that
12.6
can
that
report
CDI
Myelocytes
0.3t
CD 1 lb-positive
low-density
We
observed,
P=.21
in
cells.3’
increased
injury,
CD 1 1b expression
GM-CSF
as part
CSF
sor cells
cupro-
of the CD1 I b-P ositive
to
endothelium.27
we evaluated
trial.
also suggested
by
Promyelocytes
that
epithelium
these
in vitro,
compared
in which
patients
CD1 lb.32’33
the basis
of
shown
to enhanced
and
with
treatment
with GM-CSF.
were
shown
to have
a
that
against
new
against
On
the immature
magni-
observed
dialysis
sequesto be the
endothelial
pulmonary
CD1 lb level decreased
non was at least partially
no
aggregation,
possibly
mediated
of CDI lb, is thought
to be pathogenic.28
syndrome
hemodialysis
phane
syndromes
vein
granulocytes
granulocytes
to 24 hours
CDI1b.
Several
leads
endothelium
activated
GM-CSF
of the increase
is approximately
2.8-fold
and occurs
15 minutes.
The increased
expression
of CDllb
was
within
been
vein
has shown
TNF
umbilical
factor on granulocyte
continuous-infusion
to
with
by
cupro-
patients.3#{176} Complement-
also
of neutrophil-induced
expression
Arnaout
et al’#{176}
have
to GM-CSF
will
expression
expression
known
mediated
by the
enhanced
expression
of the CD1 lb molecule
in vivo may
have potentially
important
clinical
consequences.
Therefore,
in view of the ability
of GM-CSF
to stimulate
CD1 lb
pools
surface
that
complement
regard
to GM-CSF,
exposure
ofgranulocytes
the
increase
of CD1
ester,
tumor
CSF.’#{176}”3’26’27With
shown that in vitro
rapidly
Stimuli
expression
phorbol
intracellular
to the cell
study
by
to
by activated
monocytes
have been shown
of CDI
lb that can be rapidly
of
surface
is believed
in these
have
a recent
to pulmonary
as
lb
and pulmonary
to umbilical
induced
granulocytes
Fig 4.
CD1 1 b-positive
granulocytes
were
separated
on the
basis of surface
antigen
density.
(A) Presort
histogram
of the
CD1 1 b-positive
granulocytes
and the windows
utilized
for the
sort.
Fraction
1 represents
the lower 25th percentile.
and fraction
2 represents
the upper 25th percentile
of cells on the basis of
CD1 1 b surface antigen density.
(B) Postsort
histograms
showing
clear separation
of two
populations
of differing
CD1 1 b surface
antigen
density.
occurs
activated
expression
INTENSITY
be
produced
microaggregates
capacity
models
to
and
of the granulocytopenia.
Pulmonary
dysfunction
of hypoxemia
and hypocapnia
and a decrease
form
diffusing
minutes
expression
in CDI
is thought
Leukoaggregation
of granulocyte
mechanism
increase
components
membrane.
tration
FLUORESCENCE
granulocytes
within
in the surface
The
complement
phane
occur
increase
granulocytes.
expression
activated
counts
the peak
1.3
was
of potential
suggest
that
the
due
to enhanced
secondary
to the
Flu orescence
Intensity
Mature PMNs
54.0
±
1.0
P-.022
cell sorting.
it
pri-
27.7
±
3.3
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
SOCINSKI
696
rapid
increase
GM-CSF.
documented
CD 1 1 b surface
in
This
is similar
to
in the granulocytopenia
the
WBC
of
decreased
the peak value
of GM-CSF
treatment.
toxicity
of
is possible
GM-CSF
was
that
a second
given
that
granulocytes
have
microvasculature
potential
ig/kg/d
gain,
interest
and
a “capillary
Human
ties,
and
the
effect
pleural
and
two
four
in our study
and/or
within
endothelial
of
However,
(ie, sepsis),
the
pulmonary
damage.
patients
future
of this
trials
in humans
biologic
bone
monocyte
agent
edema,
effusions
consistent
marrow
functions
will
on
undoubtedly
vivo.
64
weight
with
focus
on
The
and neutrotoxicity
of GM-
of
enhanced
both
in vitro6
adherence
in both
the
cytotoxic
have
dde-
cells.27’#{176}GM-CSF
primates37
of superoxide
is believed
to mediate
by complement-stimulated
and
anion
endothelial
granulocytes.39
introduced
into
with
GM-CSF
response
and
These
theoretical
should
not
impede
clinical
efficacy
of GM-CSF.
awareness
humans38
by
granulo-
cell
damage
Stimuli
whose
a patient
cell damage.
heighten
their
potentially
to endothelial
in vivo
production
which
endotoxin
improve
could
activation
and
the
increases
Although
as well. Enhanced
expression
of CDI I b in
with enhanced
aggregation
of granulocytes
terious
effects
vitro correlates
and
fully.
on the surface
of mature
may serve to retain granuand
inflammation
granulocyte
cytes,
proteins
in vivo
by GM-CSF
at sites
been primed
inflammatory
quanti-
chemotherapy-induced
transplantation,
in
It is of
receiving
developed
pericardial
it
activated
leak” syndrome
after 5 days of treatment.
GM-CSF
can now be produced
in large
myelosuppression,
phil
stimulus
cause
that
of GM-CSF
in our study.
apparent
to be defined
of adhesion
granulocytes
function,
1b surface
has yet
in humans
noted at 60 minutes
following
No immediate
pulmonary
marginated
could
CSF
enhancement
locytes
CD1
from
initiation
mechanism
previously
of hemodialysis.’3
With
expression
count,
recovery
by
induced
expression
the
ET AL
such
granulocytes
as
have
may
lead to an exuberant
result
in enhanced
endothelial
concerns
trials
regarding
GM-CSF
designed
to
assess
the
serve
However,
our
results
should
of potential
toxic
effects
on the host.
to
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
1988 72: 691-697
Granulocyte-macrophage colony-stimulating factor induces the expression
of the CD11b surface adhesion molecule on human granulocytes in vivo
MA Socinski, SA Cannistra, R Sullivan, A Elias, K Antman, L Schnipper and JD Griffin
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