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

Antibodies against granule proteins activate neutrophils in vitro.

Polymorphonuclear leukocyte (PMN) respiratory burst was stimulated by heterologous antibodies against PMN granule proteins but not by control antibodi...
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