at. = 0 (Fo)/Fo] indicated the relative extent of membrane potential change. Azurophilic granule release. (a measure of degranulation) was assayed by elastase.
Neutrophil valency
functional
responses
Gregg R. Strohmeier, Elizabeth A. Simons Departments Ma.ssachusetts
of Biochemtstry
Abstract:
Ligand-induced
ceptors in their
(FcyR) on neutrophils stimulation, shown
responses
induced
(LICs)
and
and
by
was used changes tion cell have
previously
phils
respond
tions
of
immune
immune Ca2’ burst these
shown
re-
that
role the
complex concentration
(HICs) after their cytometry (IC)-elicited and initia-
in the occupancy.
subpopulations
same We
of neutro-
to su.bsaturating
saturating dosages stimulate the entire This discrepancy was not due to differin receptor occupancy. The magnitude of the
transient of HIC
HIC;
Ca2+ increase but depended
on
was independent the dose when
Kurt F. Seetoo, Center,
Boston
(FcyR).
of the dose an UC was
John
Resting
bind
the
Fc
School
neutrophils FcyRIII,
and
complex
Bernardo,
University
FcyRII
types
portions
Gary
J. WeiI,
of Medicine,
express both low
of IgG
immune
Boston,
only affinity
poorly
and
complexes
and
the FcyR receptors
those
(IC)
or
subthat
of antibody
aggregated
IgG
well [1-3]. Identification quiring the simultaneous
of functional occupancy
(i.e., cross-linking) valency immune
by the complex
multiple (HIC),
pancy of individual valency immune sued by numerous
receptors by the single Fc ends of low complex (LIC) molecules, has been purgroups and it has been demonstrated that
HICs
effective
are
[4-11].
concentra-
population. ences
on immune
(Ab)/antigen
complexes
([lAb) flow
simultaneously with FcyR
maximally
Fey
complexes
UCs in situ Multiparameter
of the oxidative and to correlate
of
Research
plays a significant here by contrasting immune
to measure in cytoplasmic
the Pulmonary
and
valency
valency
cross-linking to the cells.
addition
A. Brunkhorst,
cross-linking
by low
high
Beatrice
depend
more However,
at
the
PMN responses reof several receptors
Fc ends of a single high in contrast to the occu-
activating
requirement
PMN
than
LICs
of simultaneous
occu-
pancy of multiple receptor sites by a single activating gand rather than of a number of sites by unconnected valency stimuli to initiate all of the functional responses PMN has not of cross-linking
been fully activation
clarified. Although has been addressed
the
lilow of
mechanism by a number
used. As shown here, [JAb cross-linking elicited Ca2+ responses similar to those observed in HIC-stimulated cells. In contrast, UC elicited only minimal mtracel-
of investigators, the majority have chosen to cross-link the membrane proteins as antigens (i.e., using specific Abs to FcyRII and FcyRIII) [1, 12] rather than as modulating re-
lular
ceptors
EpH
and
no
tential changes accomplished
philic
oxidative
at by
all HIC
observed method,
tion
azurophilic
Fc’yR-mediated
cellular
potential,
Ca2+ and
slowly occupied.
J.
po-
by the prepara-
Thus,
pH,
concentration
can
to a lesser extent, Leukoc. Biol. 58:
and
by
two
approaches
Words:
lency
Fc receptors
stimulus
oxidntive
respouses
burst
cytoplusmic
receptor
intra-
occur, albeit more if single Fc’yR are 403-414; 1995.
.
flow
cross-linking
cylometry
.
cytoplusmic
elastase Vacalcium
are
the
Fc ends
of IC);
activation
likely
well
.
documented
the
mecha-
is achieved
to be very
2+
.
Abbrevmatmons:
by
different
for
these
[3].
neutrophils
and
B or
mntracellular
Jmt,
D;
2+
.
[Ca
cytochalasin
other
DCFH,
plex;
IC,
immune
IJAb,
low
after
addition immnune
valency
the
lndo-1,
complex;
Fc’yreceptor;
FcyR, buffered
ethyl)
5-(6)
complex
complex
pHi,,,
intracellularpli;
requests:
University
immnune
N’,
corn-
N’-tetraacetic
in situ
to cells;
by
LIC,
valency
neutrophils; PBS,
BCECF,
antibody
low
leukocytes,
albumin;
MCA,
diSC3(5),
N,
Ab,antibody;
serum
phosphate-
(carboxy
2’,7’-bis
(MeO-Sue-Ala-Ala-Pro-Val-methyl
3,3’-dipropylthio-dicarboeyanine;
3,3’-dipentyl-oxadicarbo-cyanine Reprint
bound
polymorphonuclear
carboxyfluorescein; arnide);
N,
cross-linked
was
,
bovine
fMLP,
valeney
1-[2-arnino-5-(6-carboxyindol-2yl)-
PMN
BSA,
saline;
high
ethane
immune
immune
CB/CD,
2’7’-dichloro-dihydrofluorescein;
phenoxy]-2-(2’-amino-5’-methylphenoxy) acid;
concentratmon;
HIC,
complex;
.
Ca
formyl-methionyl-leucyl-phenylalanine;
Boston
pH
via
neutrophil
been
coumarin
Key
activating
which
It has
some
for FcyR crosssuch as changes
intracellular
(i.e.,
nisms
in
effector functions such as and oxidative burst initia-
degranulation
membrane
membrane
was cross-linked, However, azuroby elastase re-
degranulation.
tion have an absolute requirement linking, whereas signaling functions in
or
in cells stimulated whereas the HIC
neutrophil
azurophilic
FcyR L/Ab.
as determined
degranulation,
lease, was not situ cross-linking triggered
burst
unless or by
(IiOC3(3),
iodide. Elizabeth
School
R.
Simons,
of Medicine,
Department
80 East
of
Concord
Biochemistry.
Street,
Boston,
MA
02118. Current
MA
Among leukocytes
the
address:
Pathology
INTRODUCTION agents
that
stimulate
human
polymorphonuclear
(PMN)
are
those
occupy
their
that
Fc’y receptors
Department,
02115. Current address:
125
Hartwell Received
Journal
Avenue, March
of
Leukocyte
Gregg
R.
Strohmeier,
20 Shattuck Beatrice
Thorn
A. Brunkhorst,
Lexington, 8, 1995;
Brigham Street,
revised
Biology
Fuji
and
Women’s
Building
Hospital,
1433,
Boston,
Immunopharinaceuticals,
MA 02173. June
Volume
2, 1995;
58,
accepted
October
June
1995
5, 1995.
403
cell nied
types that receptor-mediated stimulation by a transient rise in intracellular Ca2+
is accompaconcentration
linking
LIC
similar
to but
([Ca2]0t); the signal transduction mechanisms by which this is accomplished and the specific phospholipase that is activated depend on the specific receptor that is involved
by an
[13-17]. For neutrophils lation via the chemotactic
precross-linked
tions
of
we have peptide
already receptor,
shown with
that stimuconcentra-
formyl-methionyl-leucyl-phenylalanine
tor
with
Ab elicited
smaller
equivalent
dose
functions
philic
that
granules
elastase)
neutrophils
pended
in phosphate-buffered
[16,
18].
that stimulation by concentrations subsaturating to twice saturating
In contrast, of
also
we
HIC ranging led to uniform
NaCl,
equal Thus,
IC binding when HIC
but bound
their response to neutrophil
was FcyR
nil [11, 19, the propor-
tion of cells that responded depended on the dose, whereas the magnitude of that response in each cell, in terms of [Ca2+]jnm or of oxidative product formation, was dose independent. In contrast, preliminary studies with LIC mdicated
that
these
elicited
smaller population both the proportion
a lower
of cells of cells
[Ca2]nt
and
stimulated
a
to respond [1 1]. Furthermore, that responded and the magni-
to investigations
i[Ca2]1m, phils.
an early
step
in the
stimulus
response
suggested
that
rapid
26].
occurs
first
0.05-0.15
effector
U. This due
alkalinization Na/H To have situ
po-
[17,
masked
the
original
baseline
stimulated
neutrophils
on the
surface
of the
[24,
a final
by
(LIAb)
LIC
of Leukocyte
Biology
anti-BSA
stimulations
were
by using
probe,
(405
response,
a smaller
tnethod
OR)
transmembrane or degranulation.
Volume
58,
October
potenCross-
1995
described
nm)
was
Ab
valency
Chemical
major
peak
mass
material
because
of
this the
anti-BSA
of
dose
of 60
continuously
added
the
to
curve
jig/mI
gen-
LIC.
stirred
and
dose
addition
Ab
a precipitin
PMN
with
temperature
was
studies.
acetoxymethyl
ester
form
response
are
of
1-[2-
used
Initial
preincubated
VI (Sigma the by
ester
fluorescence cence
by
DCFH-IC
binding addition 02
(ex 450
nm)
(ex
500
was
nm/em
from 530
nm)
the nm)
by using
of the measured
same directly
by the
same
technique
the
a calibration
slope
of
generation [(Abs/s)(60
carboxyfluorescein as its
ratio
The
to 02’
PMN
BCECF
by c type
[1 1, 32].
5-(6)
cells
identical
of ferricytochrome
into
with
to oxi-
by observing yielded
was
Eugene, due
observation
ethyl)
loaded
published
increases
previously
of 1 jiM
calculated 530
the
to verify
Probes,
of DCFH-IC
reduction
as described
Probes)
nm/em
used
determined
generation
Abs)J/2 [32j. 2’,7’-bis (carboxy
was
of
a previously
Molecular
was
mm;
at 4#{176}C for 2 s) after
concentration pH
ratio
adaptation
M) was
using (530
Co.),
Molecular
Cytoplasmic
an
(10
a fluorescent
fluorescence
of absorbance change was converted nm of 02/min for 106 cells/mI
nm/U probe
at a final
by
IMLP
fluorescence
In addition,
Chemical
s/min)/(0.21 The pH (BCECF;
and
dismutase-inhibitable
maximal rate the equation:
[Cajint
ICs
(
