Journal
Antibodies
Against
Linda
A. Charles,
Departments
of Pathology
of Leukocyte
Granule Proteins In Vitro
Maria (LAG.,
Lucia R. Caldas, and J. Charles
M.L.R.C.,
J.C.J.)
Activate
Ronald Jennette
and Medicine
of Medicine,
(R.J.F.,
Chapel
Biology
J. Falk, R.S.T.),
(1991)
Neutrophils
Regina
University
50:539-546
S. Terrell,
of North
Carolina
School
Hill
Poiymorphonuciear leukocyte (PMN) respiratory burst was stimulated by heterologous antibodies against PMN granule proteins but not by control antibodies. Fluorescenceactivated cell sorter (FACS) analysis of activated PMN demonstrated the presence of two primary granule proteins, proteinase 3 (PR-3) and cationic protein 57 (CAP-57) at the membrane surface. The presence of myeloperoxidase (MPO) at the cell surface of primed and unprimed PMN was confirmed by immunoeiectron microscopy. Priming doses of recombinant tumor necrosis alpha (rTNFa) enhanced the rate of superoxide (02) production by these antibodies and increased the amount of surface protein accessible to these antibodies. Anti-neutrophil cytoplasmic autoantibodies (ANCA) with specificities for PMN granule proteins are present in patients with Wegener’s granulomatosis, polyarteritis nodosa, and idiopathic and crescentic glomerulonephritis. The demonstration that antibodies against granule proteins activate PMN supports the hypothesis that the vasculitis seen in these diseases is due in part to PMN mediated oxidative injury following PMN stimulation by ANCA. Key
words:
superoxide
anion,
vasculitis,
anti-neutrophil
INTRODUCTION Anti-neutrophil cytoplasmic autoantibodies (ANCA), first identified by Davies et a!. [5], are in the circulation of patients suffering from systemic vasculitis including Wegener’s granulomatosis and polyarteritis nodosa, and in patients
with
necrotizing
and
crescentic
phritis [4,8,10,12-16,18,21,23,25]. volved in the ANCA are orescence ANCA teinase
glomerulone-
ANCA
pathogenesis of these detected in serum by
may
processes. indirect
be in-
immunoflu-
microscopy [3,5,15,25]. Cytoplasmic-pattern (C-ANCA) most often have specificity for pro3 (PR-3) [4,10,13,16,18]; however, we have
recently identified with specificity
for
a small cationic
proportion protein
Perinuclear-pattern ANCA specificity
57
(P-ANCA)
for myeloperoxidase
of C-ANCA (CAP-57)
sera [11].
have
previously
as rabbit and capable
of
reported
mouse
stimulating
that
but autoantibody
ANCA
monoclonal respiratory
IgG,
anti-MPO burst
and
as well
IgG
are
degranula-
tion in human polymorphonuclear leukocytes (PMN) that are primed with recombinant tumor necrosis factor alpha (rTNFa) [9]. By fluorescence-activated cell sorter (FACS) analysis, we showed that P-ANCA and antiMPO gesting
© 1991
were bound to the that antigens that
Wiley-Liss,
Inc.
surface are
of primed
normally
autoantibodies
PMN granules are present on following priming with rTNFa. To bodies enon,
the
membrane
surface
demonstrate that the activation of PMN by antiagainst granule proteins is a generalized phenomwe now report that heterologous polyclonal and
monoclonal antibodies to a proteins cause PMN activation. now shown ies initiate
to enhance respiratory
variety of The effects
PMN granule of rTNFa are
the speed in which these antibodburst. FACS analysis demonstrat-
ing ANCA antigens PR-3 and microscopy demonstrating
CAP-57,
tron
MPO
and at the
confirm that ANCA antigens are present primed PMN where they can interact cause PMN activation.
immunoeleccell surface
at the surface with ANCA
of to
most often have
(MPO),
specificity for elastase and lactoferrin have also been observed [4,8]. We
cytoplasmic
confined
PMN
sugwithin
MATERIALS AND METHODS Cell Preparation PMN were purified from heparinized whole blood by Plasmagel (Cellular Products, Buffalo, NY) red blood cell (RBC) sedimentation followed by Ficoll-Hypaque
Received
December
4, 1990;
Reprint
requests:
J. Charles
University
of
Carolina
Bullitt
Building,
North CB
#7525,
accepted
February
Jennette, School
Chapel
Department of
Medicine,
27,
1991.
of Pathology, 815
Hill, NC 27599-7525.
Brinkhous-
540
Charles
et al.
density gradient centrifugation removed by hypotonic lysis in HEPES (10 mM) buffered tion with viability exclusion
[2] . Residual RBC were and PMN were resuspended Hank’s balanced salt solu-
calcium and magnesium (HHBSS was greater than 98% by trypan and the suspension was greater than
by LeukoStat NY). Cells (Sigma,
Wright-Giemsa were treated
St.
Louis,
with
MO)
stain 5.0
). Cell blue dye 98% PMN
prior
B
to stimula-
cells/45
106
by stimulants
mm
of
or with
was
granule of
fraction
The following were used: sheep human neutrophil CA); idase
IgG fractions anti-human elastase (The
of polyclonal cathepsin-G, Binding Site,
rabbit anti-human MPO, rabbit (lysozyme), rabbit anti-human
ulin, rabbit anti-human (DAKO, Carpinteria, Antibodies,
were
(Chemicon, the generous
used:
Temecula, gift of Dr.
Anti-human vided by
rabbit
albumin purified
anti-human
phosphatase CS (Atlantic anti-human
prior
(Cappel, West mouse monoclonal
double
stranded
CA); anti-human John Hoidal, Salt
All antibodies for 15 mm
DNA
PR-3 IgG Lake City,
was UT.
were diluted with HHBSS in an Eppendorf 5412 centri-
used
following
02 production was superoxide dismutase ferricytochrome and cytochalasin was purchased
performed (SOD)
by the inhibitable
measurement reduction
were pretreated up in quadruplicate
production ± SOD (125 pg/ml; 2 U/ml). Continuous
performed in the same with rTNFa, 2 X 106
ig/ml), assays
±
for riM)
inducing minimal O2 PMN were preincubated
by
15 mm, 37#{176}C.Ferricytochrome and HHBSS antibody,
or
and mixed leukocyte
15 mm. The cells F(ab)’., anti-mouse (Tago, Burlingame, and resuspended gen expression
in PBS/BSA. was performed
cell sorter unstimulated
was
antibody
Immunoelectron PMN equal 0.5%
resuspended with goat isothiocyanate (FITC) for 15 mm, washed,
Analysis of surface on a Coulter EPICS
at 488 nm comparing cells. Non-specific
conjugate
15 mm,
were washed, IgG-fluorescein CA), incubated
assessed
in
(10 M) were added, and PMN were incubated for another 45 mm, 37#{176}C.The absorbances of the resulting supernatants were scanned between 560 and 540 nm using a double beam spectrophotometer (Cary Model 1 l8C, Palo Alto, CA) and results are reported as nmole
antimodel
FMLP stimulated binding of the cells
stained
with
alone. Microscopy
(2 x
106/ml)
37#{176}C, as described
were
primed above.
with
To stop
rTNFa
for
the priming
an
volume of a mixture of 4% paraformaldehyde and glutaraldehyde in 0. 1 M phosphate buffer, pH 7.2, added to the cell suspension. For immunoelectron
fixative mixture for 45 mm. After the cells in phosphate buffer, a partial dehydration
C (4 mg/ FMLP
saline
(BSA) (Sigma) preincubated at
and were
pg/ or
the with rich
phosphate-buffered
1% bovine serum albumin at 0.5 x l06/ml. Cells were
was
of rTNFa to be the O2 pro-
itself (100 with rTNFa
heparin isolated
with to
manner except that after priming PMN were added to prewarmed
Fe3 cyto C and antibody. The priming dose used was determined in preliminary experiments lowest dose that enhanced antibody mediated duction while ml; 2 U/ml).
of of
at
Normal
the procedure. All washes and antibody dilutions were made with PBS/BSA. Cells were pelleted at 2,500 rpm, resuspended with a 1/50 dilution of mouse anti-human PR-3 or mouse anti-human CAP-57, and incubated for
secondary
(O2)
C [1]. SOD, fMLP, ferricytochrome C, B were obtained from Sigma. rTNFa from Genzyme, Boston, MA. In discon-
assays, 2 X 106 PMN cytochalasin B, and set 02 (100
Production
in
presence
CAP-57
stimulation.
with The
suspended
the
and
37#{176}C for 1 mm with l0_8 M FMLP or buffer and the reaction was quickly terminated with 1% paraformaldehyde which immobilized proteins at the cell surface. Leukocytes were maintained at 4#{176}C for the remainder of
FITC
of Superoxide
was
produced obtained
respectively.
to demonstrate PR-3
neutrophils
(PBS) with (PBS/BSA)
753 with
to use.
Measurement
HHBSS ml; 32.3
lac-
CAP-57 ascites was most generously proDrs. Anna Pererira and John Spitznagel,
Atlanta, GA. and centrifuged
measure ±rTNFa
ME);
rabbit anti-mouse PA). The following
antibodies
anti-human muramalpha-2-macroglob-
placental alkaline goat anti-human
Scarborough,
toferrmn, Chester,
tinuous 5 pg/ml
CA);
antibodies sheep antiSan Diego,
variabil-
the 02 the results
rTNFa,
constituents
human blood was collected Plasmagel as noted above.
Antibodies
Donor-to-donor
Studies
cytometry
the
[17].
for by subtracting plus rTNFa from
without
Flow Cytometry
surface
tion.
fuge
X
ity was compensated by buffer or buffer
Flow
(Fisher, Orangeburg, pg/ml cytochalasin
immediately
O2/2
microscopy,
post-fixation
was
performed
with
the
same
were washed was done
through a series of graded ethanol (50%, 5 mm; 70%, 5 mm; 55%, 10 mm, two changes). An intermediate step was introduced before embedding to avoid cell shrinkage: the cells were transferred to a 2:1 mixture of LR White (Polysciences, Warrington, PA) and 95% ethanol for 1 hr. After two changes in pure LR White for 1 hr each, the cells were infiltrated overnight at 4#{176}C. A last change of resin was done at room temperature for 1 hr before embedding in fresh LR White in gelatin capsules. Polymerization was Thin sections
performed of primed
under UV or unprimed
light for 18-24 PMN collected
hr. on
uncoated 300-mesh nickel grids and dried overnight were first incubated face down, floating in a 20 tl drop of normal goat serum for 30 mm. The grids were transferred
Antibodies TABLE
1. 02a Production
Antibody
by PMN
Dose
MPO
Elastase
Cathepsin
0
Lactoferrin
PR-3
Granule
(g/ml)
Alkaline
phosphatase
Abbreviations: X
bAll
PMN
were
cNot
statistically
*P